Radiopharmaceuticals For Sentinel Lymph Node Detection - Status and Trends

IAEA RADIOISOTOPES AND RADIOPHARMACEUTICALS SERIES No.
RADIOPHARMACEUTICALS
FOR SENTINEL LYMPH
NODE DETECTION:
STATUS AND TRENDS
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RADIOPHARMACEUTICALS FOR
SENTINEL LYMPH NODE DETECTION:
STATUS AND TRENDS
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AFGHANISTAN GHANA OMAN

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ALGERIA GUATEMALA PALAU
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IAEA RADIOISOTOPES AND RADIOPHARMACEUTICALS SERIES No. 6
RADIOPHARMACEUTICALS FOR
SENTINEL LYMPH NODE DETECTION:
STATUS AND TRENDS
INTERNATIONAL ATOMIC ENERGY AGENCY

VIENNA, 2015
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STI/PUB/1674
IAEA Library Cataloguing in Publication Data

Radiopharmaceuticals for sentinel lymph node detection : status and trends. —
Vienna : International Atomic Energy Agency, 2015.
p. ; 24 cm. — (IAEA radioisotopes and radiopharmaceuticals series,
ISSN 2077–6462 ; no. 6)
STI/PUB/1674
ISBN 978–92–0–109714–9
Includes bibliographical references.
1. Radioactive tracers. 2. Lymph nodes — Biopsy. 3. Cancer — Diagnosis.
4. Radiopharmaceuticals. I. International Atomic Energy Agency. II. Series.
IAEAL15–00951
FOREWORD
In a continuous effort to support and promote scientific research in cancer

diagnosis and therapy, the IAEA organized a coordinated research project (CRP)
on the Development of 99mTc Radiopharmaceuticals for Sentinel Node Detection
and Cancer Diagnosis. This project stemmed from results obtained from a
previous IAEA CRP on the Labelling of Small Biomolecules with Novel 99mTc
Cores. The CRP on Development of 99mTc Radiopharmaceuticals for Sentinel
Node Detection and Cancer Diagnosis was followed by a technical meeting on
the Current Role and Trends of Hybrid Imaging and Radioguided Surgery, which
focused on the use of radioactive imaging agents as a method for identifying
diseased tissues that require surgical removal. The most widely recognized
example of radioguided surgery, known as sentinel lymph node detection (SLND)
or sentinel lymph node biopsy, is currently employed for planning the therapeutic
treatment of superficial cancers such as breast cancer and skin melanoma. The
basic concept behind this method is that, for this type of cancer, the spread of
the metastases from the primary tumour site always occurs through lymphatic
drainage, and thus the most proximal (sentinel) lymph node will be the first to
receive the metastatic invasion. It follows that, after surgical resection of the
primary tumour, the precise localization of this sentinel lymph node allows its
removal and biopsy to assess whether the metastatic process has already affected
it. This information has proven to be crucial for planning the further therapeutic
treatment of the patient. In fact, if histological analysis of the node is negative,
i.e. devoid of cancerous cells, there is no need to resort to surgical dissection of
the lymph nodes, which is of clear benefit to the patient.
Various types of nanocolloidal particles labelled with 99mTc are currently
used as SLND imaging agents. However, they have suboptimal properties
because of their unspecific uptake in the lymph node. An alternative, molecular
uptake mechanism for this class of tracers that exploits the presence of receptors
for mannose on the outer macrophage’s membrane, has recently been proposed.
This entails the design of a multifunctional ligand that is able to accommodate
a number of mannose residues along with a suitable chelating group for the
radiometal. Dextran provides a convenient macromolecular scaffold for appending
a relatively large number of functional groups, and its effically has recently been
demonstrated through the development of a new 99mTc radiopharmaceutical for
SLND (marketed under the name Lymphoseek) that targets mannose receptors
on macrophages hosted in lymph nodes. This new compound can be considered
the first outstanding achievement of this novel approach to SLND and provides
the basis for developing alternative SLND imaging agents following the same
molecular design, but using different and more sophisticated chemical strategies
for incorporating the radiometal into the dextran mannose multifunctional
framework.
The first of three research coordination meetings (RCMs) of the CRP
was held on 12–16 November 2007 at the IAEA in Vienna, and was attended
by 18 participants from 17 countries. The second RCM was held in Athens on
18–22 May 2009, and was attended by 16 participants and 3 observers. The final
RCM took place at the IAEA Headquarters in Vienna, on 22–26 November 2010,
and was attended by 17 participants and 2 observers.
The technical meeting on radioguided surgery took place on
2–7 September 2012 at the IAEA Headquarters, with the participation of
15 international experts in the field.
The present publication is based on the extensive analysis carried out during
the CRP and technical meeting and all the contributing authors were participants
at these meetings. It is specifically focused on radiopharmaceuticals for SLND
and thus constitutes a unique example of a publication providing an updated
description of the status of the field. The invaluable contribution of R. Pasqualini
(France) in coordinating the biological work carried out during the CRP and
reviewing it in Chapter 8 of this report is gratefully acknowledged.
The IAEA officer responsible for this publication was A. Duatti of the
Division of Nuclear Sciences and Applications.
EDITORIAL NOTE
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judgement by the publisher, the IAEA, as to the legal status of such countries or territories, of
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The mention of names of specific companies or products (whether or not indicated as
registered) does not imply any intention to infringe proprietary rights, nor should it be construed
as an endorsement or recommendation on the part of the IAEA.
The authors are responsible for having obtained the necessary permission for the IAEA
to reproduce, translate or use material from sources already protected by copyrights.
CONTENTS
CHAPTER 1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2. Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3. Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.4. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
CHAPTER 2. BASIC DESCRIPTION OF

THE LYMPHATIC SYSTEM FROM
THE PERSPECTIVE OF SLN UPTAKE
OF RADIOACTIVE TRACERS . . . . . . . . . . . . . . . . . . . . . 7
R. Pasqualini, E. Janevik-Ivanovska
2.1.
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.3.
The lymphatic system: A general view . . . . . . . . . . . . . . . . . . . . 8
2.4.
Structure of the lymphatic network . . . . . . . . . . . . . . . . . . . . . . 8
2.4.1. Lymphatic capillaries . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.4.2. Lymphatic vessels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.4.3. Lymphatic trunks and collecting ducts . . . . . . . . . . . . . . 10
2.5. Lymph production and movement . . . . . . . . . . . . . . . . . . . . . . . 11
2.5.1. Role of the interstitial space . . . . . . . . . . . . . . . . . . . . . . 11
2.5.2. Formation of lymph . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.6. Lymph nodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.6.1. Structure of a lymph node . . . . . . . . . . . . . . . . . . . . . . . . 13
2.6.2. Function of lymph nodes . . . . . . . . . . . . . . . . . . . . . . . . 15
2.7. The SLN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.7.1. The SLN concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2.7.2. Physicochemical aspects of lymph node delivery
of biologically inert agents . . . . . . . . . . . . . . . . . . . . . 17
2.7.3. Receptor based radiotracers for SLND . . . . . . . . . . . . . . 24
2.8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
References to Chapter 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
CHAPTER 3. CLINICAL APPLICATIONS OF
RADIOGUIDED SLNB . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
F. Orsini, G. Mariani
3.1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.3. SLNB in breast cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.3.1. Indications and contraindications for
SLNB in breast cancer . . . . . . . . . . . . . . . . . . . . . . . . 40
3.3.2. SLNB procedures in breast cancer . . . . . . . . . . . . . . . . . 42
3.4. SLNB in CM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.4.1. Indications and contraindications to
SLNB in melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.4.2. SLNB procedures in melanoma . . . . . . . . . . . . . . . . . . . 52
3.5. Other applications of radioguided SLNB . . . . . . . . . . . . . . . . . . 56
3.5.1. SLNB in head and neck cancers . . . . . . . . . . . . . . . . . . . 57
3.5.2. SLNB in gastrointestinal cancers . . . . . . . . . . . . . . . . . . 58
3.5.3. SLNB in tumours of the female reproductive system . . 60
3.5.4. SLNB in tumours of the male reproductive system . . . . 60
CHAPTER 4. TECHNETIUM-99m TILMANOCEPT:

A SYNTHETIC RECEPTOR TARGETED
MOLECULE FOR SLNM . . . . . . . . . . . . . . . . . . . . . . . . . . 73
D.R. Vera, C.K. Hoh, D.J. Hall, C.A. Tokin,
A.M. Wallace
4.1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
4.2.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
4.2.2. Ideal sentinel node imaging agent . . . . . . . . . . . . . . . . . 75
4.2.3. Technetium-99m tilmanocept . . . . . . . . . . . . . . . . . . . . . 75
4.2.4. Lymphoseek . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
4.3. Preclinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.3.1. Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4.3.2. Injection site clearance . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.3.3. SLN accumulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.3.4. SLN retention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
4.3.5. Biodistribution, toxicity and absorbed radiation dose . . 83
4.4. Clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.4.1. Phase 1 clinical trials . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
4.5. Retrospective comparative studies . . . . . . . . . . . . . . . . . . . . . . . 87
4.5.1. Comparison to radiocolloids . . . . . . . . . . . . . . . . . . . . . . 87
4.6. Additional imaging modalities . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.6.1. Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.6.2. PET imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
4.6.3. Fluorescence imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
CHAPTER 5. A NEW CLASS OF 99mTc(I) AGENTS FOR SLND:

CHEMICAL DESIGN AND SYNTHESIS . . . . . . . . . . . . . 95
M. Morais, J.D.G. Correia, I. Santos, M. Pelecanou,
I. Primettis, M. Papadopoulos
5.1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
5.2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
5.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
CHAPTER 6. A NEW CLASS OF 99mTc(I) AGENTS FOR SLND:

LABELLING AND QUALITY CONTROL . . . . . . . . . . . . 109
M. Morais, J.D.G. Correia, I. Santos, M. Pelecanou,
I. Primettis, M. Papadopoulos
6.1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

6.2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
6.3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
6.3.1. Radiolabelling with [99mTc(CO)3]+ . . . . . . . . . . . . . . . . . 110
6.3.2. Quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
6.3.3. Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
CHAPTER 7. 4 + 1 Tc(III) COMPLEXES IN THE DESIGN AND

DEVELOPMENT OF 99mTc LABELLED DEXTRAN
MANNOSE DERIVATIVES AS POTENTIAL
RADIOPHARMACEUTICALS FOR SLND . . . . . . . . . . . 117
A. Rey, H.-J. Pietzsch
7.1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
7.2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
7.3. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
7.3.1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
7.3.2. Synthesis of dextran derivative with
isocyanopropyl arms . . . . . . . . . . . . . . . . . . . . . . . . . . 121
7.3.3. Radiolabelling and control of radiochemical purity . . . . 122
7.3.4. Biological evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
7.4. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
7.4.1. Synthesis and characterization of dextran derivatives
with isocyano propyl arms . . . . . . . . . . . . . . . . . . . . . 124
7.4.2. Radiochemical purity and stability of
99m
Tc dextran derivatives . . . . . . . . . . . . . . . . . . . . . . 124
7.5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
CHAPTER 8. BIOLOGICAL EVALUATION OF LOW VALENCE

99m
Tc DEXTRAN DERIVATIVES FOR SLND:
RESULTS OF A MULTILABORATORY STUDY . . . . . . . 133
R. Pasqualini
8.1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

8.2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
8.3. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
8.3.1. Synthesis of dextran derivatives . . . . . . . . . . . . . . . . . . . 137
8.3.2. Physical characterization of dextran derivatives . . . . . . 138
8.3.3. Radiolabelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
8.3.4. Control of radiochemical purity . . . . . . . . . . . . . . . . . . . 140
8.3.5. Determination of partition coefficient of some
99m
Tc(CO)3 dextran derivatives . . . . . . . . . . . . . . . . . 141
8.4. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
8.4.1. Synthesis and characterization of dextran derivatives . . 143
8.4.2. Physical characterization of dextran derivatives . . . . . . 143
8.4.3. Radiochemical purity and stability of 99mTc dextran
derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
8.4.4. Octanol water partition coefficients . . . . . . . . . . . . . . . . 144
8.4.5. Biodistribution studies . . . . . . . . . . . . . . . . . . . . . . . . . . 146
8.4.6. Gamma camera imaging . . . . . . . . . . . . . . . . . . . . . . . . . 151
8.5. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
8.6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
CONTRIBUTORS TO DRAFTING AND REVIEW . . . . . . . . . . . . . . . . . . 159

Chapter 1
INTRODUCTION
1.1. BACKGROUND
A sentinel lymph node (SLN) is defined as the first lymph node to which
cancer cells are most likely to spread from a primary tumour. In the case of
established cancerous dissemination, it is postulated that SLNs are the target
organs primarily reached by metastasizing cancer cells from the tumour. This
is because the spread of some forms of cancer usually follows an orderly
progression, spreading first to regional lymph nodes because the flow of lymph is
unidirectional. In these cases, if the cancer spreads, it will migrate first to lymph
nodes (lymph glands) close to the tumour before reaching other parts of the body.
SLN surgery determines if the cancer has spread to the very first draining
lymph node or not. Sentinel lymph node biopsy (SLNB) is a procedure in which
the SLN is identified, removed and examined to determine whether cancer cells
are present. If the SLN does not contain cancerous cells, then there is a high
likelihood that the cancer has not spread to any other area of the body. Hence,
a negative SLNB result suggests that the cancer has not developed the ability
to spread to nearby lymph nodes or other organs. Conversely, a positive SLNB
result indicates that the cancer is present in the SLN and may be present in other
nearby lymph nodes (called regional lymph nodes) and, possibly, other organs.
This information can help a physician determine the stage of the cancer (the
extent of the disease within the body) and develop an appropriate treatment plan.
There are various advantages to the SLN procedure. First and foremost, it
decreases unnecessary lymph node dissections where this is not necessary, thereby
reducing the risk of lymphoedema, which is a common complication of this
procedure. Increased attention to the node(s) identified as most likely to contain
metastases is also more likely to detect micrometastases and result in staging and
treatment changes. The main uses are in breast cancer and malignant melanoma
surgery, although the procedure has been used in other tumour types with some
degree of success. The average sensitivity of the SLNB is approximately 90%,
with a false negative rate of about 10%. A positive histological result indicates
that malignant cancer cells have migrated to some of the SLNs. Conversely,
SLNs that are not affected by malignant cells will give a negative result.
The basic technique of sentinel node identification involves the injection of
a tracer that identifies the lymphatic drainage pathway from a primary tumour.
The earliest applications used a vital blue dye (VBD), usually isosulphan
blue. Although isosulphan blue is generally safe, anaphylaxis can occur in up
1
to 1% of patients. Other blue dyes such as methylene blue have had similar
success rates in a small series of studies. Subsequent reports describe the use of
radioisotopes such as 99mTc bound colloids for preoperative lymphoscintigraphy
and intraoperative localization using a gamma probe. The most commonly used
radiolabelled colloid in the United States of America is 99mTc sulphur colloid,
whereas 99mTc nanocolloid and 99mTc antimony sulphide are more commonly
used in Europe and Australia, respectively.
The ideal radiotracer should show rapid transit towards sentinel nodes,
with persistent retention in the nodes. In general, the drainage, distribution and
clearance of radioactive colloids by the lymphatic system vary and are dependent
on particle size. Small particles are drained and cleared first, and large particles are
drained and cleared last, and may be retained virtually indefinitely at the injection
site. The distribution of particle sizes within each radioactive colloid preparation
is rather diverse, and is a major determinant of the kinetics of tracer clearance
and thus SLN visualization. While smaller particles allow quick visualization of
SLNs, larger particles have the advantages of longer tracer retention in the SLN,
which permits intraoperative detection the following day, and slow transit in the
lymphatic system, which minimizes visualization of lymph nodes downstream
of the SLN. Additionally, particles smaller than 4–5 nm may penetrate capillary
membranes without adequate retention in the lymph nodes. It is believed that a
particle size range of 100–200 nm is the best compromise between the conflicting
needs for efficient, fast, lymphatic drainage and satisfactory retention in the SLN.
Evidently, conventional sentinel lymph node detection (SLND) imaging agents,
such as nanocolloids, are non-specific particles or macromolecules because
their uptake mechanism is driven by passive diffusion to follow the lymphatic
drainage and accumulate within the lymph node. They generally lack the ideal
imaging properties of rapid injection site clearance and high SLN extraction.
New radiotracers have been developed for SLND with the aim of
improving performance. The molecular imaging (MI) paradigm has guided the
design of these new SLND tracers. The ultimate goal of MI is the non-invasive
localization and quantification of certain molecular events in vivo. It is the
visualization, characterization and measurement of biological processes at the
molecular and cellular levels in humans and other living systems. As with other
imaging applications, MI requires fixation of a signal emitting molecule (e.g. one
containing a radioactive, fluorescent or paramagnetic label) within the cell or
tissue where the target is expressed. There are several different mechanisms by
which this can be brought about. In the receptor–ligand model, one signal
molecule (the ligand) binds with high affinity to a specific site on a target
molecule (the receptor), which has a stationary biodistribution over the period
of imaging. The key determinants of success for this type of imaging are the
specificity and affinity of the receptor–ligand interaction, as well as the receptor
2
density itself. The receptor–ligand pair does not necessarily have to mimic a
naturally occurring interaction, but can be made to bind with high specificity to
molecules that are not considered to have any intrinsic biological activity.
Following this receptor targeted molecular approach, the design of new
and more specific SLND diagnostic tracers has been undertaken. In Chapter 4,
Vera et al. report the synthesis of 99mTc diethylenetriaminepentaacetic acid (DTPA)
mannosyl dextran, which is designed to bind to mannose receptor type C (MRC)
within the lymphoid tissues for SLN imaging. The MRC is a 165 kD membrane
glycoprotein, and MRC belongs to the C type lectin superfamily. These proteins
are involved in mediating phagocytosis of microbes and are highly expressed on
the surface of macrophages hosted in lymphoid tissues. The new SLND agent
99m
Tc DTPA mannosyl dextran consists of a polysaccharide backbone (dextran)
with an average molecular weight (MW) of 9500 g/mol, bearing pendant arms
containing 55 mannose glycosides for high affinity binding to MRCs, and 8
DTPA groups for 99mTc chelation. The resulting linear molecule has a diameter
of 7.1 nm.
In this compound, dextran plays the role of a macromolecular scaffold
to accommodate a number of different functional groups, each performing a
specific chemical or biological function (multifunctional ligand). By applying the
same strategy, in the course of the coordinated research project (CRP) entitled
Development of 99mTc Radiopharmaceuticals for Sentinel Node Detection and
Cancer Diagnosis, research efforts have been undertaken which the dextran
backbone is further functionalized using different types of chelators for
binding to different 99mTc metallic fragments including [99mTc(CO)3]+ (99mTc
carbonyl), [99mTc ≡N]2+ (99mTc nitrido), [99mTc(SNS)] (99mTc 4 + 1) and [99mTc
Hydrazinonicotinic acid] cores. These metallic cores allow the use of highly
sophisticated chemical methods for labelling the dextran derived multifunctional
mannosylated ligand in high specific activity while affording robust 99mTc
conjugates. The resulting multifunctional ligands are essentially similar to
DTPA mannosyl dextran, but differ in the coordination mode to the selected
99m
Tc fragment.
The MI paradigm is changing the approach of medicine to the treatment
of disease, particularly for cancer treatment. A living organism is viewed as
a chemical fabric where an intricate and astonishingly complex network of
biochemical processes is acting to sustain life. A fundamental switch in the
clinical perspective has been brought about by the recognition that treatment
of pathological conditions should be approached at the molecular level.
Nuclear medicine is always at the forefront of this revolution because its basic
approach is intrinsically molecular, as evidenced by the use of single molecule
radioactive probes for investigating inner cellular and subcellular processes.
Owing to its inherent molecular trait, the progress of nuclear medicine is
3
tightly bound to the constant development of new radiolabelled agents capable
of detecting increasingly smaller details of the underlying molecular substrate.
The evolution of radiolabelled diagnostic agents for SLND offers a notable
example of this transition towards more specific molecular probes, as witnessed
by the development of macrophage-receptor-specific tracers that are expected to
improve the sensitivity of this diagnostic modality. The IAEA promptly captured
this ongoing scientific advancement by promoting a CRP and a technical
meeting on this subject. Therefore, the results of these activities, as presented
in this publication, constitute a significant example of real time contribution to
the progress of a specific scientific discipline and a highly valuable reference
material for operators working with this clinical methodology.
1.2. OBJECTIVE
The scientific objectives of the CRP can be outlined as follows:
—— Design and synthesis of dextran multifunctional ligands with pendant

mannose residues for interaction with the macrophage’s receptors and
suitable chelating groups for binding to novel 99mTc cores;
—— Preparation of the corresponding 99mTc labelled conjugates with high yield
and in high specific activity;
—— Analytical characterization of the multifunctional ligands and of the
corresponding radiolabelled conjugates;
—— In vitro evaluation of stability and binding affinity for mannose
receptors and in vivo biological evaluation in animal models for sentinel
node detection;
—— Development of a ‘cold kit’ formulation for easy and reliable preparation
of the new 99mTc radiopharmaceuticals to be distributed in Member States.
1.3. SCOPE
The main outcomes of the CRP are described in this publication, along
with results of the extensive analysis of the status and perspectives of SLND
carried out during the technical meeting on Current Role and Trends of Hybrid
imaging and radioguided surgery. Thus, the present publication is intended to
provide a broad illustration of SLND methodology by covering basic principles,
clinically established applications and recent advancements brought about
after the advent of the MI paradigm and the development of dextran mannose
multifunctional ligands.
4
1.4. STRUCTURE
In Chapter 2, an overview of the biology and physiology of the lymphatic

system and of the basic uptake mechanisms underlying the use of radiolabelled
colloids as imaging agents for SLND is presented. Chapter 3 reviews the most
important clinical applications of the SLND approach with emphasis, in particular,
on radioguided surgery. Chapter 4 reports on application of the novel molecular
approach to the development of more specific and selective SLND tracers by
illustrating the preparation and diagnostic properties of the first dextran mannose
SLND imaging agent 99mTc tilmanocept (Lymphoseek). Chapters 5–8 describe
the most important results of the CRP, with particular emphasis on chemical
characterization and preclinical evaluation in animal models of three new SLND
MI agents with a similar structure based on the dextran mannose multifunctional
platform, but specifically tailored to the chemistry of the novel 99mTc cores.
5
Chapter 2
BASIC DESCRIPTION OF THE LYMPHATIC SYSTEM

FROM THE PERSPECTIVE OF SLN UPTAKE
OF RADIOACTIVE TRACERS
R. PASQUALINI
CIS bio international/IBA, Saclay, RP Innovative,
Clamart, France
E. JANEVIK-IVANOVSKA
Faculty of Medical Sciences,
Goce Delcev University,
Stip, The Former Yugoslav Republic of Macedonia
2.1. SUMMARY
The lymphatic system is part of the circulatory system. It is closely

associated with the cardiovascular system because it includes a network of vessels
that contributes to liquid transportation throughout the body. This circulatory
system is essential for the maintenance of interstitial fluid balance, uptake of
dietary fat and for body defence against invasion by disease causing agents.
Lymph nodes, which contain large numbers of B and T lymphocytes and
macrophages, are located along lymphatic pathways. They have two primary
functions: filtering and digesting potentially harmful particles from lymph before
returning it to the bloodstream and contributing to the immune surveillance
provided by lymphocytes and macrophages. In their function of filtering particles
or cell debris, lymph nodes may collect cancer cells that are breaking and
travelling away from the primary tumour. The spread of some forms of cancer
usually follows an orderly progression, spreading first to regional lymph nodes,
then the next rank of lymph nodes and so on. Therefore, the first lymph node
(the SLN) is more likely than other lymph nodes to contain cancer cells. If a
suitable radioactive tracer, generally a nanocolloid or a dye, is administrated in
the proximity of the tumour site, it will travel through the lymphatic system and
be trapped in the SLN, allowing its localization using an appropriate probe or by
visual determination. The size and the charge of the radioactive tracer will mainly
influence the extent of radioactivity remaining at the site of injection, the rate of
diffusion into the lymphatic vessels and the uptake in the SLN. Knowledge of the
7
physiology of the lymphatic system will help to identify factors influencing the
diffusion and uptake mechanism of radioactive tracers in the lymph nodes and
will assist in the design of more efficient and selective SLN seeking drugs.
2.2. INTRODUCTION
The purpose of this chapter is to provide readers with a basic description

of the structure and function of lymph nodes, with a particular interest in the
mechanisms that might mediate the uptake of radioactive tracers in these
structures. In this regard, many aspects of the physiology of the lymphatic
system, mainly those related to the formation and transport of lymphatic fluid,
will not be developed comprehensively. Several excellent articles have appeared
recently describing the microstructure and physiology of the lymphatic system
[2.1–2.6]. Readers are encouraged to consult these references, which have been
of constant help when writing the chapter that follows.
2.3. THE LYMPHATIC SYSTEM: A GENERAL VIEW
The lymphatic system is closely associated with the cardiovascular system.

Similar to the latter, it includes a network of vessels that contributes to liquid
transportation throughout the body. Lymphatic vessels transport excess fluid
away from the interstitial space between cells in most tissues and return it to the
bloodstream, as schematically depicted in Fig. 2.1.
The lymphatic system is found throughout the body, with the exception
of the central nervous system, where cerebrospinal fluid fulfils the normal role
of lymph. This circulatory system is essential for the maintenance of interstitial
fluid balance, uptake of dietary fat and the body’s defence against invasion by
disease causing agents.
2.4. STRUCTURE OF THE LYMPHATIC NETWORK
The lymphatic network begins as lymphatic capillaries merge to form

lymphatic vessels, which in turn converge to form large vessels that eventually
join the blood stream via the right lymphatic duct [2.7].
8
FIG. 2.1.  Lymphatic vessels, which are closely associated with blood vessels, transport
fluid from interstitial spaces to the bloodstream (graphical material provided by Servier
Medical Art).
2.4.1. Lymphatic capillaries
Lymphatic capillaries are closed ended tubes (see Fig. 2.2). They are
10–60 µm in diameter, and, like blood capillaries, are composed of an endothelial
cell layer. However, the gaps between the endothelial cells in the lymphatic
capillaries are larger than those found in the blood capillaries [2.8, 2.9]. These
endothelial clefts can open to dimensions of several micrometres, allowing
macromolecules, colloids, cells and cellular debris to pass unhindered, depending
on the degree of distension [2.10–2.13]. The lymphatic capillaries are thus
more permeable and larger than blood capillaries, allowing for easier entrance
of relatively large protein particles. Attached to the lymphatic capillaries are
anchoring filaments, which contain elastic fibres. These anchoring filaments
extend out from the lymphatic capillary, attaching lymphatic endothelial cells
to surrounding collagen, and prevent endothelial cells from collapsing when
interstitial fluid pressure increases.
9
FIG. 2.2.  Tissue fluid enters lymphatic capillaries through flap like valves between adjacent
cells. Anchoring filaments to tissue cells are not drawn (graphical material provided by Servier
Medical Art).
2.4.2. Lymphatic vessels
Lymphatic vessels form as lymph capillaries merge. The walls of lymphatic

vessels are similar to those of veins, although thinner. Morphologically, the walls
are composed of three layers: an endothelial lining, a middle layer of smooth
muscle and elastic fibres, and an outer layer of connective tissue. Like veins,
semilunar valves are present to prevent the backward flow of lymph.
2.4.3. Lymphatic trunks and collecting ducts
Lymphatic trunks form from the union of collecting vessels. They are named
after the regions they serve: lumbar, jugular, subclavian, bronchomediastinal and
intestinal trunks. Except for the latter trunk, each of them occurs in pairs, left and
right, for each side of the body. These lymphatic trunks then join one of the two
collecting ducts, the thoracic duct or the right lymphatic duct. These ducts empty
into the left and right subclavian veins, respectively.
Figure 2.3 is a view of the lymphatic and blood network in the region of
fluid interchanges.
10
FIG. 2.3.  Relationship between the lymphatic and blood capillaries (graphical material
provided by Servier Medical Art, modified).
2.5. LYMPH PRODUCTION AND MOVEMENT
2.5.1. Role of the interstitial space
The interstitial space located between the capillary walls and cells is
composed of two phases [2.14]:
—— An interstitial fluid containing salts and plasma proteins;

—— An extracellular matrix (ECM), which is constituted of fibrous protein
(predominantly collagen, but also elastin, fibronectin and laminin) and
glycosaminoglycans (GAGs) [2.15].
Collagen fibres provide much of the structural framework of the tissue.

There are several types of collagen, with type I being predominantly found in the
skin. All collagens consist of three polypeptide α chains with high hydroxyproline
(Hyp) content and a very common sequence Gly-Pro-X and
Gly-X-Hyp (Gly = glycine, Pro = proline, X = proline or 4-hydroxyproline) [2.16].
Collagen I possess a slightly positive charge (pI ~ 8, where pI = –logI,
I = ionic strength) at physiological pH [2.17].
Glycosaminoglycans are unbranched polysaccharide chains composed of
repeating disaccharide units. One of the two sugars is always an amino sugar
(N-acetylglucosamine or N-acetylgalactosamine), which, with the exception of
11
hyaluronic acid, is sulphated. The second sugar is usually glucoronic or iduronic
acid [2.18]. Because there are sulphate and/or carboxyl groups on most of their
sugars, GAGs are highly negatively charged. With the exception of hyaluronic
acid, these GAG chains attach to a protein core to make up larger molecules
called proteoglycans (PGs), and, in turn, PG subunits are combined to form large
aggregates, which can have MWs as large as 108 g/mol.
Collagen and PGs will restrict the space available to macromolecules in
the interstitial space by steric effects. Additionally, because of their high density
negative charge at a physiological pH, PGs will exert an electrostatic exclusion
effect on negatively charged macromolecules [2.14]. This results in the presence
of narrow aqueous tissue channels of approximately 100 nm diameter, allowing
the passage of macromolecules by diffusion [2.19]. Therefore, it seems reasonable
to speculate that the size of administered colloids should be less than 100 nm in
diameter if good drainage from the injection site has to be achieved.
2.5.2. Formation of lymph
Tissue fluid is composed of water and dissolved substances that migrate

from blood capillaries by diffusion and filtration. Tissue fluid contains, among
other small molecules and gas, some relatively small proteins. Usually, these
small proteins are not reabsorbed when water and dissolved molecules travel back
into the venule ends of these capillaries. As a result, the protein concentration of
the tissue fluid tends to rise, increasing the osmotic pressure of the fluid in the
interstitial space. This increasing interstitial pressure forces some of the tissue
fluid into the lymphatic capillaries, where it becomes lymph. Because of the high
permeability of lymphatic walls, there is no exclusion of interstitial molecules,
irrespective of their size. The protein composition of lymph is nearly equivalent
to that of interstitial fluid, which is similar to, albeit less concentrated than, that
of blood plasma, from which it originates [2.1]. The only exception occurs for
intestinal lymph, which contains a high amount of fat resorbed directly from
the intestine.
Lymph, like venous blood, is under relatively low hydrostatic pressure. Flow
through the lymphatic vessels cannot occur without the intervention of outside
help. These outside forces include contraction of skeletal muscles, pressure
changes owing to the action of breathing muscles and, for large lymphatic trunks,
contraction of the wall’s smooth muscles. Movement of the lymph in the vessels
is unidirectional because the presence of valves prevents backflow [2.20].
12
2.6. LYMPH NODES
Lymph nodes are located along the lymphatic pathways, particularly

where lymphatic vessels merge to form trunks. They contain a large
number of lymphocytes and macrophages, whose role is to combat against
invading microorganisms.
2.6.1. Structure of a lymph node
Lymph nodes may vary in size and shape, ranging from a few millimetres
to a maximum of 2.5 cm long in their normal state, and are often bean shaped.
Figure 2.4 illustrates a section of a typical lymph node.
Lymph nodes consist of multiple lymphoid lobules. This structure, which
is the basic anatomical and functional unit of a lymph node, is surrounded by
sinuses, in which lymph flows, and enclosed by a capsule. The smallest lymph
nodes may contain very few (or even one single) lymphoid lobules, while the
largest ones may comprise a great number of such structures.
Only the principal aspects of the anatomical structure and function of
lymph nodes that are related to the mechanism of uptake of radiotracers will be
FIG. 2.4.  Section of a lymph node.
13
covered here. For a more detailed description, readers are invited to refer to the
previously mentioned references [2.1–2.6].
Schematically, lymphoid lobules are arranged side by side, radiating from
the hilum, at the indented region of the lymph node, to which they are anchored,
separated from the capsule by the sinuses. The hilum is the portion through which
the blood vessels connect with the node. The afferent vessels enter separately
at various points of the convex surface, but the efferent lymphatic vessel exits
from the hilum. Because lymph nodes have fewer efferent vessels than afferent
vessels, the lymph flows slowly and stagnates somewhat within the node. This is
important because it allows time for the generation of an immune response and
for macrophages to remove debris from the lymph before it re-enters the blood
vascular system. Because each afferent vessel collects lymph from a different
drainage region, each lymphoid lobule is potentially exposed to different sets of
antigens, antigen-presenting cells and inflammatory mediators [2.21]. As a result
of this heterogeneous immunological stimulation, lobules within the same lymph
node may have different levels of immunological activity.
The entire lymph node is filled with a fine reticular meshwork composed of
spindle shaped or elongated fibroblastic reticular cells and their reticular fibres
that form the basic supporting structure of the lobules and criss-cross the lumens
of the sinuses [2.22]. This causes the lobule to subdivide into a large number
of narrow channels and interstices, 10–20 µm wide, which are occupied by
lymphocytes, macrophages and antigen-presenting cells.
In the sinuses, the reticular meshwork has thinner branches and
correspondingly larger interstices than the lobular reticular meshwork [2.23].
Macrophages, also known as sinus histiocytes, stick to this reticular meshwork
and trap bacteria, cell debris, red blood cells and other particulates suspended
in the lymph as it flows through the meshes of this biological filter. They tend
to occur in clusters; their number increases in response to the need for particle
clearance and they may completely fill the sinuses. However, an supportive
(reticular) cells in lymphoid tissue can also ingest foreign particles and cell
debris, but with less efficiency than macrophages.
Ludwig has shown that two types of structural configurations between
lymph vessels and lymph nodes may exist [2.24]. In the first type, as illustrated
in Fig. 2.4, the lymph node receives lymph from the afferent duct. The lymphatic
liquid passes through the lymph node, which filters it, and then empties into
the efferent channel. In the other configuration, the lymphatic vessel develops
through the lymph node, or over its surface, without allowing emptying of its
contents into that node. As pointed out by Tanis et al. [2.25], if the first lymph
node adopts the latter configuration, the tumour cells transported in the lymph
will not be trapped in the lymph node. This situation could be one of the
explanations of false negative SLND with radiocolloids.
14
2.6.2. Function of lymph nodes
Lymph nodes have two primary functions:
—— They prevent the spread of microorganisms and toxins that enter interstitial
fluids by filtering potentially harmful particles from lymph before returning
it to the bloodstream. Furthermore, they destroy bacteria, toxins and
particulate matter through the phagocytic action of macrophages.
—— They operate an immune surveillance, which is provided by lymphocytes
and macrophages (the immune system function will not be treated further
in this chapter).
2.6.2.1. Filtering function of lymph nodes
The rationale for SLND with radioactive colloids is based on the ability of
lymph nodes to retain foreign particles that are present in the lymphatic fluid.
As described earlier, the labyrinthine structure of the reticular meshwork,
in which lymph slowly percolates, and the presence of macrophages and reticular
cells lined on it, allows sequestration of foreign particles or products from cell
degradation. The lymph nodes appear to provide two main types of filtration:
the first is a simple mechanical passage through the reticular meshwork and the
second involves a biological reaction with the phagocytic elements. Receptors on
the phagocytes and reticular cells are able to recognize foreign colloids, either
by the nature of the particle surface or by opsonins adsorbed onto the particle
surface [2.26]. Removing the lymph of particulate matter is based on an active,
saturable phagocytosis process.
2.6.2.2. Receptor based uptake of foreign matters
In the lymph node, phagocytosis is the main process of macrophages.

These cells are derived from circulating monocytes, whose phagocytic ability is
highly enhanced by this differentiation process [2.27]. Monocytes/macrophages,
as well as neutrophils, have been referred to as ‘professional phagocytes’ because
of their high phagocytic capacity and efficiency at internalizing particles [2.28].
This property can probably be ascribed to the presence of an array of dedicated
phagocytic receptors that increases the particle range and phagocytic rate [2.29].
It should be outlined that the role of macrophages is not only to ingest and
destroy bacteria, viruses, inorganic foreign particles and cellular debris. Some of
them also facilitate cross-talk between innate and adaptive immunity by acting
as ‘antigen presenting cells’. However, the involvement of macrophage in the
immune response will not be further developed in this chapter.
15
Phagocytosis occurs through a complex mechanism that cannot be
appropriately explained on the basis of a simple single model. Despite the
complexity associated with several phagocytic mechanisms, the basic features of
the process can easily be accommodated within the following description. First,
particle internalization is initiated by the interaction of specific receptors on the
surface of the phagocyte with some ligands on the surface of the particles. Then,
a phagocytic cup is formed on the macrophage membrane around the foreign
matter, with subsequent engulfment in a vesicle that fuses with cell lysosomes
where chemical degradation takes place. Finally, non-digestible residues are
ejected from the cell by exocytosis.
Engulfment of invading microorganisms, foreign particles and apoptotic
bodies is initiated by the engagement of specialized pattern recognition
receptors, which can discriminate among numerous bacterial components such
as bacterial carbohydrates (mannose receptor, glucan receptor) and bacterial
lipids (lipopolysaccharides receptors), as well as other structures typically
found on pathogen surfaces (toll like receptors, scavenger receptors) [2.30].
Additionally, macrophages have molecules on their surface known as Fc
receptors, which are capable of binding immunoglobin G through its Fc region
onto opsonized pathogens.
The mannose receptor on macrophages recognizes mannose and fucose
on the surfaces of pathogens and mediates phagocytosis of the organisms. The
mannose receptor is a single chain receptor with a short cytoplasmic tail and an
extracellular domain including eight lectin-like carbohydrate binding domains,
a fibronectin type II domain and an ending N-terminal cysteine rich domain
[2.31]. The three types of domains at the extracellular region have different
ligand specificity:
—— The C type lectin-like domain binds glycoproteins with exposed mannose,

fucose or N-acetyl glucosamine residues [2.32–2.34];
—— The fibronectin type II domain binds to collagens I, II, III and IV with high
affinity [2.35];
—— The cysteine rich domain recognizes sulphated carbohydrates [2.36, 2.37].
In recent years, macrophage specific delivery systems have gained

much attention because macrophages act as a natural hideout for several
pathogenic microorganisms. Therefore, several research groups have explored
the carbohydrate specific glycoprotein uptake system of macrophages for
specifically targeting these drug delivery systems [2.38–2.42]. Among different
carriers, liposomes have been extensively investigated as delivery systems for
phagocyte targeted therapies because of their advantages which include low
immunogenicity, biocompatibility, cell specificity and drug protection [2.43].
16
2.7. THE SLN
2.7.1. The SLN concept
The SLN is the hypothetical first lymph node, or group of nodes, that
receives lymph collected from a primary tumour mass. If cancerous dissemination
occurs, it is postulated that the SLN will trap any metastasizing cells leaving
the tumour. If the SLN is negative for tumour metastasis, the presence of
cancerous cells in all other lymph nodes in the same lymphatic chain is highly
improbable [2.44–2.49].
Sentinel lymph node mapping (SLNM) can be performed using a dye
(patent blue or isosulphan blue), a radioactive tracer or both. The radioactive
tracers currently used in clinical routine are based on 99mTc labelled colloids
(either inorganic or of albumin origin) [2.50]. However, the new approved
dextran based tracer, 99mTc tilmanocept (see Section 2.7.3) is expected to be
gradually adopted by the nuclear medicine community.
Ideally, an SLN imaging agent would rapidly clear from the injection site to
allow easy detection of nodes that are located close to the injection site. Residence
of the activity in the SLN should be selective and high enough to allow different
protocols for detection (external acquisition or radioguided surgery). It should
be obtained with high specific activity to avoid saturation of the phagocytic
process, with consequent leakage of radioactivity to the higher echelon nodes of
the region, and should be safe.
2.7.2. Physicochemical aspects of lymph node delivery of

biologically inert agents
Basically, to be taken up by the SLN, large molecules or colloids

administered by subdermal injection should first travel into the interstitial space,
cross the membrane of lymphatic capillaries, flow freely into the lymphatic
vessels and, finally, be phagocytized by macrophages of the first lymph node
encountered on their travel.
There is no unanimous consent in defining the order of importance of the
parameters affecting the overall localization process of foreign material into
lymph nodes. However, characteristics related to the size, shape and charge of
particles are generally mentioned as the most relevant ones [2.2, 2.51, 2.52].
17
2.7.2.1. Particle size
Size is the major factor determining the behaviour of particulate materials

after subcutaneous injection. Particles that are smaller than a few nanometres will
mostly penetrate the blood capillary membrane, whereas larger particles (up to
about 100 nm) can enter the lymphatic capillaries and be transported to lymph
nodes. Larger particles will be trapped in the interstitial space for a long time
[2.53]. Additionally, particle size also governs endocyte uptake by macrophages.
However, the upper size limit for lymphatic uptake has not been strictly defined.
The optimal colloidal size for lymphoscintigraphy is believed to be approximately
50–70 nm [2.54], but the range 10–100 nm has also been proposed [2.50–2.55].
The results of recent studies correlating the particle profile of 99mTc labelled
inorganic colloids with lymph node uptake suggest that colloids with nanometric
dimensions are the best suited for a high node uptake [2.56]. As a general rule,
it can be assumed that very small nanoparticles (<10 nm) are best suited for
lymphoscintigraphy or rapid SLND, whereas large particles (>100 nm) display a
longer retention in the first encountered lymph node.
The earliest lymphoscintigraphic methods used colloidal (198Au) gold
(9–15 nm in size) [2.57]. Although promising results were obtained, confirming
the suitability of small sized colloids, the absorbed dose at the injection site
was judged to be unacceptably high (13–27 Gy/MBq) to allow safe use of this
radiocolloid. These dosimetric concerns were removed by the introduction of the
less irradiating 99mTc based radiocolloids.
There is variation in the nature and size of 99mTc labelled colloids used
worldwide for the detection of SLNs [2.2, 2.58]. Studies in the USA were initiated
with 99mTc sulphur colloid, which is a radiopharmaceutical that was initially
approved for liver and spleen scintigraphy. This radiocolloid (administered
0.22 µm filtered or unfiltered) is still used nowadays, with the US Food and Drug
Administration (FDA) approving its indication for SLNM only very recently.
In Europe, albumin colloids and a preformed rhenium sulphide colloid are
the approved agents. Of the two, albumin colloid is the most frequently used
tracer. Technetium-99m labelled antimony trisulphide is the colloid of choice in
Australia, whereas 99mTc calcium phytate is the tracer used in Japan.
The size ranges of some radiocolloids currently used in clinical practice are
shown in Table 2.1 [2.58–2.62].
18
TABLE 2.1.  SIZE OF SOME APPROVED 99mTc RADIOCOLLOIDS
Colloid composition Size range Comments References

(nm)
Human serum albumin 7–23a Registered in different countries [2.59, 2.60]

3–16a in Europe;
not approved in USA
Human serum albumin 100–600b Registered in different countries Refer to

<100b in Europe; supplier
not approved in USA brochures
Stannous/stannic hydroxide 30–200a Approved in some European [2.60]

countries
Rhenium sulphide 8–68a Registered in different countries [2.61]

in Europe
Sulphur colloid 50–1000c Registered in USA [2.50, 2.61]

0.1 µm filtered sulphur colloid 30–50c
Antimony trisulphide 2–16d Registered in Australia [2.58]

17–23a
Calcium phytate 150–200a Mainly used in Japan; [2.62]

150–1500a size depends on
Ca2+ concentration
a
Particle size measurement using dynamic light scattering.
b
Particle size measurement method not disclosed.
c
Particle size measurement using membrane filtration.
d
Particle size measurement using transmission electron microscopy.
As illustrated in Table 2.1, approved radiocolloids span quite a large

size range. Although in some guidelines, it is reported that a preparation with
the majority of particles ranging between 100 nm and 200 nm in size can be
considered the best compromise between fast lymphatic drainage and optimal
retention in the SLN [2.52], the influence of the colloid size on diagnostic
performance (sensitivity and specificity) has not yet been evaluated in large
prospective studies. Probably, one of the largest comparative studies is that
reported by Paganelli et al., in which 215 patients with operable breast carcinoma
were assigned to three different diagnostic procedures [2.63]. Group A was
treated with 99mTc antimony sulphide (particle size < 50 nm), group B was
19
treated with 99mTc colloidal albumin (particle size < 80 nm) and group C was
treated with 99mTc colloidal albumin (particle size 200 –1000 nm). In each group,
two methods of administration (peritumoural and subdermal) were evaluated.
The conclusion of this study was that larger particle sized colloids were most
successful in detecting only one or two sentinel nodes, even 14–16 h after
injection, and that subdermal administration was the preferred route. However,
the choice of the best radiocolloid has been dictated to a great extent, and still is,
by its availability as an approved radiopharmaceutical.
It must be emphasized, however, that great care must be taken to not simply
compare the size of radiocolloids measured with different sizing techniques,
mainly because most particles are not regularly shaped, accentuating the inherent
limitations of each technique. For instance, transmission electron microscopy
will give the size of inorganic cores, but often not the coating (e.g. gelatine
layers). Moreover, particles are heated by the electron beam, possibly causing
sublimation of some components of the particle (e.g. sulphur in the case of metal
sulphide), therefore altering the original size (the freeze–fracture technique
should be used). Dynamic light scattering (DLS), also referred to as photon
correlation spectroscopy, is able to give the hydrodynamic diameter of particles,
but the diameter that is obtained using this technique is the diameter of a sphere
that would have the same translational diffusion coefficient as the particle. As the
translational diffusion coefficient depends not only on the size of the particle
‘core’, but also on any surface structure, as well as the concentration and type of
ions in the medium, meaningful results are obtained only with strict control of the
experimental parameters. Additionally, the use of DLS becomes more and more
problematic when the size distribution of the sample broadens.
2.7.2.2. Particle shape
Recently, particle shape has been identified as playing an important role

in the ability of macrophages to internalize particles [2.64, 265]. Theoretical
models based on cellular internalization have already underlined the benefit
of using non-spherical particles for drug delivery [2.66]. Experimental studies
have confirmed this prediction, showing, in an alveolar macrophage model, that
the local geometry of the particle at the point of cell attachment, not the overall
particle shape, can dictate whether macrophages initiate internalization [2.67].
The effect of shape on phagocytosis has also been observed for CdTe
quantum dot cystine composites with sphere, rod and needle particle shapes. In
mouse leukaemic monocyte macrophage, the microspheres exhibit the highest
degree of internalization and the fastest phagocytosis rate, while almost no
internalization of the needle shaped species occurs, clearly indicating a significant
effect of the shape on the macrophage phagocytosis [2.68]. Therefore, the size
20
of particles will affect the completion of phagocytosis, especially when the
particle volume exceeds the cell volume, and particle shape remains important in
initiating engulfment of a particle by macrophages.
2.7.2.3. Particle charge
Although this has been less frequently investigated, there is evidence that
modifications of the surface charge of particles can influence transport in the
interstitial space and uptake by lymph node macrophages.
While for particles the size is the predominant factor governing movement
in the interstitial space, the effect of charge becomes significant for proteins and
small sized polymers. In the interstitial space, fixed negative charges, principally
because of GAGs, are expected to contribute significantly to interstitial
exclusion of charged macromolecules. In an ex vivo model of mouse tail skin,
Reddy et al. [2.69] investigated the rate of interstitial convective transport
of anionic and uncharged 3 kg/mol dextran. It was found that the anionic
dextran moved with a higher average velocity through the interstitium than the
neutral dextran, suggesting that electrostatic repulsion may serve to reduce the
interactions between a negatively charged solute and the ECM.
The influence of the surface charge on macrophage phagocytosis is less
clear. From a literature survey, general trends cannot be reliably developed
that would apply to any kind of particle, as the chemical composition of the
surface may also play a role in the phagocytic process. Two classes of synthetic
particles have been extensively used to study phagocytosis: liposomes (vesicles
composed of a lipid bilayer) and polymerized microspheres (e.g. polystyrene).
These particles offer a large flexibility for tailoring their size, shape, charge
and hydrophobicity.
Liposomes have been extensively used as a particulate model because
of their potential for carrying and delivering drugs to cells and because of the
relative ease with which their charge and size can be shaped. The drainage of
negatively charged liposomes has been shown to be faster than that for positive
liposomes [2.70]. It has also been shown that the rate of liposome localization
in the lymph nodes after subcutaneous injections to rats was in the order
negative > positive > neutral liposomes [2.71].
In a recent review article on drug delivery to monocytes and macrophages
with liposomes, Kelly et al. [2.43] report that negatively charged lipids such
as phosphatidylserine and phosphatidylglycerol are preferentially recognized
by macrophages [2.72]. Studies comparing phosphotidylcholine (neutral) and
phosphatidylserine (anionic) composed liposomes have established negative
liposome formulations to have enhanced macrophage internalization in lung
21
macrophages of mice after intravenous administration of liposomes to the
animals [2.73].
Experiments using polystyrene microspheres with macromolecule modified
surfaces produced different clearance and organ deposition patterns for negatively
or positively charged particles [2.74], suggesting that positive charges increase
phagocytic uptake while negative charges reduce uptake [2.75]. These findings
were not confirmed by the work of Tabata et al. [2.76], who studied uptake
by mouse peritoneal macrophages of modified cellulose microspheres with
different surface charges. It was found that there was no significant difference
in phagocytosis between cationic and anionic surfaces when compared at a zeta
potential of the same absolute value.
2.7.2.4. Surface hydrophobicity/hydrophilicity
Opsonins prefer to associate with hydrophobic rather than hydrophilic

surfaces [2.77]. Indeed, the phagocytosis of hydrophobic polystyrene nanospheres
was shown to be drastically reduced by the adsorption of hydrophilic block
copolymers prior to intravenous administration [2.78]. Conversely, nanospheres
conditioned with block copolymers of polyoxyethylene chains of 5–15 ethylene
oxide units are effectively opsonized in lymphatics, which is a process that
dramatically enhances sequestration by regional lymph nodes [2.79]. However,
because hydrophobicity exerts a negative effect on particle drainage from the
injection site, an appropriate coating should be found that allows optimization
between the extent of drainage and the uptake process by macrophages.
2.7.2.5. Molecular weight
As the MW increases, there is a decreased ability for the molecule to

penetrate blood capillaries, with a consequent increase in preference to enter into
lymphatic vessels.
It has been shown that for water soluble compounds with MWs ranging
from 246 g/mol to 19 000 g/mol, a linear relationship exists between the MW of
a compound administered subcutaneously and the fraction of the dose absorbed
by the lymphatic draining. Compounds with a MW greater than 16 000 g/mol are
absorbed mainly by the lymphatics that drain the application site [2.80]. This was
the rationale for the use, either for lymphoscintigraphy or for SLND, of
non-particulate agents such as 99mTc human serum albumin (HSA)
(MW = 67 000 g/mol) [2.81], 99mTc dextran (average MW = 110 000 g/mol) [2.82]
and 99mTc hydroxyethyl starch (average MW = 450 000 g/mol) [2.83].
22
The suitability of 99mTc dextran (average MW = 82 000 g/mol) as a lymph
node imaging agent was determined in rabbits and dogs by comparison with
99m
Tc antimony sulphide colloid. In both species, in spite of a rapid uptake rate,
total popliteal lymph node sequestration of 99mTc dextran was significantly lower
than that observed for colloidal 99mTc antimony sulphide [2.84]. These results
were confirmed in a more recent study in which mice were used as the animal
model. SLN uptake of 99mTc dextran (average MW = 70 000 g/mol) was lower
than that observed for 99mTc albumin colloid and 99mTc antimony sulphide [2.85].
Despite these findings in animal models, 99mTc dextran has been
used, with apparent success, to detect SLN in small cohorts of patients with
breast carcinoma [2.86–2.88]. Promising results were also reported with
99m
Tc hydroxyethyl starch in a small group of normal volunteers and in patients
with breast carcinoma [2.83].
The labelling process of dextran and hydroxyethyl starch with stannous-
reduced 99mTc deserves comment. The two compounds are equally made of a
large number of glucose units, although they are differently assembled. Because
only weak chelating hydroxyl groups are present on the molecules, colloidal
Tc may form during the labelling step, thus contaminating the preparation with
radioactive particulate species. In addition, loss of labelling may also take place
ex vivo, inducing alteration of the expected radioactivity distribution. Moreover,
because the labelling process of dextran is performed in the presence of high
amounts of stannous chloride, concomitant formation of 99mTc-labelled tin colloid
cannot be totally excluded. To obviate these limitations, a modified dextran,
incorporating cysteamine as the chelator for 99mTc, has been synthesized and
tested in animals. This dextran derivative has shown higher chelation stability
against DTPA than 99mTc dextran, enabling clear visualization of axillary lymph
nodes after intradermal injection in rats [2.89].
Technetium-99m HSA has also been proposed as a radioactive
non-particulate agent for the detection of SLNs. Comparative studies with
other lymphoscintigraphic agents (99mTc sulphur colloid, 99mTc albumin colloid
and 99mTc phytate) have been performed in animals and in humans. In mice,
99m
Tc HSA tends to accumulate in the SLN less efficiently than 99mTc sulphur
colloid and disperses more rapidly to the next echelon nodes and to the systemic
circulation [2.90].
The diagnostic performance of 99mTc HSA has been compared to that of
99m
Tc albumin colloid and filtered 99mTc sulphur colloid. In one clinical study,
51 patients with stage I or stage II melanoma were equally assigned to one of
the three treatment groups. Early images with all three agents provided reliable
identification of SLNs. As expected, 99mTc HSA demonstrated faster washout
rates from injection sites and better definition of lymph channels than either
23
particulate agent, whereas the particulate agents were retained longer in nodes
and demonstrated more nodes in delayed images than in early images [2.91].
A similar study was conducted in patients with cutaneous melanoma (CM)
using unfiltered 99mTc sulphur colloid as the comparator. In this study, the majority
of patients (85 out of 106) received 99mTc albumin. A few of them (4) received
both tracers. In this study, SLNM was similar in both HSA and sulphur colloid
cohorts. However, it should be pointed out that acquisition of images was limited
to 1 h after administration of tracers, which is a relatively short period of time, in
which the 99mTc HSA activity has not yet migrated from the node [2.92].
The analysis of a retrospective study including 533 patients with breast
carcinoma, with half of them explored with 99mTc HSA and the remainder with
99m
Tc phytate, showed that the identification rate of SLNs was significantly
higher in the phytate group than in the HSA group. Most importantly, the highest
radioactivity of SLNs per case was more than five times higher in the phytate
group than in the HSA group, demonstrating a better diagnostic performance of
99m
Tc phytate [2.93].
Therefore, although no rigorous studies can be advocated to confirm the
inferior performance of 99mTc HSA and 99mTc dextran, it seems unlikely that these
two tracers would, in the near future, replace the well established radiocolloids
for SLND.
2.7.3. Receptor based radiotracers for SLND
Very recently, cell mannose receptors have been used to target resident
lymph node macrophages with a radioactive agent for SLND. The radiotracer is a
synthetic macromolecule composed of a dextran backbone and multiple subunits
of DTPA (for 99mTc chelation) and mannose (for receptor binding) [2.94]. This
receptor based approach has shown excellent results in animals and humans
for detecting SLNs [2.95, 2.96]. This dextran derivative, named 99mTc labelled
tilmanocept, has been approved by the FDA for use in patients with breast
cancer or melanoma who are undergoing surgery to remove tumour draining
lymph nodes. At the time of writing, the drug had recently been approved by the
European Medicines Agency. An extensive discussion of this new tracer is given
by Vera et al. in Chapter 4.
Receptor mediated uptake by lymph nodes has also been reported by
Takagi et al. [2.97], who have compared 99mTc-labelled HSA and 99mTc-labelled
mannosyl neoglycoalbumin to a commercial 99mTc rhenium sulphide colloid in
mice. Similar to the results obtained with the mannosyl dextran derivatives [2.94],
the introduction of mannose residues on albumin increases the uptake and the
residence time of this radiotracer in the lymph nodes. Indeed, the added mannose
24
residues allow the protein to be recognized and internalized by macrophages,
thus confirming the efficacy of the receptor mediated process of phagocytosis.
2.8. CONCLUSIONS
Having a basic understanding of what is occurring at the microscopic level

in the lymphatic system may allow for better appreciation of the parameters that
influence the biological properties of current SLND tracers and, eventually, help
to raise suggestions for the design of an ideal agent. This chapter has therefore
attempted to briefly outline the anatomy and physiology of the lymphatic system
and to illustrate how the functioning of this circulatory system can affect the fate
of exogenous materials in the body. Some oversimplifications of the complex
mechanism underlying transport in the lymph and phagocytosis have been
necessary, but hopefully without affecting the scientific value of the text.
For biological inert radiocolloids, not only size, but also shape and
charge, play important roles in the overall uptake process by lymph nodes. It is
interesting to note that most of the radiocolloids that are now approved for SLND
were developed either for lymphoscintigraphy or for liver and spleen imaging.
From a scientific perspective, these radiocolloids do not display the optimal
characteristics required for a diagnostic agent.
Impressive progress has been achieved in very recent years in designing
new agents whose uptake by lymph nodes is mediated by a receptor–ligand
interaction mechanism. With the use of this kind of new agent, nuclear medicine
has further strengthened its leading position as an MI discipline.
REFERENCES TO CHAPTER 2
[2.1] SWARTZ, M.A., The physiology of the lymphatic system, Adv. Drug. Deliv. Rev. 50
(2001) 3–20.
[2.2] SZUBA, A., SHIN, W.S., STRAUSS, H.W., ROCKSON, S., The third circulation:
radionuclide lymphoscintigraphy in the evaluation of lymphoedema, J. Nucl. Med. 44
(2003) 43–57.
[2.3] SCHULTE-MERKER, S., SABINE, A., PETROVA, T.V., Lymphatic vascular
morphogenesis in development, physiology, and disease, J. Cell Biol. 1193 (2011)
607–618.
[2.4] ALITALO, K., The lymphatic vasculature in disease, Nat. Med. 17 (2011) 1371–1380.
[2.5] SKOBE, M., DETMAR, M., Structure, function, and molecular control of the skin
lymphatic system, J. Investig. Dermatol. Symp. Proc. 5 (2000) 14–9.
[2.6] WILLARD-MACK, C.L., Normal structure, function, and histology of lymph nodes,
Toxicol. Pathol. 34 (2006) 409–424.
25
[2.7] SHIER, D., BUTLER, J., LEWIS, R., Holes’s Human Anatomy and Physiology,
12th edn, McGraw-Hill Science/Engineering/Math, New York (2009).
[2.8] BALUK, P., et al., Functionally specialized junctions between endothelial cells of
lymphatic vessels, J. Exp. Med. 204 (2007) 2349–2362.
[2.9] DEJANA, E., ORSENIGO, F., MOLENDINI, C., BALUK, P., McDONALD, D.M.,
Organization and signaling of endothelial cell-to-cell junctions in various regions of the
blood and lymphatic vascular trees, Cell Tissue Res. 335 (2009) 17–25.
[2.10] CASTENHOLZ, A., “Structure of initial and collecting lymphatic vessels”,
Lymph Stasis: Pathophysiology, Diagnosis and Treatment (OLSZEWSKI, W.L., Ed.),
CRC Press, Boca Raton, FL (1991), 15–41.
[2.11] IKOMI, F., SCHMID-SCHONBEIN, G., Lymph transport in the skin, Clin. Dermatol.
13 (1995) 419–427.
[2.12] LEAK, L.V., Lymphatic removal of fluids and particles in the mammalian lung,
Environ. Health Perspect. 35 (1980) 55–76.
[2.13] CASTENHOLZ, A., Strukturbild und Wirkungsweise der “initialen lymphbahn”,
Z. Lymphol. 81 (1984) 55–64.
[2.14] WIIG, H., SWARTZ, M.A., Interstitial fluid and lymph formation and transport:
physiological regulation and roles in inflammation and cancer, Physiol. Rev. 92 (2012)
1005–1060.
[2.15] FRANTZ, C., STEWART, K.M., WEAVER, V.M., The extracellular matrix at a glance,
J. Cell Sci. 123 (2010) 4195–4200.
[2.16] KADLER, K.E., BALDOCK, C., BELLA, J., BOOT-HANDFORD, R.P., Collagens at
a glance, J. Cell. Sci. 120 (2007) 1955–1958.
[2.17] LI, S.T., KATZ, E.P., An electrostatic model for collagen fibrils. The interaction of
reconstituted collagen with Ca2+, Na+, and Cl−, Biopolymers 15 (1976) 1439–1460.
[2.18] SCOTT, J.E., Structure and function in extracellular matrices depend on interactions
between anionic glycosaminoglycans, Pathol. Biol. 49 (2001) 284–289.
[2.19] CASLEY-SMITH, J.R., The fine structure and functioning of tissue channels and
lymphatics, Lymphology 13 (1980) 120–129.
[2.20] ZAWIEJA, D.C., Contractile physiology of lymphatics, Lymphat. Res. Biol. 7 (2009)
87–96.
[2.21] SAINTE-MARIE, G., PENG, F.S., BELISLE, C., Overall architecture and pattern of
lymph flow in the rat lymph node, Am. J. Anat. 164 (1982) 275–309.
[2.22] CLARK, S.L., Jr., The reticulum of lymph nodes in mice studied with the electron
microscope. Am. J. Anat. 110 (1962) 217–257.
[2.23] LUK, S.C., NOPAJAROONSRI, C., SIMON, G.T., The architecture of the normal
lymph node and hemolymph node. A scanning and transmission electron microscopic
study, Lab. Invest. 29 (1973) 258–265.
[2.24] LUDWIG, J., Über Kurschlusswege der Lymphbahnen und ihre Beziehungen zur
lymphogen Krebsmetastasierung, Pathol. Microbiol. 25 (1962) 329–334.
[2.25] TANIS, P.J., NIEWEG, O.E., VALDÉS OLMOS, R.A., KROON, B.B., Anatomy and
physiology of lymphatic drainage of the breast from the perspective of sentinel node
biopsy, J. Am. Coll. Surg. 192 (2001) 399–409.
26
[2.26] FRIER, M., “Phagocytosis”, Progress in Radiopharmacology (COX, P.H., Ed.),
Elsevier, Amsterdam (1981) 249–260.
[2.27] GUYTON, A.C., Textbook of Medical Physiology, 7th edn, W.B. Saunders (1986).
[2.28] RABINOVITCH, M., Professional and non-professional phagocytes: an introduction,
Trends Cell. Biol. 5 (1995) 85–87.
[2.29] ADEREM, A., UNDERHILL, D.M., Mechanisms of phagocytosis in macrophages,
Ann. Rev. Immunol. 17 (1999) 593–623.
[2.30] TAYLOR, P.R., et al., Macrophage receptors and immune recognition, Ann. Rev.
Immunol. 23 (2005) 901–944.
[2.31] TAHL, P.D., EZEKOWITZ, R.A., The mannose receptor is a pattern recognition
receptor involved in host defense, Curr. Opin. Immunol. 10 (1998) 50–55.
[2.32] STAHL, P.D., RODMAN, J.S., MILLER, M.J., SCHLESINGER, P.H., Evidence
for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal
glycosidases by alveolar macrophages, Proc. Natl. Acad. Sci. USA 75 (1978)
1399–1403.
[2.33] SHEPHERD, V.L., LEE, Y.C., SCHLESINGER, P.H., STAHL, P.D., L-Fucose-
terminated glycoconjugates are recognized by pinocytosis receptors on macrophages,
Proc. Natl. Acad. Sci. USA 78 (1981) 1019–1022.
[2.34] HUBBARD, A.L., WILSON, G., ASHWELL, G., STUKENBROK, H., An electron
microscope autoradiographic study of the carbohydrate recognition systems in rat liver.
I. Distribution of 125I-ligands among the liver cell types, Cell Biol. 83 (1979) 47–64.
[2.35] MARTINEZ-POMARES, L., et al., Carbohydrate-independent recognition of collagens
by the macrophage mannose receptor. Eur. J. Immunol. 36 (2006) 1074–1082.
[2.36] FIETE, D.J., BERANEK, M.C., BAENZIGER, J.U., A cysteine-rich domain of the
“mannose” receptor mediates GalNAc-4-SO4 binding, Proc. Natl. Acad. Sci. USA 95
(1998) 2089–2093.
[2.37] LETEUX, C., et al., The cysteine-rich domain of the macrophage mannose receptor
is a multispecific lectin that recognizes chondroitin sulfates A and B and sulfated
oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated
N-glycans of lutropin, J. Exp. Med. 191 (2000) 1117–1126.
[2.38] YOO, M.K., et al., Superparamagnetic iron oxide nanoparticles coated with mannan for
macrophage targeting. J. Nanosci. Nanotechnol. 8 (2008) 5196–5202.
[2.39] NAHAR, M., JAIN, N.K., Preparation, characterization and evaluation of targeting
potential of amphotericin B-loaded engineered PLGA nanoparticles, Pharm. Res. 26
(2009) 2588–2598.
[2.40] NIMJE, N., et al., Mannosylated nanoparticulate carriers of rifabutin for alveolar
targeting, J. Drug Target. 17 (2009) 777–787.
[2.41] PRAKASH, J., et al., Tumor-targeted intracellular delivery of anticancer drugs through
the mannose-6-phosphate/insulin-like growth factor II receptor, Int. J. Cancer 126
(2010) 1966–1981.
[2.42] JAIN, N.K., MISHRA, V., MEHRA, N.K., Targeted drug delivery to macrophages,
Expert Opin. Drug Deliv. 10 (2013) 353–367.
[2.43] KELLY, C., JEFFERIES, C., CRYAN, S.A., Targeted liposomal drug delivery to
monocytes and macrophages. J. Drug Deliv. 2011 (2011), 727241.
27
[2.44] CABANAS, R.M., An approach for the treatment of penile carcinoma, Cancer 39
(1977) 456–466.
[2.45] ALAZRAKI, N.P., et al., Lymphoscintigraphy, the sentinel node concept, and the
intraoperative gamma probe in melanoma, breast cancer, and other potential cancers,
Semin. Nucl. Med. 27 (1997) 55–67.
[2.46] VERONESI, U., et al., Sentinel-node biopsy to avoid axillary dissection in breast
cancer with clinically negative lymph nodes, Lancet 349 (1997) 1864–1867.
[2.47] KESHTGAR, M.R.S., ELL, P.J., Sentinel lymph node detection and imaging, Eur. J.
Nucl. Med. 26 (1999) 57–67.
[2.48] MARIANI, G., et al., Radioguided sentinel lymph node biopsy in breast cancer surgery,
J. Nucl. Med. 42 (2001) 1198–1215.
[2.49] BUSCOMBE, J., et al., Sentinel node in breast cancer procedural guidelines, Eur. J.
Nucl. Med. Mol. Imaging 34 (2007) 2154–2159.
[2.50] WILHELM, A.J., MIJNHOUT, G.S., FRANSSEN, E.J., Radiopharmaceuticals in
sentinel lymph-node detection: an overview, Eur. J. Nucl. Med. 26 (1999) S36–S42.
[2.51] HAWLEY, A.E., DAVIS, S.S., ILLUM, L., Targeting of colloids to lymph nodes:
influence of lymphatic physiology and colloidal characteristics, Adv. Drug Deliv. Rev.
17 (1995) 129–148.
[2.52] MITRAGOTRI, S., LAHANN, J., Physical approaches to biomaterial design, Nat.
Mater. 8 (2009) 15–23.
[2.53] MOGHIMI, S.M., BONNEMAIN, B., Subcutaneous and intravenous delivery of
diagnostic agents to the lymphatic system: applications in lymphoscintigraphy and
indirect lymphography, Adv. Drug Deliv. Rev. 37 (1999) 295–312.
[2.54] BERGQVIST, L., STRAND, S.E., PERSSON, B.R., Particle sizing and biokinetics of
interstitial lymphoscintigraphic agents, Semin. Nucl. Med. 13 (1983) 9–19.
[2.55] UREN, R.F., HOWMAN-GILES, R.B., THOMPSON, J.F., Regarding sentinel lymph
node localization in early breast cancer, J. Nucl. Med. 40 (1999) 1403–1406.
[2.56] NÚÑEZ, E.G., et al., Influence of colloid particle profile on sentinel lymph node
uptake, Nucl. Med. Biol. 36 (2009) 741–747.
[2.57] HULTBORN, K.A., LARSSO, L.-G., RAGNHULT, I., The lymphodrainage from the
breast to the axillary and parasternal lymph nodes, studied with the aid of colloidal
Au198, Acta Radiol. 43 (1955) 52–64.
[2.58] TSOPELAS, C., Particle size analysis of 99mTc-labeled and unlabeled antimony
trisulfide and rhenium sulfide colloids intended for lymphoscintigraphic application, J.
Nucl. Med. 42 (2001) 460–466.
[2.59] GOMMANS, G.M.M., et al., Further optimisation of 99mTc-Nanocoll sentinel node
localisation in carcinoma of the breast by improved labeling, Eur. J. Nucl. Med. 28
(2001) 1450–1455.
[2.60] JIMENEZ, I.R., et al., Particle sizes of colloids to be used in sentinel lymph node
radiolocalization, Nucl. Med. Commun. 29 (2008) 166–172.
[2.61] HUNG, J.C., et al., Filtered technetium-99m-sulfur colloid evaluated for
lymphoscintigraphy, J. Nucl. Med. 36 (1995) 1895–1901.
[2.62] HIGASHI, H., et al., Particle size of tin and phytate colloid in sentinel node
identification, J. Surg. Res. 121 (2004) 1–4.
28
[2.63] PAGANELLI, G., et al., Optimized sentinel node scintigraphy, Q. J. Nucl. Med. 42
(1998) 49–53.
[2.64] CHAMPION, J.A., KATARE, Y.K., MITRAGOTRI, S., Particle shape: a new
design parameter for micro- and nanoscale drug delivery carriers, J. Control. Rel.
121 (2007) 3–9.
[2.65] MITRAGOTRI, S., In drug delivery, shape does matter, Pharm. Res. 26 (2009) 232–234.
[2.66] DECUZZI, P., PASQUALINI, R., ARAP, W., FERRARI, M., Intravascular delivery of
particulate systems: does geometry really matter? Pharm. Res. 26 (2009) 235–243.
[2.67] CHAMPION, J.A., MITRAGOTRI, S., Role of target geometry in phagocytosis, Proc.
Natl. Acad. Sci. USA 103 (2006) 4930–4934.
[2.68] LU, Z., QIAO, Y., ZHENG, X.T., CHAN-PARKA, M.B., LI, C.M., Effect of particle
shape on phagocytosis of CdTe quantum dot–cystine composites, Med. Chem.
Commun. 1 (2010) 84–86.
[2.69] REDDY, S.T., BERK, D.A., JAIN, R.K, SWARTZ, M.A., A sensitive in vivo model
for quantifying interstitial convective transport of injected macromolecules and
nanoparticles, J. Appl. Physiol. 101 (2006) 1162–1169.
[2.70] HIRANO, K., HUNT, C.A., STRUBBE, A., MacGREGOR, R.D., Lymphatic transport
of liposome encapsulated drugs following intraperitoneal administration-effect of
liposome composition, Pharm. Res. 6 (1985) 271–278.
[2.71] PATEL, H.M., BOODLE, K.M., VAUGHAN-JONES, R., Assessment of the potential
uses of liposomes for lymphoscintigraphy and lymphatic drug delivery: failure of
99m
technetium marker to represent intact liposomes in lymph nodes, Biochim. Biophys.
Acta 801 (1984) 76–86.
[2.72] AHSAN, F., RIVAS, I.P., KHAN, M.A., TORRES SUAREZ, A.I., Targeting to
macrophages: role of physicochemical properties of particulate carriers – liposomes and
microspheres – on the phagocytosis by macrophages, J. Control. Rel. 79 (2002) 29–40.
[2.73] FIDLER, I.J., RAZ, A., FOGLER, W.E., Design of liposomes to improve delivery
of macrophage-augmenting agents to alveolar macrophages, Cancer Res. 40
(1980) 4460–4466.
[2.74] WILKINS, D.J., MYERS, P.A., Studies on the relationship between the electrophoretic
properties of colloids and their blood clearance and organ distribution in the rat, Br. J.
Exp. Pathol. 47 (1966) 568–576.
[2.75] AYHAN, H., TUNCEL, A., BOR, N., PISKIN, E., Phagocytosis of monosize
polystyrene-based microspheres having different size and surface properties,
J. Biomater. Sci. Polym. 7 (1995) 329–342.
[2.76] TABATA, Y., IKADA, Y., Effect of the size and surface charge of polymer microspheres
on their phagocytosis by macrophage, Biomaterials 9 (1988) 356–362.
[2.77] PATEL, H.M., Serum opsonins and liposomes: their interaction and opsonophagocytosis,
Crit. Rev. Ther. Drug Carrier Syst. 9 (1992) 39–90.
[2.78] ILLUM, L., DAVIS, S.S., The organ uptake of intravenously administered colloidal
particles can be altered using a non-ionic surfactant (Poloxamcr 338), FEBS Lett. 167
(1984) 79–82.
29
[2.79] MOGHIMI, S.M., et al., Surface engineered nanospheres with enhanced drainage into
lymphatics and uptake by macrophages of the regional lymph nodes, FEBS Lett. 344
(1994) 25–30.
[2.80] SUPERSAXO, A., HEIN, W.R., STEFFEN, H., Effect of molecular weight on
the lymphatic absorption of water-soluble compounds following subcutaneous
administration, Pharm. Res. 7 (1990) 167–169.
[2.81] McNEILL, G.C., et al., Whole-body lymphangioscintigraphy: preferred method for
initial assessment of the peripheral lymphatic system, Radiology 172 (1989) 495–502.
[2.82] HENZE, E., et al., Lymphoscintigraphy with Tc-99m-labeled dextran, J. Nucl. Med. 23
(1982) 923–929.
[2.83] SADEK, S., OWUNWANNE, A., ABDEL-DAYEM, H.M., YACOUB, T., Preparation
and evaluation of Tc-99m hydroxyethyl starch as a potential radiopharmaceutical for
lymphoscintigraphy: comparison with Tc-99m human serum albumin, Tc-99m dextran,
and Tc-99m sulfur microcolloid, Lymphology 22 (1989) 157–166.
[2.84] JUMA, N., ANDREY, T., EGE, G.N., Comparison as a lymphoscintigraphic agent
between 99Tcm dextran and 99Tcm antimony sulphide colloid, Br. J. Radiol. 58 (1985)
325–330.
[2.85] EDREIRA, M.M., COLOMBO, L.L., PEREZ, J.H., SAJAROFF, E.O.,
DE CASTIGLIA, S.G., In vivo evaluation of three different 99mTc-labelled
radiopharmaceuticals for sentinel lymph node identification, Nucl. Med. Commun. 22
(2001) 499–504.
[2.86] KARAYALCIN, B., ARAS, G., ERBAY, G., ERCAN, M.T., Parasternal
lymphoscintigraphy using 99Tcm-dextran, Nucl. Med. Commun. 9 (1988) 657–662.
[2.87] OFFODILE, R., et al., Minimally invasive breast carcinoma staging using lymphatic
mapping with radiolabeled dextran, Cancer 82 (1998) 1704–1708.
[2.88] BARROS, A., CARDOSO, M.A., SHENG, P.Y., COSTA, P.A., PELIZON, C.,
Radioguided localisation of non-palpable breast lesions and simultaneous sentinel
lymph node mapping, Eur. J. Nucl. Med. Mol. Imaging. 29 (2002) 1561–1565.
[2.89] MATSUNAGA, K., et al., Technetium labeling of dextran incorporating cysteamine as
a ligand, Nucl. Med. Biol. 32 (2005) 279–285.
[2.90] NATHANSON, S.D., ANAYA, P., KARVELIS, K.C., ECK, L., HAVSTAD, S.,
Sentinel lymph node uptake of two different technetium-labeled radiocolloids, Ann.
Surg. Oncol. 4 (1997) 104–110.
[2.91] GLASS, E.C., ESSNER, R., MORTON, D.L., Kinetics of three lymphoscintigraphic
agents in patients with cutaneous melanoma, J. Nucl. Med. 39 (1998) 1185–1190.
[2.92] BEDROSIAN, I., et al., 99mTc-human serum albumin: an effective radiotracer for
identifying sentinel lymph nodes in melanoma, J. Nucl. Med. 40 (1999) 1143–1148.
[2.93] TAKEI, H., et al., 99mTc-phytate is better than 99mTc-human serum albumin as a
radioactive tracer for sentinel lymph node biopsy in breast cancer, Surg. Today 36
(2006) 219–224.
[2.94] VERA, D.R., WALLACE, A.M., HOH, C.K., MATTREY, R.F., A synthetic
macromolecule for sentinel node detection: (99m)Tc-DTPA-mannosyl-dextran, J. Nucl.
Med. 42 (2001) 951–959.
30
[2.95] SONDAK, V.K., et al., Combined analysis of phase III trials evaluating [99mTc]
tilmanocept and vital blue dye for identification of sentinel lymph nodes in clinically
node-negative cutaneous melanoma, Ann. Surg. Oncol. 20 (2013) 680–688.
[2.96] WALLACE, A.M., et al., Comparative evaluation of [(99m)Tc]tilmanocept for sentinel
lymph node mapping in breast cancer patients: results of two phase 3 trials, Ann. Surg.
Oncol. 20 (2013) 2590–2599.
[2.97] TAKAGI, K., et al., 99mTc-labeled mannosyl-neoglycoalbumin for sentinel lymph node
identification, Nucl. Med. Biol. 31 (2004) 893–900.
31
Chapter 3
CLINICAL APPLICATIONS OF RADIOGUIDED SLNB
F. ORSINI, G. MARIANI
Regional Center of Nuclear Medicine,
University of Pisa Medical School,
Pisa, Italy
3.1. SUMMARY
A survey of the most important clinical applications of SLNB, which is

accomplished using radiolabelled nanocolloidal particles, is presented in this
chapter. In particular, emphasis is given to the diagnostic value of this procedure
for the treatment of various degenerative diseases.
3.2. INTRODUCTION
The most advanced diagnostic techniques of preoperative imaging

(computed tomography (CT), magnetic resonance imaging, ultrasound, single
photon emission tomography (SPECT), positron emission tomography (PET)
and PET/CT) provide information of increasing quality regarding topographical
location and extent of neoplastic disease. The surgeon can thus rely on such
information for planning the optimal surgical approach tailored to each individual
patient, with the final goal of radically removing the tumour, while, at the same
time, limiting the aggressiveness of surgery, within the increasingly common
scenario for oncological surgery to become less and less mutilating and to adopt
minimally invasive surgical procedures.
Intraoperatively, radioguidance provides the surgeon with an additional
interaction modality that enables exploration of the surgical field by relying not
only on the traditional modalities of direct visual inspection and palpation, but also
on the possibility of identifying the tissue to be removed by preoperative labelling
of the target tissue with a proper radiopharmaceutical. This additional guidance
requires the use of an intraoperative gamma probe for detecting and localizing the
site of accumulation of a radioactive compound (the radiopharmaceutical) at the
target tissue, guiding the surgeon through an acoustic signal that is proportional
to the amount of radioactivity detected by the probe. This combined procedure
is termed radioguided surgery, and is sometimes also aided by the acquisition
of intraoperative scintigraphic images using a dedicated, small-field-of-view
33
portable gamma camera. Although several different approaches based on
radioguided surgery have been developed or are in various phases of clinical
validation, the most widely utilized applications of this procedure concern SLNB
in patients with solid epithelial cancers [3.1].
The popularity of radioguided surgery is increasing, not only in highly
specialized and/or academic environments, but also in peripheral hospitals;
this trend is especially obvious in the case of SLNB, or alternatively SLND,
in patients with breast cancer or malignant CM, which are the two applications
where the effectiveness of radioguided surgery has most definitely been proven.
For these two cancers, the validity of the SLN concept is now also recognized
by the most recent version of the manual for tumour staging developed by the
American Joint Committee on Cancer, the so-called TNM system (where T stands
for status of the primary tumour, N for status of locoregional lymph nodes and
M for distant metastases) [3.2]. In fact, both for breast cancer and for melanoma,
the N parameter includes specific notations for the presence of macrometastasis,
micrometastasis or isolated tumour cells in the SLN. In this regard, because no
imaging modality is able to detect microscopic metastasis (nor the presence of
isolated tumour cells), such information must be based on histopathological
analysis of the SLN. Therefore, SLNB emerges as the only reliable method for
identifying micrometastatic disease in regional lymph nodes, and is now the
accepted standard of care in various clinical conditions, in particular, for patients
with early stage breast cancer and CM. Nevertheless, despite its established
benefits as a minimally invasive approach for nodal staging, SLNB is still
relatively underutilized in patients with tumours other than breast cancer and CM.
In the past 5 years, there has been momentum in technology and in clinical
research, thus opening new avenues to novel strategies for radioguided surgery.
In this setting, the ongoing integration of ‘virtual reality’ approaches with
standard approaches in some well defined conditions is increasing, facilitating
the use of minimally invasive surgeries. Indeed, MI—in particular, radionuclide
imaging — is becoming essential for optimizing surgical approaches that are
tailored to the disease-specific conditions encountered. This synergy has fostered
growing interactions between surgical specialties (mostly oncological surgery)
and nuclear medicine. In this new perspective, different medical specialties
cooperate to achieve a common goal of improved patient management and
overall benefit [3.3].
Key concepts of SLNB include the following:
—— SLNB is a specific application of radioguided surgery.

—— Labelling a tissue with a radioactive compound provides the surgeon with
the opportunity to identify the target tissue to be removed.
34
—— A gamma probe is able to localize the site of accumulation of the
radiopharmaceutical at the target tissue, guiding the surgeon through an
acoustic signal.
—— SLNB can be applied to solid epithelial cancers and, in particular, SLNB
has been validated in breast cancer and melanoma.
—— SLNB allows detection of micrometastases and isolated tumour cells by
detailed histopathological analysis.
3.3. SLNB IN BREAST CANCER
Breast cancer is the most frequent type of cancer diagnosed in women

worldwide. In the USA, more than 209 000 new cases were diagnosed in 2009,
with more than 40 000 deaths from this malignancy [3.4].
The rationale for SLNB in the surgical treatment of breast cancer is based
on the fact that, in the case of lymphatic metastasis, the tumour spreads through
the lymphatic system following an orderly progression, from the first level to
higher levels (Fig. 3.1). Therefore, the first lymph node encountered by the lymph
in its route from the site of the primary tumour to the basin of lymphatic drainage
(i.e. the SLN) is the most likely to be the first site of metastasis, and is therefore
to be considered as the ‘lymph nodal sentinel’ for the detection of early lymphatic
metastasis; conversely, the absence of metastasis in the SLN stands for absent
metastatic involvement of all other lymph nodes in the same lymphatic chain.
Thus, in breast cancer as in other solid tumours, the SLN is a lymph node
that first receives direct lymphatic drainage from the breast neoplasm. Usually,
there are one or two SLNs, but occasionally, more nodes can be identified by
lymphoscintigraphic mapping and during an intraoperative search with a
gamma probe. International meetings on SLNB have reached consensus over
the procedures to be employed, and many national and international scientific
societies have published their own recommendations [3.5−3.20].
Once identified (either pre- or intraoperatively through lymphoscintigraphy),
the SLN can be surgically removed and histologically analysed for prognostic and
therapeutic strategy purposes. In fact, when the SLN does not harbour metastasis,
this procedure avoids unnecessary radical axillary lymph node dissection. Only
when metastasis is found in the SLN should radical lymph node dissection
be performed.
Preoperatively, the axillary lymph node status can be assessed by clinical
examination (with rather low sensitivity), by ultrasound (more sensitive,
especially if including fine needle aspiration cytology, if necessary) [3.21] and
by 18F fluorodeoxyglucose PET/CT (>95% specificity, but low sensitivity for
lymph nodes < 5–8 mm in size). Detection of axillary lymph node metastasis by
35
FIG. 3.1.  Schematic representation of the migration of cancerous cells from primary lesion to
SLNs following lymphatic flow.
any of the above evaluation techniques mandates de novo axillary lymph node
dissection. Therefore, these patients are not candidates for SLNB.
Nevertheless, given the limited sensitivity of imaging techniques for
evaluating the axillary lymph node status, the axillary lymphatic basin must be
explored surgically. In fact, according to the TNM approach, the locoregional
lymph node status is an essential component for prognostic purposes and for
selecting the most appropriate treatment strategy; in this regard, the status of
axillary lymph nodes remains the most important prognostic factor for survival
[3.22].
Depending on the status of the axilla, different therapeutic strategies
are adopted in terms of surgery, adjuvant chemotherapy, radiotherapy and
hormonal therapy. In addition, axillary lymph node dissection provides excellent
locoregional control of disease in the case of metastasis.
About 30%–40% of patients without palpable lymph nodes will develop
macroscopic axillary recurrence if axillary lymph node dissection is not
performed, regardless of adjuvant chemotherapy. However, it should be noted
that in prospective randomized trials, a significant increase of overall survival has
never been demonstrated for patients treated with axillary dissection compared to
those who have not undergone the procedure.
Radical de novo axillary lymph node dissection, in addition to excision
of the primary tumour, has, for some decades, represented the standard surgical
36
procedure for patients with breast cancer. However, this surgical procedure
has considerable impact on the wellbeing of patients because a non-negligible
proportion of them (5%–20%) suffer from significant complications such as
prolonged wound healing, pain, lymphoedema and motor sensory impairment.
On the other hand, the fraction of patients who, after lymph node
axillary dissection, are found to actually have axillary metastasis at the time
of lumpectomy is directly related to the diameter of the primary tumour, and
only amounts to approximately 20%–30% of the cases in early stage breast
cancer. Thus, on average, at least 70% of these patients undergo radical axillary
dissection without proof that they actually need such an aggressive procedure
(yet with the same associated morbidity experienced by patients for whom
axillary dissection is potentially beneficial). In this regard, it should be noted that
the SLNB procedure entails significantly reduced morbidity compared to radical
axillary dissection [3.23].
In the perspective of reducing the number of such unnecessary de novo
axillary dissections, Giuliano et al. [3.6, 3.7] first described, in 1994, their
experience with the use of a vital dye (Evans blue) injected around the primary
tumour site to find SLNs in patients with breast cancer. This technique of
intraoperative lymphatic mapping then evolved (and actually improved) with
the adoption of 99mTc labelled colloids for lymphatic mapping, and with the
introduction of an intraoperative gamma probe for identifying the source of
radioactivity (the SLN(s) that had trapped the radiocolloid injected interstitially
at the tumour site) in the surgical field.
SLNM and SLNB can be performed using the blue dye, the radiocolloid
or both. SLNM with radiocolloids alone leads to successful SLN identification
in more than 97% of cases; the blue dye technique alone has a lower success
rate (75%–80%). The use of both techniques can further increase the success
rate to 99%, especially when associated with lymphoscintigraphy. The combined
procedure has the advantage of also identifying massively metastatic SLNs,
an occurrence where extensive replacement of the normal tissue (in particular,
macrophages) with tumour cells may prevent efficient radiocolloid accumulation
in the affected lymph node [3.24, 3.25].
When lymphatic mapping is not successful, full axillary lymphadenectomy
is generally necessary to allow assessment of the status of the axillary
lymph nodes.
It should be emphasized that adequate training of a multidisciplinary team
is required to reduce the number of unsuccessful SLNM procedures, as well as the
potentially associated morbidity. In fact, correctly performing radioguided SLNB
is based on the collaboration of different specialists, including surgeons, nuclear
physicians and pathologists. Incorporation of radiologists and radiotherapists into
such a multidisciplinary team should also be encouraged [3.18, 3.26].
37
—— Radical axillary lymph node dissection has, for some decades, represented
the standard surgical procedure for patients with breast cancer.
—— Only in 20%–30% of cases of early stage breast cancer is lymphatic
metastasis detected by radical axillary dissection.
—— Approximately 5%–20% of patients undergoing radical axillary dissection
suffer from disabling complications.
—— SLNB is a procedure for identifying those lymph nodes that are most likely
to be the first sites of lymphatic metastasis.
—— The absence of metastasis in the SLNs represents absent metastatic
involvement of all other lymph nodes in the same lymphatic chain.
—— Only when metastasis is found in the SLN should radical axillary lymph
node dissection be performed.
—— The false negative rate (i.e. tumour recurrence in an axilla that had been
classified as negative for metastasis based on SLNB) is 3%–5%.
—— Histopathological analysis is more accurate when focused on only one
(or a few) lymph node(s) and combined with complete sectioning and
staining of the whole lymph node, rather than only two to three sections
per node.
—— The rates of detection and intraoperative harvesting of internal mammary
chain SLNs are much lower than those for axillary SLNs, and the clinical
significance and impact of internal mammary chain SLNB on outcome are
still being debated.
When the SLNB procedure is correctly performed and the SLN is found to
be free from metastasis, the negative predictive value of this finding for metastatic
involvement of other axillary lymph nodes is very high, ~95%–97%. This means
that only in a low percentage of cases (3%–5%) may the SLNB technique yield
false negative results. In this regard, the false negative rate can be defined as
the proportion of patients with negative SLNB who actually have disease in the
axillary lymph nodes, as shown either by completion axillary lymphadenectomy
(as was initially done in early clinical validation studies) or during follow-up.
The current gold standard to assess the false negative rate for SLN axillary
staging is long term follow-up (assessing for appearance of axillary metastasis
in an axilla that had been classified as negative on the basis of the SLNB). In
fact, sufficient experience has now been published to compare the axilla disease-
free survival time after SLNB and after lymphadenectomy [3.27]. More recently,
the survival benefit of SLNB was addressed in the National Surgical Adjuvant
Breast and Bowel Project trial B-32 (NSABP-32) that randomized 5611 women
to SLNB versus SLNB + axillary lymphadenectomy. After a mean follow-up of
38
95.6 months, the SLNB arm displayed a 90.3% survival rate at 8 years versus
91.8% in the axillary lymphadenectomy arm (with the difference being not
statistically significant) [3.28]. These findings, based on long term follow-up,
therefore confirm earlier data from the same clinical protocol suggesting that, in
the case of negative SLNB, there are no significant differences regarding disease-
free survival, overall survival and local control of disease between patients
undergoing de novo axillary lymph node dissection and those not undergoing
axillary dissection because of a negative SLNB [3.29].
Nevertheless, it should be noted that false negative SLNB procedures are
higher in patients with higher pathological grading of the tumour (grade 3),
as well as in patients in whom only one SLN was retrieved intraoperatively,
compared to patients in whom multiple SLNs were retrieved [3.30]. Regarding
analysis of the SLN, it should be emphasized that with this approach, the attention
of the pathologist is focused on only one (or a few) SLN(s), rather than on the up
to 20–25 lymph nodes that are typically retrieved during full axillary dissection.
Therefore, this low nuber of SLNs can be analysed more accurately, for instance,
with complete sectioning and staining of the whole lymph node rather than with
only two to three sections per node. Immunohistochemistry (and, to a greater
degree, molecular analysis using the technique of reverse transcriptase polymerase
chain reaction (RT-PCR)), further improves the sensitivity of analysis, possibly
leading to the identification of micrometastases and even isolated tumour cells,
which cannot be assessed with conventional haematoxylin eosin staining. In this
regard, micrometastasis, which is observed in approximately 10%–20% of the
SLNs so extensively analysed, entails a certain risk of metastatic involvement of
upper echelon lymph nodes. Therefore, SLNB has improved the overall accuracy
of the histopathological staging of the axilla in breast cancer patients because it
enables the detection of minimal nodal metastatic involvement (micrometastases)
in patients with clinically negative axilla.
More recently, the results of a multicentric randomized study have
suggested that radical axillary lymph node dissection may even be unnecessary
for women with early breast cancer (T1 and T2) and axillary isolated tumour
cells or micrometastases in the SLN (detected by haematoxylin eosin staining).
In fact, the local recurrence rate was 2.5% in the SLNB-only group (i.e. patients
who had not been submitted to axillary lymph node dissection despite having had
a positive SLNB) versus 3.6% in the group of patients who had been submitted
to axillary lymphadenectomy because of the positive SLNB finding. These
results demonstrate that SLNB offers an excellent regional control in early breast
cancer patients, even without axillary lymphadenectomy, as well as sufficient
information to select adequate adjuvant therapy [3.31].
39
Another issue that deserves consideration concerns the patterns of
radiocolloid migration to the internal mammary chain (IMN) observed in
different patients. Although early studies showed great variation in the frequency
of scintigraphic IMN visualization, it was later found that the detection rate of
IMN SLNs is significantly affected by the depth of radiocolloid injection, in
that lymphoscintigraphic mapping of the IMN requires deep injection of the
radiocolloid, either peritumourally or intratumourally [3.32].
The rates of detection and intraoperative harvesting of IMN SLNs are
much lower than those for axillary SLNs. In particular, IMN SLNs have been
detected in about one third of patients with breast cancer, of which approximately
63%–92% could be harvested during surgery, and, of those, 11%–27% were
metastatic [3.33–3.36].
However, the clinical significance and the impact of IMN SLNB on
outcome are still being debated. In fact, even if there is some evidence that IMN
mapping can lead to upstage migration and modification of treatment planning
for radiotherapy and systemic therapy, there is no evidence supporting that
SLNM of the IMN will improve the outcome of treatment and survival [3.37].
3.3.1. Indications and contraindications for SLNB in breast cancer
Basic recommendations for SLNB include an early stage biopsy of proven

breast cancer (T1 and T2, up to 5 cm in maximum diameter) and appropriate
selection of patients to undergo the SLNB procedure. In particular, preoperative
diagnostic imaging of the axilla must be negative, and must include fine needle
aspiration cytology in the case of suspicious lymph nodes [3.20]. In the case
of preoperative findings indicating metastatic involvement of axillary lymph
nodes, the patient should not undergo SLNB but rather full axillary lymph
node dissection.
The emergence of molecular biology techniques for SLN analysis and
the introduction of newer imaging techniques during SLNM, although posing
new challenges, also enable the resolution of conditions that had been initially
defined as contraindications for performing SLNB in patients with breast cancer.
In fact, several meta-analyses and guidelines have been presented, and have
led to general acceptance of the SLNB procedure in a wider spectrum of cases
than initially accepted, such as in cases of multicentric/multifocal tumours, prior
diagnostic or excisional breast biopsy, ductal carcinoma in situ, after neoadjuvant
therapy or after previous breast surgery (including plastic surgery) [3.38−3.40].
For patients who have had previous breast surgery, lymph drainage may
be changed (and follow unexpected patterns) in patients who have undergone
prior breast or axillary surgical procedures, because non-axillary drainage has
been identified more often in reoperative SLNB than in primary SLNB. In
40
73% of patients, migration to the regional nodal drainage basins has been noted
in ipsilateral axillary, supraclavicular, internal mammary, interpectoral and
contralateral axillary nodes [3.41].
Multifocal breast cancer is defined as separate foci of ductal carcinoma
more than 2 cm apart within the same quadrant, while multicentric breast
cancer indicates the presence of separate independent foci of carcinoma in
different quadrants [3.42]. Until a few years ago, SLNB was considered to be
contraindicated in patients with multicentric and multifocal breast cancer because
it was believed that it was difficult to localize the true SLN, and a negative SLNB
result would not exclude, in these cases, the possibility of metastasis being
present in a lymph node draining from other regions of the breast. However, the
majority of the breast can actually be considered as a single unit with lymph
drainage to only a few designated lymph nodes in the axilla [3.43, 3.44].
Several studies have demonstrated an efficacy of radioguided SLNB in
these patients equal to that commonly observed in patients with unicentric breast
cancer. The presence of lymph node metastasis is significantly higher in SLNs,
as well as in non-SLNs in patients with multicentric breast cancer; however, the
sensitivity, false negative rate and overall accuracy of SLNB are similar in both
conditions, although there are some discrepancies between the results reported by
different groups [3.45−3.47].
Debate is still ongoing as to whether SLNB is accurate enough after
neoadjuvant chemotherapy, or whether it should be performed before starting
chemotherapy in patients with locally advanced breast cancer. Performing SLNB
before or after neoadjuvant systemic treatment has advantages and disadvantages
in each case. Before neoadjuvant chemotherapy (as accepted by American
Society of Clinical Oncology guidelines), SLNB yields a more precise axillary,
staging reliable information about the axillary lymph node status. However, it
can delay the beginning of the treatment, and two surgeries can be needed in
the same patient (i.e. SLNB before starting neoadjuvant chemotherapy, then
surgery of the primary tumour). On the other hand, SLNB performed after such
primary chemotherapy can assess the response at lymphatic level, but may lead
to the underestimation of the initial stage [3.48]. Furthermore, after neoadjuvant
chemotherapy, the SLND rate decreases, the rate of false negative cases increases
and the long term local recurrence rate in patients in whom lymphadenectomy
has not been performed because of a negative SLNB has yet to be determined.
This group of patients was previously regarded as ineligible for SLNB, under
the assumption that, after neoadjuvant chemotherapy, the lymph drainage pattern
may not represent the pattern occurring in the tumour basin before chemotherapy,
therefore possibly leading to false negative results. The available data show that
there are no significant differences in the success rate of SLNB according to
clinical tumour size or clinical nodal status, and that the false negative rate is
41
not affected by tumour response to chemotherapy. Recently, a systematic review
of 24 clinical SLNB trials in patients with breast carcinoma after neoadjuvant
chemotherapy was undertaken. The global SLN identification rate was 89.6%,
with metastatic lymph node involvement being observed in 37% of the patients;
the overall false negative rate was 8.4% [3.49−3.51].
Although pregnancy has traditionally been considered as a contraindication
to performing radioguided SLNB, the radiodosimetric burden to patients is very
low, and the benefit should be considered in those presenting early lesions. In
pregnant patients with early lesions and clinically/ultrasound negative axilla,
the possibility of performing a 1 day procedure including breast surgery and
radioguided SLNB with administration of a low dose of radiocolloid should be
considered. In fact, the available data indicate that radioguided SLNB can be
performed safely and successfully in pregnant women with breast cancer, as it
entails minimum risk to the fetus [3.52−3.54].
Radiation exposure of the fetus from the administered radiocolloid is very
low and does not increase the risk of prenatal death, congenital malformation or
mental impairment. On the other hand, blue dyes should not be used in pregnant
patients [3.20].
Finally, some conditions that were previously considered as formal
contraindications to SLNB have changed to possible applications, on a patient by
patient basis [3.2, 3.18, 3.38]. These conditions include large or locally advanced
invasive breast cancers (T3), ex situ ductal carcinoma, prior non-oncological
breast surgery or axillary surgery, and the presence of suspicious palpable axillary
lymph nodes.
—— Indications for SLNB consist of tumour diameters of less than 5 cm and
a negative preoperative diagnostic imaging of the axilla (including fine
needle aspiration in suspected axillary lymph nodes).
—— Absolute contraindications of SLNB include clinical evidence of lymph
node metastasis.
—— Other relative contraindications such as pregnancy, multicentric/multifocal
tumours, prior diagnostic or excisional breast biopsy, ductal carcinoma in
situ, previous neoadjuvant therapy or previous breast surgery (including
plastic surgery) are now not completely accepted, and each case has to be
discussed individually.
3.3.2. SLNB procedures in breast cancer
The SLNB procedure consists of several sequential phases: radiocolloid

injection, lymphoscintigraphy, intraoperative detection using a gamma probe
42
(aided or not by intraoperative imaging) and, finally, pathological analysis of the
resected SLNs.
Lymphoscintigraphy for SLNM is usually performed approximately 3 h to
24 h before surgery, depending on logistics and/or the scheduled time of surgery.
The radiopharmaceutical most commonly used in Europe consists of colloidal
particles of HSA, ranging in size between 20 nm and 80 nm (Nanocoll), labelled
with 99mTc. A radiocolloid activity of 7–30 MBq is generally injected in a volume
of 0.2–0.8 mL, by means of a 25 gauge needle. In the case of obese patients, the
activity to be administered is increased to 15–55 MBq. Following administration,
the patient is invited to gently massage the breast for a few minutes to facilitate
and accelerate lymphatic drainage.
There are different modalities of radiocolloid administration: intratumoural,
peritumoural, intradermal, subdermal or perisubareolar.
Intratumoural injection requires higher volumes and activities of the
radiopharmaceutical to overcome the high intratumoural pressure owing to
abnormal lymphoneoangiogenesis; such high volumes of the radiocolloid cause
some extravasation towards the peritumoural lymphatic vessels, thus initiating
migration of the radiocolloid, which can be depicted by lymphoscintigraphy.
Because of the reduced efficiency of lymphatic drainage within the tumour mass,
the SLN is often visualized quite late after intratumoural radiocolloid injection.
Peritumoural (or intraparenchymal) administration consists of multiple
injections of the radiocolloid around the tumour, under mammographic or
ultrasound guidance in the event of non-palpable lesions. As observed after
intratumoural injection, this technique results in good visualization of the axillary
lymphatic drainage, but also of the IMN displaying the internal mammary lymph
nodes (in 10%–30% of cases); however, the rate of radiocolloid migration from
the injection site to the SLN is still quite slow.
Subdermal or intradermal injection of the radiocolloid on the skin projection
of the breast tumour allows fast SLN visualization, by virtue of the remarkable
development of the lymphatic network at that level. In fact, under physiological
conditions, most of the lymphatic flow of the breast converges into the subareolar
plexus of Sappey; lymph flow then proceeds from the deep zones to the more
superficial subcutaneous plexus, which drains mostly to the anterior (or pectoral)
group of axillary lymph nodes. With this technique, lymphoscintigraphic
visualization of IMN SLNs is very rare (~1%–2%).
Finally, perisubareolar injection has more recently been proposed, based
on the presence of the lymphatic plexus of Sappey, towards which lymph from
virtually the entire breast seems to drain. It is the simplest method of injection
and could be particularly appropriate in specific cases, such as non-palpable or
multicentric/multifocal tumours, or in the case of previous lumpectomy.
43
Numerous studies have shown that, irrespective of the different modalities
of radiocolloid injection described above (peritumoural, intradermal, subareolar),
in virtually all patients, lymphoscintigraphy shows migration to the same SLN
or group of SLNs. These findings therefore suggest that lymph from different
regions of the breast reaches the same axillary lymph nodes.
Lymphoscintigraphy can be performed the day before surgery, or at least
2–3 h prior to surgery. A gamma camera equipped with a parallel hole, low
energy, high resolution collimator is generally used. The patient is positioned
supine, with the arms abducted (in the same position as during surgery), placing
the collimator as close as possible to the axillary region. A 57Co flood source
can be positioned underneath the patient’s body, to obtain some reference
anatomical landmarks in the scintigraphic image. Alternatively, the body contour
can be identified by moving a 57Co point source along the patient’s body during
scintigraphic acquisition.
In the current protocol, a sequence of three static planar scans (in the
oblique anterior, lateral and anterior projections) is acquired at least 5–10 min
after radiocolloid administration (Figs 3.2 and 3.3). Dynamic acquisition can also
be performed, starting immediately after radiocolloid injection, and it usually
consists of sequential sets of a few minutes each. Such a dynamic imaging mode
can be useful (although not always strictly required) to assess the initial kinetics of
radiocolloid migration and to visualize the lymphatic routes leading to the SLNs.
Using a point source of 57Co, it is possible to mark the cutaneous projection of
the SLN, to help the surgeon identify the best surgical approach. In the case of
low/absent migration of the radiocolloid, late acquisitions are performed at 3 h
or 18 h postinjection. In addition to identifying the SLNs, lymphoscintigraphy
is also useful for identification of any unusual lymph draining basins, as well as
radiocolloid migration to IMN SLNs, or even to intramammary, interpectoral or
infraclavicular lymph nodes.
Major advances in the whole process of SLNM in the preoperative
phase have been made possible by the use of SPECT/CT imaging [3.55, 3.56].
This is generally performed after acquiring delayed planar imaging (mostly
2–4 h after radiocolloid administration). SPECT/CT does not replace planar
lymphoscintigraphy, but should rather be considered as a complementary imaging
modality. The use of SPECT/CT acquisitions is particularly important for the
identification of uptake ‘foci’ in cases of unusual lymph node draining basins,
thus obviating the problem of identifying anatomical landmarks as a reference
for anatomotopographic localization of the SLN(s) [3.57–3.59].
44
FIG. 3.2.  Oblique anterior (top, left), lateral (top, right) and anterior (bottom) static
projections, acquired 5–10 min after radiocolloid administration, showing exclusive migration
to the first SLN (larger spot corresponds to the injection site).
FIG. 3.3.  Oblique anterior (top, left), lateral (top, right) and anterior (bottom) static
projections, acquired 5–10 min after radiocolloid administration, also showing migration
of radioactivity to lymph nodes adjacent to the first SLN (larger spot corresponds to the
injection site).
45
An additional useful application of SPECT/CT imaging is in patients in
whom the SLN is not visualized on planar imaging. Multiplanar reconstruction
(MPR) enables two dimensional display of fusion images in relation to CT and
SPECT, with the use of cross-reference lines allowing navigation between axial,
coronal and sagittal views. At the same time, this tool enables correlation of the
radioactive SLNs seen on the fused SPECT/CT images with the lymph nodes
seen on CT images. This information may be helpful during the intraoperative
procedure, as well as for assessing the completeness of excision using portable
gamma cameras or probes. Fused SPECT/CT images may also be displayed using
maximum intensity projection. This tool enables three dimensional (3-D) display,
and improves anatomical localization of SLNs by summing up various slices.
When using volume rendering for 3-D display, different colours are assigned
to anatomical structures such as muscle, bone and skin (Figs 3.4 and 3.5). This
facilitates better identification of anatomical reference points and incorporates an
additional dimension in the recognition of SLNs.
FIG. 3.4.  Fused SPECT/CT image showing a thoracic lymph node (left) and the corresponding
3-D volume and colour rendering (right) showing bone and muscle anatomical structures.
FIG. 3.5.  Fused SPECT/CT image showing an axillary lymph node (left) and the corresponding
3-D volume and colour rendering (right) showing bone anatomical structures.
46
Intraoperative SLND is facilitated by a preliminary exploration of the axilla,
with a gamma probe, to confirm the correct location of the skin mark placed on
the skin projection of the SLN during lymphoscintigraphy. The gamma probe
does not produce any scintigraphic image, but it yields both a numerical readout
and an audible signal that is proportional to the counting rate, thus guiding the
surgeon to the foci with the highest target/background ratios. A small incision
is then performed in the skin region with maximum radioactive counts, along
the ideal line for axillary dissection. When the tumour is located in the upper
outer quadrant, access to the SLN can occur through the same surgical incision
used for tumour excision. An intraoperative search of the SLN is performed by
moving the gamma probe in different directions to precisely localize areas of
maximum radioactivity accumulation, corresponding to the SLN [3.60].
Once the SLN has been removed, the gamma probe is utilized to obtain an
ex vivo counting rate (to confirm that it is actually the SLN), and the surgical
field is explored again with the probe, assessing residual radioactivity to confirm
removal of the hot node(s). If necessary, the search continues for possible further
radioactive lymph nodes. The SLN and any other nodes so identified are then
sent for complete histopathological analysis.
During the past decade, dedicated portable gamma cameras for real time
intraoperative imaging probes have become commercially available; they are
generally employed not for actual radioguidance during surgery, but to confirm
completeness of SLN removal [3.61−3.63]. Several recent reports outline
the added value of imaging with portable gamma cameras in clinical and
experimental settings for radioguided SLNB [3.64−3.69].
There is no delay between image acquisition and display (real time
imaging), with the possibility of continuous monitoring and spatial orientation
on the screen. However, because non-imaging gamma probes are still the
standard equipment for radioguided surgery, the role of intraoperative imaging
is generally limited, at least so far, to that of an additional aid to the surgeon
for SLN identification. In particular, the usefulness of non-imaging probes in
patients with breast cancer has been ascertained in particular cases with difficult
lymphatic drainage or extra-axillary drainage, in cases with only faint lymph
node radiocolloid uptake, when the SLN is located very close to the injection
site, or finally, when no conventional gamma camera is available.
Novel technological advances have also made it possible to combine a
spatial localization system and two tracking targets to be fixed on the patient’s
body and on a conventional hand held gamma probe, resulting in a new modality
of 3-D localization of the traditional acoustic signal of the gamma probe. This
feature, together with the real time information on depth that the system may
provide, expands the application of radioguided SLNB in oncology, particularly
for malignancies with deep lymphatic drainage patterns [3.70, 3.71].
47
On the other hand, the possibility of combining the current
radiopharmaceuticals with other agents opens new avenues for exploration
in SLNB. In this regard, a radiolabelled nanocolloid agent has been combined
with indocyanine green, a fluorescent agent, for sentinel node detection in robot
assisted lymphadenectomy [3.72−3.74].
For all these new intraoperatives modalities, the preoperative anatomical
SPECT/CT acquisition remains the crucial starting point for optimal
surgical planning.
—— The SLNB procedure consists of several sequential phases: radiocolloid

injection, lymphoscintigraphy (aided by SPECT/CT or not), intraoperative
detection with a gamma probe (aided by intraoperative imaging or not) and,
finally, pathological analysis of the resected SLNs.
—— Radiocolloid administration can be performed with different modalities:
intratumoural, peritumoural, intradermal, subdermal or perisubareolar.
—— Lymphoscintigraphy is a mandatory step of SLNM performed by a gamma
camera to visualize the lymphatic route and to mark the skin projection of
SLNs with a cutaneous marker.
—— SPECT/CT, MPR and volume rendering for 3-D display allows
better identification of anatomical landmarks as a reference for
anatomotopographic localization of the SLN(s).
3.4. SLNB IN CM
CM, which is potentially the most dangerous form of skin tumour, causes
90% of skin cancer mortality, and it can present rather differently from patient to
patient. It is often highly aggressive, and usually fatal if diagnosed in an advanced
stage. Better prognosis is observed in the early stages, when the probability of
distant metastasis is low and resection of the primary tumour and regional lymph
nodes may be curative. The 5 year mortality is approximately 20% in patients
with stages I–II CM, but it is dramatically higher in stage III (35%) and stage IV
(90%) [3.75, 3.76].
The incidence of CM has increased about threefold over the last 30–40 years.
The main risk factors are represented by age, family history, immunosuppression
and exposure to the sun. In the very early stages of disease (with a Breslow
thickness of 0.76–1.5 mm), the incidence of metastatic involvement of the SLNs
is only about 5%, but it increases up to 20% for melanomas with a thickness
between 1.5 mm and 4 mm. With a Breslow thickness greater than 4 mm, the
incidence of metastases in SLNs rises to about 50% [3.77]. The presence of
48
lymph node metastases decreases the survival rate by about half, with the 5 year
mortality being around 70%–75% in the case of nodal metastasis. Furthermore,
for patients with N1 disease, the number of involved lymph nodes and the
extension of lymph nodal metastatic involvement (microscopic or macroscopic)
are the two most important prognostic factors. Regardless of the Breslow
thickness, the 5 year survival of patients with non-ulcerated melanoma and a
single lymph nodal metastasis is significantly higher than in patients with four or
more lymph nodal metastases (70% versus 27%).
The radioguided SLNB procedure in malignant CM was first reported in
1993 [3.78] using intradermally injected 99mTc sulphur colloid. Since that time,
the use of radioguided SLNB has largely surpassed the use of VBD alone for the
surgical evaluation of at-risk nodal basins in patients with malignant CM [3.79].
The SLN status is included in the latest version of the TNM staging system
for CM, reflecting its importance for staging and treatment [3.80, 3.81]. The
procedure can, in fact, identify patients who may benefit from postoperative
adjuvant therapy and also provides a means of homogeneous stratification of
patients for and within randomized clinical trials [3.12].
Similar to breast cancer patients, in patients with CM, the presence of
an SLN that is free from metastasis obviates the need for lymphadenectomy,
while the presence of metastasis mandates it [3.82, 3.83]. Radical lymph node
dissection is also recommended when SLNB shows micrometastasis, because
approximately 5%–12% of these patients will have metastatic involvement of
non-sentinel nodes. At variance with breast cancer, locoregional lymph node
dissection in the case of a positive SLNB is performed not only for staging/
prognostic stratification, but also for improving overall survival of patients.
Overall, SLNB identifies those 20%–25% of patients who present with
clinically occult regional disease, by guiding the pathologist to perform extensive
analysis of the lymph node(s) most likely to contain metastatic disease. It
can also minimize the morbidity associated with elective lymphadenectomy
(e.g. lymphoedema), by identifying those patients most likely to benefit from
lymphadenectomy after a minor procedure. Moreover, similar to breast cancer,
SLNB in patients with melanoma allows the pathologist to analyse the SLN, using
multiple sections and different staining techniques, much more accurately than is
possible when analysing the numerous lymph nodes retrieved during locoregional
lymph node dissection. After conventional staining with haematoxylin eosin, it
is important to perform immunohistochemistry employing specific antibodies
against the melanoma associated S-100 and human melanoma black 45 antigens
[3.84–3.90].
Molecular biology RT-PCR techniques to detect the messenger ribonucleic
acid of melanoma related molecules such as tyrosinase (the key enzyme in the
synthesis of melanin) and the melanoma antigens recognized by T lymphocytes
49
(melanoma antigen recognized by T cells 1 (MART-1 and MART-2) are
also gaining popularity [3.91]. These techniques are characterized by high
sensitivity and specificity, and have high prognostic values. In particular, they
allow identification of patients who, despite a negative SLN by conventional
histopathology, have a tumour recurrence risk between that of patients with
negative molecular analysis and those whose sentinel nodes contain metastasis
by conventional histopathological evaluation [3.92–3.97].
Metastatic spread through the lymphatic system starts with the infiltration
of neoplastic cells through the basal lamina in the connective tissue. After
infiltrating the endothelium of lymphatic vessels, cancer cells follow the
lymphatic flow and home in on the first lymph node encountered (the SLN),
proliferating in the sinusoids and remaining confined in the subcapsular space to
subvert the normal lymph node structure. Accurate staging of lymph node status
is therefore of paramount importance in the management of patients with early
stage melanoma (clinical stages I and II). In fact, SLNB probably has maximum
clinical impact in the early to intermediate stages of the disease (with a Breslow
thickness of less than 4 mm) [3.98–3.101].
Prophylactic lymph nodal dissection in the basin where a metastatic SLN
was found and postoperative adjuvant therapy with interferon type 2β result in
increasing overall survival (especially in the case of micrometastasis).
The success of SLN identification with gamma probe guidance is very
high in patients with CM (~98%), higher than that obtained using the blue
dye technique alone (75%–80%). When combining these two techniques
(radiocolloid and blue dye), the success rate for SLN identification is higher than
99% [3.75, 3.76, 3.102].
While in breast cancer patients, axillary lymphadenectomy performed in
the case of positive SLNB is mainly used to better stratify patients for prognostic
purposes, in CM patients, locoregional lymphadenectomy represents per se an
effective therapy for patients with SLN metastasis. In fact, lymphadenectomy
reduces mortality from about 70% to about 50% in these patients.
In early studies on SLNB in CM patients, complete lymphadenectomy was
performed, regardless of the presence or absence of SLN metastasis, to determine
the accuracy of SLNB. In these early studies, false negative results were observed
in as few as 2% of patients [3.103]. Subsequent studies based on the appearance
of metastasis in lymphatic basins that had been classified as negative by SLNB,
and in which, therefore, lymphadenectomy had not been performed, have
instead reported higher false negative values of between 4% and 16% [3.104].
However, in these studies, the SLN had only been analysed with intraoperative
frozen section histology, which is now known to have a low sensitivity. In some
studies, only haematoxylin eosin staining (without immunohistochemistry or
without multiple sections) had been performed. Therefore, such apparently
50
high false negative rates of SLNB may have been related to the accuracy of the
histopathological analysis of SLNs [3.84]. On the other hand, molecular analysis
may ensure maximum accuracy of the technique, and in fact reduces false
negative SLNB rates [3.97].
—— SLNB is also employed in patients with melanoma to ascertain

TNM staging.
—— SLNB identifies those 20%–25% of patients who present with clinically
occult lymph node disease.
—— The presence of an SLN that is free from metastasis obviates the need for
lymphadenectomy, while the presence of metastasis mandates it.
—— In the case of a positive SLNB, locoregional lymph node dissection is
performed, not only for staging/prognostic stratification, but also for
improving the overall survival of patients.
—— Similar to breast cancer, SLNB in patients with melanoma allows the
pathologist to analyse accurately the SLN using multiple sections and
different staining techniques.
—— The success of SLN identification with gamma probe guidance is very high
(~98%), and is greater than that obtained with the blue dye technique alone
(75%–80%).
3.4.1. Indications and contraindications to SLNB in melanoma
Patients with high risk lesions between 0.75 mm and 0.99 mm in thickness
should be considered for SLNB if their melanoma is Clark level IV or V,
ulcerated, shows a vertical growth phase, or has lymphatic invasion or a high
mitotic rate. On the other hand, a contraindication to SLNB in CM is a lesion of
Breslow thickness greater than 4 mm, because in these cases, the probability of
locoregional lymph node metastasis is high, therefore, regional lymphadenectomy
may be indicated, regardless of the SLN status. However, even in these cases,
lymphoscintigraphy can identify unexpected patterns of lymphatic drainage, for
instance, towards distant or contralateral lymph node basins, and SLNB may
identify patients who are node negative and may be spared dissection [3.76].
Prior wide excision with large skin flaps, which may distort the pattern of
lymphatic drainage, may be a contraindication to radioguided SLNB because
lymphoscintigraphy could show unreliable lymphatic drainage, which is different
from that followed by cancer cells prior to surgery.
Lymph from CM lesions localized in the extremities flows from the site
of the primary tumour predictably to the ipsilateral axilla or to the ipsilateral
inguinal lymphatic basins; nevertheless, SLNs may also be found in aberrant
51
areas of drainage, such as the epitrochlear nodal basin in the upper extremities
and the popliteal nodal basin in the lower extremities.
On the contrary, the patterns and direction of lymphatic drainage are very
variable and often unpredictable for melanomas in the median and paramedian
regions of the trunk, as well as in the head and neck regions. In these locations,
lymphatic drainage is often ambiguous, with drainage being possible to both
axillary and inguinal regions, or to the contralateral neck [3.105–3.109].
Several studies have demonstrated that lymphatic drainage from melanomas
of the head, neck and trunk cannot be reliably predicted based on the classic
anatomical notions of Sappey. Instead, lymphoscintigraphy directly visualizes
drainage from these sites to SLNs in aberrant locations. This possibility highlights
the importance of preoperative lymphoscintigraphy for performing radioguided
SLNB in these patients.
For melanomas of the trunk, preoperative lymphoscintigraphic localization
to multiple nodal basins has been demonstrated in 17%–32% of patients
undergoing radioguided SLNB [3.110−3.112].
Likewise, preoperative lymphoscintigraphy identifies in-transit (interval,
aberrant) SLNs in 3%–10% of patients. Metastatic disease is found in
approximately 18% of these in-transit SLNs, which is similar to the rate found in
conventional lymph node basins, and the status of one basin does not predict the
status of the other basin [3.113−3.115].
—— Indications for SLNB in melanoma are patients with a Breslow thickness

ranging between 1 mm and 4 mm, or high risk lesions with a Breslow
thickness between 0.75 mm and 0.99 mm (with Clark levels IV or V,
ulcerated, showing a vertical growth phase, or with lymphatic invasion or a
high mitotic rate).
—— An absolute contraindication is a lesion of Breslow thickness greater than
4 mm, because in these cases, the probability of locoregional lymph node
metastasis is high (therefore, lymph node dissection should be performed
de novo).
—— A relative contraindication is prior wide excision with large skin flaps
because lymphoscintigraphy could show unreliable lymphatic drainage,
which is different from that followed by cancer cells prior to surgery.
3.4.2. SLNB procedures in melanoma
There is general consensus that the optimal modality of radiocolloid

administration in patients with melanoma is intra/subdermal injection. This is
usually performed as multiple administrations around the primary tumour or
52
around the recent surgical scar, which is the most common occurrence because
the diagnosis and thickness of melanomas are usually determined by excisional
biopsy. In order not to alter the lymphatic drainage pattern or kinetics, as
could be caused by an excessive increase in interstitial pressure, it is generally
recommended to inject small volumes of radiocolloid, about 0.05–0.2 mL per
aliquot, with each aliquot containing approximately 5 MBq of 99mTc radiocolloid.
The total administered activity is generally higher, usually between 74 MBq and
100 MBq, than that used for breast cancer [3.102, 3.104]. For melanomas of
the extremities, a single injection could be performed perilesionally on the side
towards the axilla or groin.
Lymphoscintigraphy is generally performed using a wide-field-
of-view gamma camera, equipped with a high resolution collimator. Unlike
lymphoscintigraphy for breast cancer, dynamic scintigraphic acquisition should
always be performed for melanoma [3.116]. The dynamic images (one every
30 s or 60 s) are usually acquired during the first 20–30 min from injection, after
which static images of the lymphatic basin reached by the radiocolloid are usually
acquired. Planar static images are acquired in different projections, depending on
the pattern of lymphatic drainage and location of the SLN(s), to identify and mark
with a dermographic pen the skin projection of the SLNs. Lymphoscintigraphy
frequently shows more than one SLN. The most radioactive node may not be
the first one visualized along the pathway(s) of lymphatic drainage, i.e. the
SLN. Dynamic acquisition is especially useful to highlight lymphatic drainage
and to visualize the correct progressive order of lymph nodes along the drainage
pathway (Figs 3.6 and 3.7).
Tomographic imaging with hybrid SPECT/CT equipment is especially
useful when performing lymphoscintigraphy for patients with CM because
it allows better evaluation of the anatomic topographical coordinates of the
lymph nodes and of the surrounding structures. As in breast cancer, SPECT/CT
constitutes a valuable aid to the surgeon for more rapid intraoperative localization
of the SLNs (Fig. 3.8) [3.56, 3.117]. It is useful for providing important additional
information to planar imaging, especially when the sentinel node is located in
uncommon locations that require unusual surgical access.
53
FIG. 3.6.  Planar SPECT dynamic images showing the drainage flow connecting adjacent
lymph nodes (a–b); an enlarged view of a single frame (c) and the corresponding SPECT-CT
3-D volume rendering display showing bone and muscle anatomical structures (d).
FIG. 3.7.  Planar dynamic acquisition showing the migration of radioactivity from the injection
site to the lymph nodes (a). Enlarged view of a single dynamic frame (b).
54
FIG. 3.8.  SPECT/CT lymphoscintigraphy of a patient with breast cancer showing uptake in
axillary lymph nodes.
SLNB in CM patients is performed within a few hours of

lymphoscintigraphy, on the same day or on the next day, depending on the
institutional preference. The gamma probe is inserted through the incision made
on the cutaneous marker, and it is slowly moved to explore the entire lymphatic
basin. After identification and excision, the SLN should be counted ex vivo
with the gamma probe. The resulting counts are important to confirm that the
excised node is the SLN. Furthermore, the counts constitute a reference for other
radioactive lymph nodes in the basin. The basin should be explored again with
the gamma probe, looking for any other radioactive lymph nodes. These should
be removed if their ex vivo counting rate is higher than 10%–20% of the ex vivo
counting rate of the SLN [3.118, 3.119].
The role of SPECT/CT imaging for SLNB in melanomas is similar to that
already mentioned for breast cancer. Conventional planar lymphoscintigraphy
offers a clear visualization of SLNs, providing practical information on their
number and location for skin marking and showing unexpected, aberrant and
deeper nodes. However, some important aspects, such as depth and anatomical
relationship of the SLNs with surrounding tissues, cannot be obtained using
planar imaging alone. There are also instances where false negative interpretation
can occur, for instance, when one SLN is obscured by another, or by the nearby
55
radiocolloid injection site, when the SLN contains too little radioactivity or
when two adjacent lymph nodes are thought to be a single node [3.120]. In this
regard, the introduction of SPECT/CT [3.121–3.124] has overcome some of
the limitations of planar scintigraphic mapping. SPECT/CT fused images show
SLNs in an anatomical 3-D volume rendering scenario, thus providing a real
anatomical map through which the surgeon can navigate for searching the SLNs
more easily [3.56, 3.125]. Accurate SLN localization leads to improved patient
management owing to changes in treatment or surgical approach. SPECT/CT is
particularly helpful for localizing SLNs draining from primary tumours high on
the trunk, or in the head and neck region.
—— The SLNB procedure consists of several sequential phases: radiocolloid

injection, lymphoscintigraphy (aided or not by SPECT/CT), intraoperative
detection with a gamma probe (aided or not by intraoperative imaging) and,
finally, pathological analysis of the resected SLNs.
—— Radiocolloid administration is generally performed peritumourally or
around the surgical scar, with at least four aliquots of radiocolloid.
—— Lymphoscintigraphy is a mandatory step of SLNM, performed using a
gamma camera to visualize lymphatic routes and to identify the SLNs of
each lymphatic basin.
—— SPECT/CT, MPR and volume rendering for 3-D display allow
better identification of anatomical landmarks as a reference for
anatomotopographic localization of the SLN(s).
—— In the intraoperative phase, the surgeon is radioguided to localize the SLNs
by a gamma probe (with an acoustic sound) or by a portable gamma camera
(with real time images).
3.5. OTHER APPLICATIONS OF RADIOGUIDED SLNB
As mentioned earlier, SLNB procedures in patients with either breast

cancer or CM have already achieved the status of standard of care for N-staging
purposes, so that further management of cancer patients after primary surgery can
adequately be planned. Nevertheless, potential benefits of this approach can also
be envisaged for other solid cancers, with the rationale of accurately mapping the
status of locoregional lymph nodes while avoiding aggressive procedures based
on extensive lymph node dissection.
Numerous clinical trials have explored the feasibility and accuracy of
SLNB in several oncological conditions, including, among others, tumours of the
head and neck region, the gastrointestinal tract and the genitourinary apparatus.
56
Nevertheless, in all these fields, SLNM should still be considered within well
designed experimental clinical trials, as further investigations are required for
defining the clinical impact of this procedure. In this section, the main potential
applications of SLNB in tumours other than breast cancer and melanoma are
briefly listed.
3.5.1. SLNB in head and neck cancers
In addition to melanomas located in the head and neck, the most important
applications of SLNB for tumours of this region concern cancers of the oral cavity.
In these patients, radioguided SLNB is emerging as a highly promising modality
to stage the clinical and radiological N0 neck, considering the poor sensitivity
of other commonly used radiological and nuclear medicine staging modalities.
A positive SLN is considered to be an unfavourable prognostic factor [3.126].
However, the role of SLNB in oral cancer is not as well established as it is
for breast cancer and melanoma, and some controversy still persists regarding
the appropriate indication. The procedure has been used for stage T1 or T2
lesions [3.127, 3.128], although several investigators have found selective neck
dissection more appropriate in view of the high risk of nodal metastasis [3.129].
In general, patients with transorally resectable T1–T2 tumours and with negative
lymph node assessment on clinical and radiological examination (including fine
needle aspiration cytology) are considered for SLNB.
SLNB in the head and neck region may be complex owing to difficult
anatomical relations and unpredictable lymphatic drainage. Generally, lymphatic
drainage is expected to occur towards the homolateral lymph nodes of the neck,
but in cancers of the oral cavity, lymphatic drainage can be highly unpredictable,
and contralateral drainage is often seen, even in well lateralized malignancies of
the tongue or floor of the mouth [3.130, 3.131].
Early results regarding the accuracy of SLNB in patients with oral cavity
malignancies, in which both the SLNB procedure and a complete selective neck
dissection were performed, showed accurate staging of regional lymph nodes,
with a 96% negative predictive value [3.132].
Lymphoscintigraphy for SLNM is generally performed some hours before
the scheduled time for surgery. After peritumoural or intratumoural radiocolloid
injection, both planar lymphoscintigraphy and SPECT/CT images are generally
acquired. Intraoperatively, the search for the radioactive SLN is usually
performed using a conventional hand held gamma probe. However, the advent
of new techniques such as the use of a portable gamma camera for intraoperative
SLND or the concomitant injection of radiolabelled and fluorescent colloidal
agents may improve the accuracy of SLNB in head and neck cancers, especially
when the injection site is close to the SLN basin; such combined imaging may, in
57
fact, help the surgeon to localize the SLNs in relation to the anatomical level in
the neck [3.133, 3.134].
For thyroid cancer, SLNB may display some benefits for accurate nodal
staging, particularly for detecting metastatic lymph nodes outside the central
neck, and for selecting patients who would benefit from complete neck dissection
and optimized radioiodine ablation therapy. Currently, however, there is no
direct evidence that SLNB is associated with long term clinical and survival
benefits in patients with thyroid cancer. The procedure includes peritumoural
or intratumoural radiocolloid administration and accurate preoperative imaging
to optimally plan lymph node dissection. Well controlled prospective clinical
trials will determine the clinical significance of occult metastases and their early
detection by SLNB in patients with thyroid cancer [3.134].
3.5.2. SLNB in gastrointestinal cancers
Procedures for SLNM and SLNB in gastrointestinal cancers have gradually

expanded in recent years. Lymphatic drainage of the gastrointestinal tract is much
more complicated than other sites, with skip metastasis being rather frequent
because of aberrant lymphatic drainage outside the basin [3.135]. At present, the
applicability of the SLN concept in cancers of the gastrointestinal tract has still to
be established through well designed prospective multicentre studies. The aim of
SLNB varies according to the tumour site.
In cases of oesophageal cancer, SLNB can minimize surgical invasiveness,
but still allow accurate analysis of the lymph node status. In fact, considering that
cancers of the distal oesophagus can metastasize to cervical lymph nodes, while
cancers of the cervical oesophagus can metastasize to celiac lymph nodes, SLNB
is expected to allow for a more selective approach [3.136].
The extent of lymphadenectomy in gastric cancer has been a source of
controversy for decades. Because of a lack of consensus regarding the surgical
approach to lymph node dissection, individualized approaches based on SLNB
are still being debated, and could represent an opportunity worth exploring,
especially in patients with early gastric cancer (T1–T2). In this regard,
laparoscopic function, preserving resection of the primary gastric tumour, in
conjunction with lymphatic basin dissection, guided by SLNB, could become
the standard for treating early gastric cancer. In the event of uninvolved SLNs,
transabdominal surgery could be avoided [3.137].
The most accurate SLNB approach combines both blue dye and radiocolloid
administration (i.e. endoscopic submucosal injection of radiocolloid the day
prior to surgery and endoscopic submucosal injection of 1% isosulphan blue
intraoperatively) [3.138].
58
Moreover, SLNB can disclose unexpected and/or aberrant sites of drainage,
thus guiding surgeons to perform a regional dissection approach tailored to the
individual patient [3.139−3.142].
The potential advantages of SLNM in the case of colorectal cancer consist
of the identification of aberrant lymphatic drainage leading to modification
of the intended surgical approach (an event occurring in 5%–8% of patients).
Moreover, following standardized surgical procedure (including selective
lymphadenectomy), SLNB can identify the crucial node(s) to be submitted to
extensive analysis with immunohistochemistry to search for micrometastases.
The overall success rate for radioguided SLNB is generally high. Most
of the failures reported in patients with rectal cancer are most likely because of
local submucosal lymphatic fibrosis induced by neoadjuvant radiation therapy
administered prior to surgery [3.143]. Furthermore, the overall accuracy for
predicting lymph node metastases is generally high (~95%), and the false
negative rate is low.
The modality of radiocolloid administration varies according to the tumour
site, although in most cases, it can be performed during preoperative endoscopy;
as is the case for other tumours, the blue dye is instead injected intraoperatively.
Use of the two agents in combination is reported to lead to better results.
Usually, radiocolloid injection is performed between 2 h and 24 h before surgery,
either submucosally around the tumour (during endoscopy prior to surgery) or
subserosally (during open or laparoscopic surgery) [3.144].
Because of a recurrence rate as high as 30% in node negative colon cancer
patients treated only with surgery and locoregional lymphadenectomy, SLNB
could more appropriately allow for selective focused sampling techniques to
ultrastage patients and ultimately identify a subset of patients who will benefit
from adjuvant chemotherapy [3.145, 3.146]. With this approach, lymph nodal
upstaging occurs in about 20% of the cases. Nevertheless, the clinical impact of
micrometastasis in terms of prognosis and need for adjuvant chemotherapy has
still to be clarified.
In rectal cancer, SLNM could be useful to select patients who would benefit,
after standard surgery, from a selective approach with lateral pelvic lymph node
dissection, which becomes necessary in about 15% of the cases [3.146, 3.147].
Radioguided SLNB has also been applied to patients with anal
cancer [3.148−3.154]. SLN visualization is generally demonstrated in 75%–100%
of the patients. There are three common pathways of lymphatic drainage from
anal cancers (i.e. drainage to the inguinal, iliac and mesorectal lymphatic basins).
The very frequent finding of bilateral lymphatic drainage emphasizes the
importance of preoperative lymphoscintigraphy for most effectively performing
radioguided SLNB. The inguinal region is the predominant lymphatic drainage
pathway, representing the site of localization most accessible to perform a
59
minimally invasive radioguided SLNB. However, large scale prospective clinical
trials assessing the clinical impact of radioguided SLNB in patients with anal
cancer are still lacking.
3.5.3. SLNB in tumours of the female reproductive system
There has also been increasing interest in the use of radioguided SLNB in
gynaecological cancers, because this procedure has been shown to be accurate
for evaluating nodal basins for metastatic disease and is associated with reduced
short term and long term morbidity when compared with complete lymph
node dissection.
Because of the high morbidity rates of lymph nodal dissection, recent
literature continues to support the safety and feasibility of SLNB for early
stage vulvar cancer because the procedure is associated with low recurrence
rates, excellent survival, lower morbidity and shorter hospital stay compared
to classical inguinal dissection. SLNB therefore promises to provide adequate
staging with less treatment related morbidity, especially in selected vulvar cancer
patients in whom it can be considered an alternative surgical approach to full
lymph node dissection [3.155].
For endometrial and cervical cancer, SLNB has been described with
encouraging results, although this application requires further investigation, given
some ambiguity in published reports. In particular, reports on SLNB only include
small groups of patients, and the SLND rates vary depending on the injection
sites/modalities and the agents employed [3.156]. For endometrial cancer, most
studies have reported low false negative rates, with variable sensitivities and
generally low SLND rates. Radioguided SLNB is also promising in early stage
cervical cancer, with detection rates and sensitivities greater than 90%.
3.5.4. SLNB in tumours of the male reproductive system
For cancers of the male reproductive system, the feasibility of radioguided

SLNB is increasingly being explored because of the high incidence of morbidity,
such as lymphoedema and infections, after standard surgery with full lymph
node dissection. Moreover, the introduction of microinvasive surgical techniques
implies the possibility to achieve proper evaluation of the lymph node status,
even with a small surgical incision. This goal can be reached by analysing
SLNs with serial sectioning and extensive histopathological analysis, including
immunohistochemical staining.
There is no consensus on the management of patients with clinically node
negative penile carcinoma, in whom radical inguinal lymph node dissection is
routinely performed, although it turns out to be unnecessary in approximately
60
75%–80% of patients [3.157]. In addition, this procedure is associated with
substantial morbidity. However, since its clinical introduction, there have been
reservations about the use of SLNB for penile cancer because of the supposedly
long learning curve associated with the procedure and because of possible false
negative cases (up to 21% of procedures) [3.158]. After analysis of the possible
causes of false negative cases, several modifications were introduced to increase
the sensitivity of the SLNB procedure [3.159]. Thus, radioguided SLNB has
currently evolved into a reliable minimally invasive staging technique with
an associated high sensitivity (90%–95%) and low morbidity in experienced
centres [3.160]. However, there are still important differences in the clinical
protocols employed worldwide; harmonization of the different phases of the
SLNB procedure in patients with penile cancer (including how to implement the
learning curve) is therefore needed.
For prostate cancer, where the incidence of complications post lymph
node dissection rises up to 25% with 20 lymph nodes dissected, the main
advantage of SLNB is the possibility to reduce the incidence of such unnecessary
complications [3.161]. Accurate laparoscopic radioguided localization of SLNs
in the pelvis can be challenging, especially when the SLNs are located near the
intraprostatic injection site (because of the high radioactive background) or in
the case of aberrantly located SLNs [3.162]. Original validation of radioguided
SLNB for prostate cancer was based on open surgery and on the use of a
gamma probe to guide detection of the radioactive SLNs (with only 5.5% false
negative cases being reported) [3.163]. SLNB has recently been validated using
a laparoscopic gamma probe during minimally invasive surgery with a similarly
high detection rate (94%) and sensitivity (~95%) [3.164].
Large scale randomized clinical studies are necessary to assess and validate
the added benefit of SLNB in patients with testicular cancers. In this regard, only
a few studies [3.165–3.167] have been published, all with small size populations
(<25 patients per study), which are therefore without the statistical power and
follow-up data to assess sensitivity/false negative rates. On the other hand, in
these patients, radioguided SLNM and SLNB was found to be potentially useful
to detect aberrant lymphatic drainage [3.165–3.167] and to identify those selected
patients who would benefit from adjuvant treatment after orchidectomy.
[3.1] MARIANI, G., GIULIANO, A.E., STRAUSS, H.W., Radioguided Surgery:

A Comprehensive Team Approach, Springer, New York (2008).
[3.2] EDGE, S.B., et al. (Eds), AJCC Cancer Staging Manual, 7th edn, Springer, New York
(2009) 347–369.
61
[3.3] ZAKNUN, J.J, et al., Changing paradigms in radioguided surgery and intraoperative
imaging: the GOSTT concept, Eur. J. Nucl. Med. Mol. Imaging 39 (2012) 1–3.
[3.4] SIEGEL, R., NAISHADHAM, D., JEMAL, A., Cancer statistics 2012, CA Cancer J.
Clin. 62 1 (2012) 10–29.
[3.5] KRAG, D.N., et al., Surgical resection and radiolocalization of sentinel lymph node in
breast cancer using a gamma probe, Surg. Oncol. 2 (1993) 335–339.
[3.6] GIULIANO, A.E., KIRGAN, D., GUENTHER, J.M., Lymphatic mapping and
sentinel lymphadenectomy for breast cancer, Ann. Surg. 220 (1994) 391–401.
[3.7] GIULIANO, A.E., et al., Improved axillary staging of breast cancer with sentinel
lymphadenectomy, Ann. Surg. 222 (1995) 394–401.
[3.8] NOGUCHI, M., KATEV, N., MIYAZAKI, I., Diagnosis of axillary lymph node
metastases in patients with breast cancer, Breast Cancer Res. Treat. 40 (1996) 283–293.
[3.9] TAYLOR, A., Jr., et al., Dynamic lymphoscintigraphy to identify the sentinel and
satellite nodes, Clin. Nucl. Med. 21 (1996) 755–758.
[3.10] MEIJER, S., et al., Less axillary dissection necessary due to sentinel node biopsy in
patients with breast carcinoma, Ned. Tijdschr. Geneeskd. 140 (1996) 2239–2243.
[3.11] ALBERTINI, J.J., et al., Lymphatic mapping and sentinel node biopsy in the patient
with breast cancer, JAMA 276 (1996) 1818–1822.
Semin. Nucl. Med. 27 (1997) 55–67.
[3.13] PIJPERS, R., et al., Impact of lymphoscintigraphy on sentinel node identification with
technetium-99m-colloidal albumin in breast cancer, J. Nucl. Med. 38 (1997) 366–368.
[3.14] GIULIANO, A.E., Lymphatic mapping and sentinel node biopsy in breast cancer.
JAMA 277 (1997) 791–792.
[3.15] REINTGEN, D., et al., The role of selective lymphadenectomy in breast cancer,
Cancer Control 4 (1997) 211–219.
cancer with clinically negative lymph-nodes. Lancet 349 (1997) 1864–1867.
[3.17] MARIANI, G., et al., Radioguided sentinel lymph node biopsy in breast cancer
surgery, J. Nucl. Med. 42 (2001) 1198–1215.
[3.18] LYMAN, G.H., et al., American Society of Clinical Oncology guideline
recommendations for sentinel lymph node biopsy in early-stage breast cancer, J. Clin.
Oncol. 23 (2005) 7703–7720.
[3.19] BENSON, J.R., DELLA ROVERE, G.Q., AXILLA MANAGEMENT CONSENSUS
GROUP, Management of the axilla in women with breast cancer, Lancet Oncol 8
(2007) 331–348.
[3.21] BONNEMA, J., et al., Ultrasound guided aspiration biopsy for detection of non
palpable axillary node metastases in breast cancer patients: new diagnostic method,
World J. Surg. 21 (1997) 270–274.
[3.22] CARLSON, R.W., et al., Breast cancer: clinical practice guidelines in oncology,
J. Natl. Compr. Canc. Netw. 7 (2009) 122–192.
62
[3.23] PURUSHOTHAM, A.D., et al., Morbidity after sentinel node biopsy in primary
breast cancer: results from a randomized controlled trial, J. Clin. Oncol. 23
(2005) 4312–4321.
[3.24] ESTOURGIE, S.H., et al., Eight false negative sentinel lymph node procedures in
breast cancer: what went wrong? Eur. J. Surg. Oncol. 29 (2003) 336–340.
[3.25] LEIJTE, J., et al., Visualization of tumor blockage and rerouting of lymphatic drainage
in penile cancer patients by use of SPECT/CT, J. Nucl. Med. 50 (2009) 364–367.
[3.26] NATIONAL COMPREHENSIVE CANCER NETWORK, Clinical Practice
Guidelines in Oncology. Breast Cancer VI (2010),
www.nccn.org /professionals/physician_gls/f_guidelines.asp
[3.27] VERONESI, U., et al., A randomized comparison of sentinel-node biopsy with routine
axillary dissection in breast cancer, New Engl. J. Med. 349 (2003) 546–553.
[3.28] KRAG, D.N., et al., Sentinel lymph node resection compared with conventional
axillary-lymph node dissection in clinically node-negative patients with breast cancer:
overall survival findings from the NSABP-32 randomised phase 3 trial, Lancet Oncol.
11 (2010) 927–933.
[3.29] KRAG, D.N., et al., Technical outcomes of sentinel-lymph node resection and
conventional axillary lymph-node dissection in patients with node-negative breast
cancer: results from the NSABP B32 randomised phase III trial, Lancet Oncol. 8
(2007) 881–888.
[3.30] GOYAL, A., NEWCOMBE, R.G., CHABRA, A., MANSEL, R.E., ALMANAC
TRIALISTS GROUP, Factors affecting failed localization and false-negative rates
of sentinel node biopsy in breast cancer–results of the ALMANAC validation phase,
Breast Cancer Res. Treat. 99 (2006) 203–208.
[3.31] GIULIANO, A.E., et al., Axillary dissection versus no axillary dissection in women
with invasive breast cancer and sentinel node metastasis. A randomized clinical trial,
JAMA 305 (2011) 569–575.
[3.32] KRYNYCKYI, B.R., et al., Factors affecting visualization rates of internal mammary
sentinel nodes during lymphoscintigraphy, J. Nucl. Med. 44 (2003) 1387–1393.
[3.33] PAREDES, P., et al., Clinical relevance of sentinel lymph nodes in the internal
mammary chain in breast cancer patients, Eur. J. Nucl. Med. Mol. Imaging 32 (2005)
1283–1287.
[3.34] ESTOURGIE, S.H., et al., Should the hunt for internal mammary chain sentinel nodes
begin? An evaluation of 150 breast cancer patients, Ann. Surg. Oncol. 10 (2003)
935–941.
[3.35] VAN DER ENT, F.W., et al., Halsted revisited: internal mammary sentinel lymph
node biopsy in breast cancer, Ann. Surg. 234 (2001) 79–84.
[3.36] BOURRE, J.C., et al., Can the sentinel lymph node technique affect decisions to
offer internal mammary chain irradiation? Eur. J. Nucl. Med. Mol. Imaging 36 (2009)
758–764.
[3.37] VERONESI, U., MARUBINI, E., MARIANI, L., VALAGUSSA, P., ZUCALI, R.,
The dissection of internal mammary nodes does not improve the survival of
breast cancer patients. 30-year results of a randomised trial, Eur. J. Cancer 35
(1999) 1320–1325.
63
[3.38] GOLDHIRSCH, A., et al., Thresholds for therapies: highlights of the St Gallen
International expert consensus on the primary therapy of early breast cancer 2009,
Ann. Oncol. 20 (2009) 1219–1229.
[3.39] KAUFMANN, M., Locoregional treatment of primary breast cancer, Cancer 116
(2010) 1184–1191.
[3.40] RODRIGUEZ-FERNANDEZ, J., et al., Sentinel node biopsy in patients with previous
breast aesthetic surgery, Ann. Surg. Oncol. 16 (2009) 989–992.
[3.41] TABACK, B., et al., Sentinel lymph node biopsy for local recurrence of breast cancer
after breast-conserving therapy, Ann. Surg. Oncol. 13 (2006) 1099–1104.
[3.42] KUMAR, R., et al., Retrospective analysis of sentinel node localization in multifocal,
multicentric, palpable, or nonpalpable breast cancer, J. Nucl. Med. 44 (2003) 7–10.
[3.43] KLIMBERG, V.S., et al., Subareolar versus peritumoral injection for location of the
sentinel lymph node, Ann. Surg. 229 (1999) 860–864.
[3.44] NIEWEG, O.E., ESTOURGIE, S.H., VAN RIJK, M.C., KROON, B.B., Rationale
for superficial injection techniques in lymphatic mapping in breast cancer patients, J.
Surg. Oncol. 87 (2004) 153–156.
[3.45] KNAUER, M., et al., Multicentric breast cancer: a new indication for sentinel node
biopsy – a multi-institutional validation study, J. Clin. Oncol. 24 (2006) 3374–3380.
[3.46] GOYAL, A., et al., Sentinel lymph node biopsy in patients with multifocal breast
cancer, Eur. J. Surg. Oncol. 30 (2004) 475–479.
[3.47] GIARD, S., et al., Feasibility of sentinel lymph node biopsy in multiple unilateral
synchronous breast cancer: results of a French prospective multi-institutional study
(IGASSU 0502), Ann. Oncol. 21 (2010) 1630–1635.
[3.48] VERONESI, P., GENTILINI, O., RODRÍGUEZ-FERNANDEZ, J., MAGNONI, F.,
Breast conservation and sentinel lymph node after neoadjuvant systemic therapy,
Breast 18 (2009) 590–592.
[3.49] MAMOUNAS, E.P., et al., Sentinel node biopsy after neoadjuvant chemotherapy
in breast cancer: results from National Surgical Adjuvant Breast and Bowel Project
Protocol B-27, J. Clin. Oncol. 23 (2005) 2694–2702.
[3.50] KELLY, A.M., DWAMENA, B., CRONIN, P., CARLOS, R.C., Breast cancer sentinel
node identification and classification after neoadjuvant chemotherapy — systematic
review and metaanalysis, Acad. Radiol. 16 (2009) 551–563.
[3.51] PIÑERO, A., GIMENEZ, J., VIDAL-SICART, S., INTRA, M., Selective sentinel
lymph node biopsy and primary systemic therapy in breast cancer, Tumori 96
(2010) 17–23.
[3.52] PANDIT-TASKAR, N., DAUER, L.T., MONTGOMERY, L., et al., Organ and fetal
absorbed dose estimates from 99mTc-sulfur colloid lymphoscintigraphy and sentinel
node localization in breast cancer patients, J. Nucl. Med. 47 (2006) 1202–1208.
[3.53] KHERA, S.Y., et al., Pregnancy-associated breast cancer patients can safely undergo
lymphatic mapping, Breast J. 14 (2008) 250–254.
[3.54] GENTILINI, O., et al., Sentinel lymph node biopsy in pregnant patients with breast
cancer, Eur. J. Nucl. Med. Mol. Imaging 37 (2010) 78–83.
[3.55] MARIANI, G., BRUSELLI, L., KUWERT, T., et al., A review on the clinical uses of
SPECT/CT, Eur. J. Nucl. Med. Mol. Imaging 37 (2010) 1959–1985.
64
[3.56] VERMEEREN, L., VAN DER PLOEG, I.M., VALDES-OLMOS, R.A., et al.,
SPECT/CT for preoperative sentinel node localization, J. Surg. Oncol. 101
(2010) 184–190.
[3.57] EVEN-SAPIR, E., et al., Lymphoscintigraphy for sentinel node mapping using a
hybrid SPECT/CT system, J. Nucl. Med. 44 (2003) 1413–1420.
[3.58] LERMAN, H., et al., Lymphoscintigraphic sentinel node identification in patients
with breast cancer: the role of SPECT/CT, Eur. J. Nucl. Med. Mol. Imaging 33
(2006) 329–337.
[3.59] LERMAN, H., et al., Improved sentinel node identification by SPECT/CT in
overweight patients with breast cancer, J. Nucl. Med. 48 (2007) 201–206.
[3.60] MATHELIN, C.E., GUYONNET, J.L., Scintillation crystal or semiconductor
gamma-probes: an open debate, J. Nucl. Med. 47 (2006) 373.
[3.61] HOFFMAN, E.J., et al., Intra-operative probes and imaging probes, Eur. J. Nucl.
Med. 26 (1999) 913–935.
[3.62] SCOPINARO, F., et al., High-resolution, hand-held camera for sentinel-node
detection, Cancer Biother. Radiopharm. 23 (2008) 43–52.
[3.63] MATHELIN, C., et al., Precise localization of sentinel lymph nodes and estimation
of their depth using a prototype intraoperative mini gamma-camera in patients with
breast cancer, J. Nucl. Med. 48 (2007) 623–629.
[3.64] DUCH, J., Portable gamma cameras: the real value of an additional view in the
operating theatre, Eur. J. Nucl. Med. Mol. Imaging 38 (2011) 633–635.
[3.65] VIDAL-SICART, S., Added value of intraoperative real-time imaging in searches for
difficult-to-locate sentinel nodes, J. Nucl. Med. 51 (2010) 1219–1225.
[3.66] CARDONA-ARBONIES, J., et al., Contribution of the portable gamma camera to
detect the sentinel node in breast cancer during surgery, Rev. Esp. Med. Nucl. 31
(2012) 130–134.
[3.67] VIDAL-SICART, S., BROUWER, O.R., VALDES-OLMOS, R.A., Evaluation of the
sentinel lymph node combining SPECT/CT with the planar image and its importance
for the surgical act, Rev. Esp. Med. Nucl. 30 (2011) 331–337.
[3.68] VIDAL-SICART, S., et al., The use of a portable gamma camera for preoperative
lymphatic mapping: a comparison with a conventional gamma camera, Eur. J. Nucl.
Med. Mol. Imaging 38 (2011) 636–641.
[3.69] WENDLER, T., et al., First demonstration of 3-D lymphatic mapping in breast cancer
using freehand SPECT, Eur. J. Nucl. Med. Mol. Imaging 37 (2010) 1452–1461.
[3.70] RIEGER, A., et al., First experiences with navigated radio-guided surgery using
freehand SPECT, Case Rep. Oncol. 4 (2011) 420–425.
[3.71] VAN DER POEL, H.G., et al., Intraoperative laparoscopic fluorescence guidance
to the sentinel lymph node in prostate cancer patients: clinical proof of concept of
an integrated functional imaging approach using a multimodal tracer, Eur. Urol. 60
(2011) 826–833.
[3.72] KEEREWEER, S., et al., Optical image-guided surgery – where do we stand? Mol.
Imaging Biol. 13 (2011) 199–207.
[3.73] POLOM, K., et al., Current trends and emerging future of indocyanine green usage in
surgery and oncology: a literature review, Cancer 117 (2011) 4812–4822.
65
[3.74] THOMPSON, J.F., SCOLYER, R.A., KEFFORD, R.F., Cutaneous melanoma, Lancet
365 (2005) 687–701.
[3.75] GERSHENWALD, J.E., et al., “Sentinel lymph node biopsy in cutaneous melanoma”,
Radioguided Surgery: A Comprehensive Team Approach (MARIANI, G.,
GIULIANO, A.E., STRAUSS, H.W., Eds), Springer, New York (2008) 92–110.
[3.76] ROSS, M., “Sentinel node mapping for early-stage melanoma”, Educational
Book (PERRY, M.C., Ed.), American Society of Clinical Oncology, Alexandria,
VA (1997) 326–330.
[3.77] ALEX, J.C., KRAG, D.N., The gamma-probe-guided resection of radiolabeled
primary lymph nodes, Surg. Oncol. Clin. North Am. 5 (1996) 33–41.
[3.78] TAYLOR, A., Jr., et al., Dynamic lymphoscintigraphy to identify the sentinel and
satellite nodes, Clin. Nucl. Med. 21 (1996) 755–758.
[3.79] EDGE, S.B. (Eds), AJCC Cancer Staging Manual, 7th edn, Springer, New York
(2009) 325–340.
[3.80] GERSHENWALD, J.E., SOONG, S.J., BALCH, C.M., AMERICAN JOINT
COMMITTEE ON CANCER (AJCC) MELANOMA STAGING COMMITTEE.
2010 TNM staging system for cutaneous melanoma and beyond, Ann. Surg. Oncol.
17 (2010) 1475–1477.
[3.81] ALOIA, T.A., GERSHENWALD, J.E., Management of early-stage cutaneous
melanoma, Curr. Probl. Surg. 42 (2005) 460–534.
[3.82] BELHOCINE, T.Z., et al., Role of nuclear medicine in the management of cutaneous
malignant melanoma, J. Nucl. Med. 47 (2006) 957–967.
[3.83] GIBBS, J.F., et al., Accuracy of pathologic techniques for the diagnosis of metastatic
melanoma in sentinel lymph nodes, Ann. Surg. Oncol. 6 (1999) 699–704.
[3.84] COCHRAN, A.J., The pathologist’s role in sentinel lymph node evaluation, Semin.
Nucl. Med. 30 (2000) 11–17.
[3.85] KOOPAL, S.A., TIEBOSH, A.T., ALBERTUS PIERS, D., Frozen section analysis of
sentinel lymph nodes in melanoma patients, Cancer 89 (2000) 1720–1725.
[3.86] COCHRAN, A.J., et al., Current practice and future directions in pathology and
laboratory evaluation of the sentinel node, Ann. Surg. Oncol. 8 (2001) 13–17.
[3.87] PRIETO, V.G., CLARK, S., Processing of sentinel lymph nodes or detection of
metastatic melanoma, Ann. Diagn. Pathol. 6 (2002) 257–264.
[3.88] COOK, M.G., et al., The development of optimal pathological assessment of sentinel
lymph nodes for melanoma, J. Pathol. 200 (2003) 314–319.
[3.89] SCOLYER, R.A., THOMPSON, J.F., McCARTHY, S.W., Sentinel lymph nodes in
malignant melanoma: extended histopathologic evaluation improves diagnostic
precision, Cancer 1 (2004) 2141–2142 (author reply: 2142–2143).
[3.90] VIALE, G., MAZZAROL, G., MAIORANO, E., “Histopathology of sentinel lymph
nodes”, Radioguided Surgery: A Comprehensive Team Approach (MARIANI, G.,
GIULIANO, A.E., STRAUSS, H.W., Eds), Springer, New York (2008) 190–200.
[3.91] GOYDOS, J.S., et al., Minimally invasive staging of patients with melanoma: sentinel
lymphadenectomy and detection of the melanoma specific proteins MART-1 and
tyrosinase by reverse transcriptase polymerase chain reaction, J. Am. Coll. Surg. 187
(1998) 182–188.
66
[3.92] GOYDOS, J.S., et al., Patterns of recurrence in patients with melanoma and
histologically negative but RT-PCR-positive sentinel lymph nodes, J. Am. Coll. Surg.
196 (2003) 196–204.
[3.93] RIBUFFO, D., et al., Prognostic significance of reverse transcriptase-polymerase
chain reaction-negative sentinel nodes in malignant melanoma, Ann. Surg. Oncol. 10
(2003) 396–402.
[3.94] KAMMULA, U.S., et al., Serial follow-up and the prognostic significance of reverse
transcriptase-polymerase chain reaction-staged sentinel lymph nodes from melanoma
patients, J. Clin. Oncol. 22 (2004) 3989–3996.
[3.95] ROMANINI, A., et al., Molecular staging of the sentinel lymph node in melanoma
patients: correlation with clinical outcome, Ann. Oncol. 16 (2005) 1832–1840.
[3.96] MOCELLIN, S., et al., Sentinel lymph node molecular ultrastaging in patients with
melanoma: a systematic review and meta-analysis of prognosis, J. Clin. Oncol. 25
(2007) 1588–1595.
[3.97] BALCH, C.M., et al., A comparison of prognostic factors and surgical results in
1,786 patients with localized (stage I) melanoma treated in Alabama, USA, and
New South Wales, Australia, Ann. Surg. 196 (1982) 677–684.
[3.98] REINTGEN, D.S., et al., Efficacy of elective lymph node dissection in patients with
intermediate thickness primary melanoma, Ann. Surg. 198 (1983) 379–385.
[3.99] BALCH, C.M., et al., Efficacy of an elective regional lymph node dissection of 1
to 4 mm thick melanomas for patients 60 years of age and younger, Ann. Surg. 224
(1996) 255–263.
[3.100] LENS, M., Sentinel lymph node biopsy in melanoma patients, J. Eur. Acad. Dermatol.
Venereol. 24 (2010) 1005–1012.
[3.101] CHAKERA, A.H., et al., EANM-EORTC general recommendations for sentinel node
diagnostics in melanoma, Eur. J. Nucl. Med. Mol. Imaging 36 (2009) 1713–1742.
[3.102] THOMPSON, J.F., et al., Sentinel node biopsy for melanoma: where have we been
and where are we going? Ann. Surg. Oncol. 11 (2004) 147S–151S.
[3.103] MARIANI, G., et al., Radioguided sentinel lymph node biopsy in malignant cutaneous
melanoma, J. Nucl. Med. 43 (2002) 811–827.
[3.104] UREN, R.F., HOWMAN-GILES, R., THOMPSON, J.F., Lymphatic drainage to
triangular intermuscular space lymph nodes in melanoma on the back, J. Nucl. Med.
37 (1996) 964–966.
[3.105] UREN, R.F., HOWMAN-GILES, R., THOMPSON, J.F., Lymphatic drainage from
the skin of the back to retroperitoneal and paravertebral lymph nodes in melanoma
patients, Ann. Surg. Oncol. 5 (1998) 384–387.
[3.106] UREN, R.F., THOMPSON, J.F., HOWMAN-GILES, R., Sentinel nodes, interval
nodes, lymphatic lakes, and accurate sentinel node identification, Clin. Nucl. Med. 25
(2000) 234–236.
[3.107] THOMPSON, J.F., UREN, R.F., Anomalous lymphatic drainage patterns in patients
with cutaneous melanoma, Tumori 87 (2001) 54–56.
[3.108] UREN, R.F., HOWMAN-GILES, R., THOMPSON, J.F., Patterns of lymphatic
drainage from the skin in patients with melanoma, J. Nucl. Med. 44 (2003) 570–582.
67
[3.109] ALAZRAKI, N., et al., Procedure guideline for lymphoscintigraphy and the use of
intraoperative gamma probe for sentinel lymph node localization in melanoma of
intermediate thickness, J. Nucl. Med. 43 (2002) 1414–1418.
[3.110] LEONG, S.P., et al., Heterogeneous patterns of lymphatic drainage to sentinel lymph
nodes by primary melanoma from different anatomic sites, Clin. Nucl. Med. 30 (2005)
150–158.
[3.111] FEDERICO, A.C., et al., Effect of multiple-nodal basin drainage on cutaneous
melanoma, Arch. Surg. 143 (2008) 632–637.
[3.112] VIDAL-SICART, S., et al., Is the identification of in-transit sentinel lymph nodes in
malignant melanoma patients really necessary? Eur. J. Nucl. Med. Mol. Imaging 31
(2004) 945–949.
[3.113] THOMPSON, J.F., et al., Location of sentinel lymph nodes in patients with cutaneous
melanoma: new insights into lymphatic anatomy, J. Am. Coll. Surg. 189 (1999)
195–204.
[3.114] SCARSBROOK, A.F., GANESHAN, A., BRADLEY, K.M., Pearls and
pitfalls of radionuclide imaging of the lymphatic system. Part 1: sentinel node
lymphoscintigraphy in malignant melanoma, Br. J. Radiol. 80 (2007) 132–139.
[3.115] MARIANI, P., et al., Radioguided sentinel lymph node biopsy in patients with
malignant cutaneous melanoma: the nuclear medicine contribution, J. Surg. Oncol. 85
(2004) 141–151.
[3.116] UREN, R.F., SPECT/CT lymphoscintigraphy to locate the sentinel lymph node in
patients with melanoma, Ann. Surg. Oncol. 16 (2009) 1459–1460.
[3.117] McMASTERS, K.M., et al., Sentinel lymph node biopsy for melanoma: how many
radioactive nodes should be removed? Ann. Surg. Oncol. 8 (2001) 192–197.
[3.118] MANCA, G., et al., Optimal detection of sentinel lymph node metastases by
intraoperative radioactive threshold and molecular analysis in patients with melanoma,
J. Nucl. Med. 49 (2008) 1769–1775.
[3.119] UREN, R.F., Lymphatic drainage of the skin, Ann. Surg. Oncol. 11 (2004) 179S–185S.
[3.120] STATIUS MULLER, M.G., et al., Unpredictability of lymphatic drainage patterns in
melanoma patients, Eur. J. Nucl. Med. Mol. Imaging 29 (2002) 255–261.
[3.121] VALDES-OLMOS, R., VIDAL-SICART, S., NIEWEG, O., SPECT-CT and real-time
intraoperative imaging: new tools for sentinel node localization and radioguided
surgery? Eur. J. Nucl. Med. Mol. Imaging 36 (2009) 1–5.
[3.122] EVEN-SAPIR, E., et al., Lymphoscintigraphy for sentinel node mapping using a
hybrid SPECT/CT system, J. Nucl. Med. 44 (2003) 1413–1420.
[3.123] ISHIHARA, T., et al., Management of sentinel lymph nodes in malignant skin
tumors using dynamic lymphoscintigraphy and the single-photon-emission computed
tomography/computed tomography combined system, Int. J. Clin. Oncol. 11 (2006)
214–220.
[3.124] MAR, M.V., et al., Evaluation and localization of lymphatic drainage and sentinel
lymph nodes in patients with head and neck melanomas by hybrid SPECT/CT
lymphoscintigraphic imaging, J. Nucl. Med. Technol. 35 (2007) 10–16.
68
[3.125] KOVÁCS, A.F., et al., Positive sentinel lymph nodes are a negative prognostic factor
for survival in T1-2 oral/oropharyngeal cancer — a long-term study on 103 patients,
Ann. Surg. Oncol. 16 (2009) 233–239.
[3.126] CIVANTOS, F., et al., Sentinel node biopsy for squamous cell carcinoma of the head
and neck, J. Surg. Oncol. 97 (2008) 683–690.
[3.127] PALERI, V., et al., Sentinel node biopsy in squamous cell cancer of the oral cavity and
oral pharynx: a diagnostic meta-analysis, Head Neck 27 (2005) 739–747.
[3.128] FERLITO, A., SILVER, A., RINALDO, A., Elective management of the neck in oral
cavity squamous carcinoma: current concepts supported by prospective studies, Br. J.
Oral Maxillofac. Surg. 47 (2009) 5–9.
[3.129] SHOAIB, T., et al., The nodal neck level of sentinel lymph nodes in mucosal head and
neck cancer, Br. J. Plast. Surg. 58 (2005) 790–794.
[3.130] CALABRESE, L., et al., “Sentinel lymph node biopsy in cancer of the head and
neck”, Radioguided Surgery: A Comprehensive Team Approach (MARIANI, G.,
GIULIANO, A.E., STRAUSS, W.H., Eds), Springer, New York (2008) 120–129.
[3.131] CIVANTOS, F.J., et al., Sentinel lymph node biopsy accurately stages the regional
lymph nodes for T1-T2 oral squamous cell carcinomas: results of a prospective
multi-institutional trial, J. Clin. Oncol. 28 (2010) 1395–1400.
[3.132] VERMEEREN, L., et al., A portable gamma-camera for intraoperative detection of
sentinel nodes in the head and neck region, J. Nucl. Med. 51 (2010) 700–703.
[3.133] VAN DEN BERG, N.S., et al., Concomitant radio- and fluorescence-guided
sentinel lymph node biopsy in squamous cell carcinoma of the oral cavity using
ICG-99mTc-nanocolloid, Eur. J. Nucl. Med. Mol. Imaging 39 (2012) 1128–1136.
[3.134] ROH, J.L., KOCH, W.M., Role of sentinel lymph node biopsy in thyroid cancer,
Expert Rev. Anticancer. Ther. 10 (2010) 1429–1437.
[3.135] KOSAKA, T., et al., Lymphatic routes of the stomach demonstrated by gastric
carcinomas with solitary lymph node metastasis, Surg. Today 29 (1999) 695–700.
[3.136] TAKEUCHI, H., KITAGAWA, Y., Sentinel node navigation surgery for esophageal
cancer, Gen. Thorac Cardiovasc. Surg. 56 (2008) 393–396.
[3.137] OTANI, Y., et al., New method of laparoscopy-assisted function-preserving surgery
for early gastric cancer: vagus-sparing segmental gastrectomy under sentinel node
navigation, J. Am. Coll. Surg. 198 (2004) 1026–1031.
[3.138] KITAGAWA, Y., SAHA, S., KUBO, A., Sentinel node for gastrointestinal
malignancies, Surg. Oncol. Clin. North Am. 16 (2007) 71–80.
[3.139] HAYASHI, H., et al., Sentinel lymph node mapping for gastric cancer using a dual
procedure with dye- and gamma probe-guided techniques, J. Am. Coll. Surg. 196
(2003) 68–74.
[3.140] GRETSCHEL, S., et al., Efficacy of different technical procedures for sentinel lymph
node biopsy in gastric cancer staging, Ann. Surg. Oncol. 14 (2007) 2028–2035.
[3.141] KITAGAWA, Y., et al., Radioguided sentinel node detection for gastric cancer, Br. J.
Surg. 89 (2002) 604–608.
[3.142] EUNOSONO, Y., et al., Detection of sentinel nodes and micrometastases using
radioisotope navigation and immunohistochemistry in patients with gastric cancer, Br.
J. Surg. 92 (2005) 886–889.
69
[3.143] BILCHIK, A., et al., Aberrant drainage of missed micrometastases: the value of
lymphatic mapping and focused analysis of sentinel lymph nodes in gastrointestinal
neoplasms, Ann. Surg. Oncol. 8 (2001) 82–85.
[3.144] KITAGAWA, Y., et al., “Sentinel lymph node mapping in esophageal and gastric
cancer — impact on individualized minimally invasive surgery”, Selective Sentinel
Lymphadenectomy for Human Solid Cancer (LEONG, S., KITAJIMA, M.,
KITAGAWA, Y., Eds), Springer, New York (2005) 123–139.
[3.145] STOJADINOVIC, A., et al., Prospective randomized study comparing sentinel
lymph node evaluation with standard pathologic evaluation for the staging of colon
carcinoma: results from the United States Military Cancer Institute Clinical Trials
Group Study GI-01, Ann. Surg. 245 (2007) 846–857.
[3.146] BEMBENEK, A.E., et al., Sentinel lymph node biopsy in colon cancer: a prospective
multicenter trial, Ann. Surg. 245 (2007) 858–863.
[3.147] KITAGAWA, Y., et al., Sentinel node mapping for colorectal cancer with radioactive
tracer, Dis. Colon Rectum 45 (2002) 1476–1480.
[3.148] FUJITA, S., YAMAMOTO, S., AKASU, T., Lateral pelvic lymph node dissection for
advanced lower rectal cancer, Br. J. Surg. 90 (2003) 1580–1585.
[3.149] KESHTGAR, M.R., et al., The sentinel node in anal carcinoma, Eur. J. Surg. Oncol.
27 (2001) 113–114.
[3.150] PÉLEY, G., et al., Inguinal sentinel lymph node biopsy for staging anal cancer, Scand.
J. Surg. 91 (2002) 336–338.
[3.151] PERERA, D., et al., Sentinel node biopsy for squamous-cell carcinoma of the anus
and anal margin, Dis. Colon Rectum 46 (2003) 1027–1031.
[3.152] ULMER, C., BEMBENEK, A., GRETSCHEL, S., Sentinel node biopsy in anal cancer —
a promising strategy to individualize therapy, Onkologie 26 (2003) 456–460.
[3.153] DAMIN, D.C., ROSITO, M.A., SCHWARTSMANN, G., Sentinel lymph node in
carcinoma of the anal canal: a review, Eur. J. Surg. Oncol. 32 (2006) 247–252.
[3.154] GRETSCHEL, S., et al., Lymphatic mapping and sentinel lymph node biopsy in
epidermoid carcinoma of the anal canal, Eur. J. Surg. Oncol. 34 (2008) 890–894.
[3.155] MISTRANGELO, M., et al., Feasibility of the sentinel node biopsy in anal cancer,
Q. J. Nucl. Med. Mol. Imaging 53 (2009) 3–8.
[3.156] RABBITI, P., et al., Sentinel lymph node biopsy for squamous cell carcinoma of the
anal canal, ANZ J. Surg. 72 (2002) 651–654.
[3.157] AKRIVOS, N., et al., Detection and credibility of sentinel node in vulvar cancer: a
single institutional study and short review of literature, Arch. Gynecol. Obstet. 284
(2011) 1551–1556.
[3.158] ROBISON, K., HOLMAN, L.L., MOORE, R.G., Update on sentinel lymph node
evaluation in gynecologic malignancies, Curr. Opin. Obstet. Gynecol. 23 (2011) 8–12.
[3.159] WESPES, E., The management of regional lymph nodes in patients with penile
carcinoma and reliability of sentinel node biopsy, Eur. Urol. 52 (2007) 5–6.
[3.160] SADEGHI, R., et al., Accuracy of sentinel lymph node biopsy for inguinal lymph
node staging of penile squamous cell carcinoma: systematic review and meta-analysis
of the literature, J. Urol. 187 (2012) 25–31.
70
[3.161] KROON, B.K., et al., How to avoid false-negative dynamic sentinel node procedures
in penile carcinoma, J. Urol. 171 (2004) 2191–2194.
[3.162] LEIJTE, J.A.P., et al., Reliability and safety of current dynamic sentinel node biopsy
for penile carcinoma, Eur. Urol. 52 (2007) 170–177.
[3.163] MEINHARDT, W., et al., Laparoscopic sentinel lymph node biopsy for prostate
cancer: the relevance of locations outside the extended dissection area, Prostate
Cancer 2012 (2012) 751753.
[3.164] VERMEEREN, L., et al., Intraoperative imaging for sentinel node identification in
prostate carcinoma: its use in combination with other techniques, J. Nucl. Med. 52
(2011) 741–744.
[3.165] HOLL, G., et al., Validation of sentinel lymph node dissection in prostate cancer:
experience in more than 2,000 patients, Eur. J. Nucl. Med. Mol. Imaging 36 (2009)
1377–1382.
[3.166] SADEGHI, R., et al., Sentinel node mapping in the prostate cancer, Nuklearmedizin
50 (2011) 107–115.
[3.167] BROUWER, O.R., et al., SPECT/CT and a portable gamma-camera for image-guided
laparoscopic sentinel node biopsy in testicular cancer, J. Nucl. Med. 52 (2011)
551–554.
71
Chapter 4
TECHNETIUM-99m TILMANOCEPT:
A SYNTHETIC RECEPTOR TARGETED MOLECULE
FOR SLNM
D.R. VERA, C.K. HOH, D.J. HALL
UCSD Molecular Imaging Program,
Division of Nuclear Medicine, Department of Radiology
C.A. TOKIN, A.M. WALLACE

Divisions of Oncologic Surgery and Plastic and Reconstructive Surgery,
Department of Surgery
UCSD Moores Cancer Center, University of California,
San Diego, La Jolla, California, USA
4.1. SUMMARY
Tilmanocept is a synthetic molecular radiopharmaceutical that was designed

to minimize the limitations of currently used agents for SLN identification.
Optimal size and specific binding properties allow for improved lymphatic uptake
and high retention in the sentinel node, ultimately allowing for an improvement
in the accuracy of cancer staging in diseases that utilize SLNM. The use of
tilmanocept avoids dangers associated with the use of human or animal derived
substances, and has a superior safety profile, avoiding the adverse reactions
associated with many of the other agents used today. Furthermore, its chemical
properties allow for the attachment of additional imaging reporters, permitting
future multimodal imaging of the SLN via a molecular target.
4.2. INTRODUCTION
The SLN is the first draining lymph node in a lymphatic basin and is,
therefore, the most likely lymph node to harbour metastatic disease. The concept of
the SLN was first introduced by Cabanas in 1977 [4.1]. In 1992, Morton et al. [4.2]
developed a dye based technique for intraoperatively finding the SLN. If the SLN
is negative for tumour metastasis, the procedure can be used in lieu of complete
regional lymphadenectomy [4.3], minimizing morbidity.
SLNB has been used for many solid tumours and has been validated in
breast cancer and melanoma as a strong predictor of lymph node status and
73
long term patient outcome [4.4, 4.5]. While SLNB has been proven to be feasible
and reliable, there is still a question as to the optimal agent for SLN identification.
The procedure was conceived using VBD that could enter and image lymphatics,
and has been validated and improved upon by combining the VBD with a
radiopharmaceutical [4.1, 4.6].
Current radiotracers were not specifically designed for SLN imaging
and rely on passive uptake. Technetium-99m tilmanocept has been designed
to address the shortcomings of the older lymphatic radiopharmaceuticals. On
13 March 2013, the FDA approved 99mTc tilmanocept for intraoperative lymph
node mapping for breast cancer or melanoma.
4.2.1. Background
Identifying the SLN with high precision is crucial for accurate staging and
for monitoring disease progression. It is most commonly used in breast cancer
and melanoma; however, it has also been used in various gastrointestinal [4.7] and
gynaecological cancers [4.8], as well as in cancers of the head and neck [4.9]. The
current technique of SLNB involves the injection of a lymphatic mapping agent,
which is most commonly a combination of a VBD and a radiopharmaceutical
Tc labelled colloid [4.10]. Dissection is carried out until the SLN is identified
intraoperatively via visualization of a blue hue or an acoustic signal from a hand
held gamma probe.
Blue dye and colloid based radiotracers are not specifically designed
for rapid and specific uptake into the lymphatic system, or for retention in the
sentinel node. Their biological properties are largely dependent on passive
diffusion governed by particle size. The speed at which a particle enters and stays
within a lymph node is related to the size of the particle [4.6]. While particles
less than 4–5 nm easily enter lymph channels, their small sizes do not permit
long SLN retention times. Larger colloids are taken up by lymphatics at a slower
rate, but are retained in lymph nodes for longer periods of time, presumably via
phagocytosis by recticuloendothelial macrophages [4.6].
Technetium-99m labelled sulphur colloid was introduced in the mid-1960s
for use in hepatic imaging, but has become the most commonly used colloidal
SLN imaging agent in the USA [4.11]. A filter can be applied to theoretically
limit the particle size to 50–200 nm; however, its relatively large overall size
results in slow entry into the lymphatic system, as well as slow injection site
clearance. Other preferred radiotracers are Nanocoll (GE Healthcare, Milan,
Italy) and 99mTc HSA nanocolloid (4–80 nm), which are most commonly used
in Europe, and Lymph-Flo (Royal Adelaide Hospital, Adelaide, Australia) and
99m
Tc colloidal antimony sulphide (~10 nm), which are most commonly used in
Australia [4.12, 4.13].
74
Blue dyes also rely on passive diffusion to enter the lymphatic system,
and, owing to their small molecular size, they clear quickly from the lymph
nodes [4.6, 4.14]. Isosulphan blue (Lymphazurin) is the most commonly
used VBD for intraoperative lymphatic mapping in the USA. It is injected
intraoperatively, with visualization of the afferent lymphatics possible within
minutes. VBDs have been associated with rare, but significant adverse reactions,
including anaphylaxis and cardiopulmonary arrest [4.15, 4.16].
4.2.2. Ideal sentinel node imaging agent
An ideal SLN imaging agent would exhibit rapid injection site clearance,
while not interfering with detection of sentinel nodes in close proximity to the
injection site. The tracer should exhibit rapid SLN accumulation with sustained
SLN retention. High SLN uptake with low leakage to higher echelon nodes
allows for more flexibility in the scheduling of imaging. The agent must also have
a favourable safety profile, specifically, with no associated adverse reactions.
In addition, the agent must be synthetic and provide for future development.
These features are not exhibited by the current agents, which are colloidal
forms of albumin, sulphur and/or antimony. Nanocoll is composed of albumin,
which is derived from human blood. This has a serious disadvantage during
production, when the chemistry manufacturing and control protocols must be
constantly updated for continuous monitoring of viral contamination. Although
the probability of a viral ‘breakthrough’ into the final radiopharmaceutical kit
is extremely low, the cost of screening the donated blood and the associated
regulatory compliance is very high [4.17]. Currently, the donated blood used to
produce Nanocoll is tested for hepatitis B surface antigen, HIV antibodies and
antibodies to the hepatitis C virus [4.18]. The sulphur and antimony sulphide
colloids do not offer a path for future development. These agents consist of
chemical constituents that are not amenable to chemical modification. Examples,
which are not possible for these agents, would be the attachment of fluorophores
for hybrid imaging or chelation systems, to which PET or therapeutic
radioisotopes can be attached.
4.2.3. Technetium-99m tilmanocept
Technetium-99m tilmanocept (Fig. 4.1) [4.19] is a synthetic receptor

binding radiopharmaceutical that was designed [4.20] to address the shortcomings
of commonly used SLNM agents. Tilmanocept is the generic name for DTPA
mannosyl dextran. It is a macromolecule composed of a dextran backbone and
multiple subunits of DTPA and mannose. Figures 4.2–4.4 illustrate the chemical
synthesis of tilmanocept, which culminates in the attachment of DTPA and
75
FIG. 4.1.  Technetium-99m labelled tilmanocept, 99mTc DTPA mannosyl dextran, is composed
of repeated units of mannose (green) and units of DTPA (red) attached to a 10 kg/mol chain
of glucose.
FIG. 4.2.  Production of tilmanocept starts with the attachment of aminoterminated leashes,

which uses a two step process to prevent cross-linking of the dextran.
mannose to residues on the dextran backbone. The molecule has an average size
of 7 nm, which allows for rapid entry into lymphatic channels and rapid injection
site clearance [4.20]. Production yields 200 g of tilmanocept. The synthesis
begins with the attachment of the aminoterminated leashes, which is a two step
process that prevents dextran cross-linking. This is necessary to produce a high
density of attachment sites, and consequently, the ability to attach a high number
of substrates and chelators or imaging reporters. The high density of receptor
76
FIG. 4.3. The ratio of starting reactants, 2-imino-2-methoxyethyl-1-thio-D-manno-pyranoside,
and the number of amino terminated side chains of the conjugated dextran control the mannose
density (average number of mannose groups per dextran) of the product.
FIG. 4.4.  The mixed anhydride method [4.21] was used to attach DTPA. This method prevents
cross-linking of the dextran backbone.
77
substrates enables multivalency, which increases receptor affinity [4.22]. A high
density of imaging reporters is a significant consideration during the design of
magnetic resonance and CT based contrast agents [4.23, 4.24].
The DTPA side chains enable efficient radiolabelling with 99mTc [4.20].
Typical radiolabelling yields were in excess of 98% [4.20], and remain stable
at room temperature for at least 20 h (Fig. 4.5). This is in contrast to filtered
99m
Tc sulphur colloid, which typically [4.25] loses stability after filtration
(Fig. 4.6). The selection of DTPA as the chelator was based on clinical experience
with 99mTc galactosyl neoglycoalbumin [4.26], which is commercially available
in Japan as Galactoscint (Nihon Mediphysics, Tokyo). This agent successfully
uses DTPA as the chelation system; it exhibited a plasma clearance equivalent
to that of 131I albumin [4.27]. A nitrogen sulphur tetradentate chelation system,
mercaptoacetyltriglycine (MAG3), which provided similar radiochemical yields
and stability [4.28], was explored, but the development was discontinued when
it was realized that the FDA would require preclinical toxicology studies with
the radiolabelled agent. This is because the chemical structure of the chelation
system changes during radiolabelling as the low pH and heat remove the benzoyl
protecting group to expose the SH group. Another disadvantage of MAG3 is its
inability to chelate +3 cations, which would prevent radiolabelling with 111In,
67
Ga or 68Ga.
FIG. 4.5. Stability of the 99mTc radiolabel at room temperature.
78
FIG. 4.6. Filtration of 99mTc labelled sulphur colloid destabilizes the radiolabel.
The mannose units act as ligands for the mannose binding protein
receptor [4.29], which has been designated CD206. This receptor resides at the
cell surface macrophages and dendritic cells in high concentrations. Figure 4.7
is a Scatchard plot of 99mTc tilmanocept binding to J774E cells, which are
macrophages that express CD206. The equilibrium dissociation constant KD was
0.36nM [4.30]. Low KD values denote high receptor affinity. As a general rule,
receptor binding radiopharmaceuticals [4.31] designed for high tissue extraction
require dissociation constants in the subnanomolar range. High binding affinity
allows for improved retention in sentinel nodes and diminished leakage into
higher echelon nodes.
Initially, an SLN agent was constructed with a polylysine
backbone [4.32]. The molecule DTPA mannosyl polylysine, consisting of 18
DTPAs, 82 mannosyl groups attached to a chain of 100 lysines, provided receptor
specific images of rabbit popliteal and axillary lymph nodes that gave a mean
lymph node accumulation of 2.9% of dose 24 h after injection into each foot-pad.
A non-specific agent, DTPA polylysine, delivered one tenth of the radioactivity
at the same time after injection. Although the polylysine construct demonstrated
receptor specific delivery, it was elected to design the agent based on a dextran
backbone. This decision was based on three reasons: polylysine is very expensive,
79
FIG. 4.7. High density of mannose substrates, 55 per dextran, produced a receptor binding
radiopharmaceutical with high binding affinity. The slope of the line within the inset graph
provides a measure of the equilibrium dissociation constant KD (0.36nM).
it has a very limited human use database and it is relatively hydrophobic. Dextran
is inexpensive and has a well established safety record [4.33]. Additionally,
clinical grade dextran is available in highly defined molecular sizes, and, most
significantly, is very hydrophilic.
4.2.4. Lymphoseek
Lymphoseek (Navidea Biopharmaceuticals, Dublin, OH) is a formulation

that contains tilmanocept. The radiolabelling of tilmanocept with 99mTc employs
an ‘instant’ kit method. The Lymphoseek kit is composed of two vials: one
containing a lyophilized drug product, tilmanocept, and one containing a dilutant
buffer solution. Lymphoseek contains tilmanocept (0.25 mg, 16 738 g/mole),
trehalose dehydrate (20 mg), glycine (0.5 mg), sodium ascorbate (0.5 mg)
and stannous chloride (75 µg). The average mannose and DTPA densities
of the Lymphoseek preparation are 16.8 mannose units per dextran and
4.4 DTPA units per dextran, respectively. The dilutant buffer solution was
0.2M phosphate buffered saline (PBS). The tilmanocept synthesis, Lymphoseek
80
product formulation and vial preparation were performed using current good
manufacturing practice guidelines and a chemical and manufacturing control
package registered with the FDA.
Radiolabelling was conducted via the following steps. A sterile, apyrogenic
and oxidant free solution of sodium pertechnetate 99mTc in injectable isotonic
saline was added to the vial containing the lyophilized drug product. Isotonic
injectable saline was then added to the vial to produce a 0.5 mL volume. The vial
was then gently swirled to produce a clear, colourless solution, and was placed
in a lead lined container to stand at room temperature for at least 30 min. The
procedure was completed by adding 1.0 mL of PBS from the kit dilutant vial.
A patient dose for a single intradermal administration consisted of 0.1 mL of
labelled product within a 0.3 mL insulin syringe with a fixed 27 gauge needle.
Quality control consisted of instant thin layer chromatography (ITLC) with
Whatman 31ET as the stationary phase and dry acetone as the mobile phase.
Technetium-99m tilmanocept migrated with the solvent front. During the phase 3
clinical trials, the radiochemical purity typically exceeded 98%. All patients
received 3 nmol of tilmanocept. If SLNM was performed on the same day as
the injection, the total activity administered was 18.5 MBq. If mapping was
performed on the next day, the dose was 37 MBq.
4.3. PRECLINICAL STUDIES
The preclinical studies of tilmanocept were designed to optimize synthesis

and purification, evaluate drug pharmacokinetics and establish biodistribution
and safety profiles.
4.3.1. Pharmacokinetics
The preclinical studies concluded that tilmanocept possessed three ideal

pharmacokinetic properties: rapid clearance from the injection site, appropriate
accumulation in the sentinel node and high sentinel node retention. These
properties allow for improved SLN visualization secondary to reduced injection
site scatter, thus improving the quality of the diagnostic image obtained. Rapid
accumulation allows tilmanocept to be effective intraoperatively, while high
retention in the sentinel node with reduced washout to higher echelon nodes
increases precision and may reduce the number of nodes excised.
81
4.3.2. Injection site clearance
Injection site clearance studies were performed in rabbits and compared to

filtered 99mTc sulphur colloid. Tilmanocept was injected subdermally into the feet
pads of rabbits, with a resultant clearance half-life of 2.21 ± 0.27 h. This was
significantly faster than filtered 99mTc sulphur colloid at both 1 h and 3 h time
points, which was confirmed after calculation of the percentage of injected dose
(% ID) (p < 0.05) [4.20].
This was also true after endoscopic injection of tilmanocept [4.34] into
the gastrointestinal tract using a porcine model, again, as compared to filtered
99m
Tc sulphur colloid. After endoscopic submucosal injection into either the
stomach or colon, tilmanocept revealed clearance half-lives of 3.83 ± 1.18 h and
2.56 ± 1.04 h, respectively. This revealed a statistically significant improvement
in injection site clearance (p = 0.03) compared with filtered 99mTc sulphur
colloid [4.35].
4.3.3. SLN accumulation
Rapid accumulation in the sentinel node was demonstrated in porcine

animal studies, where sentinel node uptake of tilmanocept was not statistically
different to uptake of filtered 99mTc sulphur colloid after injection into either the
gastric or colonic submucosa. Tilmanocept exhibited high sentinel node uptake
within 10 min of submucosal injection (0.13%–4.5% ID for gastric lymph nodes
and 0.54%–2.4% ID for colonic lymph nodes) [4.34]. Rapid accumulation within
the sentinel node was also exhibited after peritumoural injection of tilmanocept
in porcine prostate cancer models following prostatectomy [4.36].
4.3.4. SLN retention
Tilmanocept exhibited improved and prolonged retention in SLNs

compared to filtered 99mTc sulphur colloid in rabbit studies comparing sentinel
nodes to distal nodes. Tilmanocept was injected proximal to the popliteal fossa,
and popliteal lymph nodes were compared to distal inguinal lymph nodes to
evaluate retention in the sentinel nodes. At both 1 h and 3 h postinjection time
points, retention for tilmanocept in the popliteal nodes was higher (p < 0.05) than
filtered 99mTc sulphur colloid, representing less leakage of the imaging agent to
the distal inguinal nodes. This was further demonstrated in porcine prostatectomy
studies where tilmanocept was sustained in the SLN for up to 3 h without washing
out to distal nodes [4.35, 4.36].
82
4.3.5. Biodistribution, toxicity and absorbed radiation dose
Preclinical biodistribution and toxicity studies have shown that

tilmanocept possesses an acceptable biodistribution profile and favourable
safety profile [4.37, 4.38]. Preclinical safety studies included single dose
acute toxicity in rats and rabbits (50× and 500× scaled human dose) and
dogs (170×, 780× and 1700×), perivascular irritation studies in rabbits (100×),
a repeat dose study in rats and dogs (14 consecutive days at 42×, 85× and
170×), cardiac safety pharmacology studies in dogs (1700× and 3400×),
a sensitization study in guinea pigs (56×, 113× and 1130×), a lymphoma
mutagenesis study in mice (300×), an in vitro reverse mutation study in bacteria
(0.3 nmol/mL, 0.9 nmol/mL, 3 nmol/mL, 9 nmol/mL and 90 nmol/mL) and an in
vitro micronucleus study in mice (2.5×, 5× and 10×).
The biological half-life of tilmanocept is 2.21 ± 0.27 h, and the effective
dose is half the value of the albumin based nanocolloid, thereby minimizing
radiation dose and exposure to the patient as well as to clinical staff [4.39]. No
direct toxicity, including anaphylactic reactions or increased mortality, were noted
in any of the preclinical animal studies, regardless of dose or route of injection.
4.4. CLINICAL TRIALS
4.4.1. Phase 1 clinical trials
Physician sponsored phase 1 clinical trials, each supported by peer reviewed

extramural funding of the University of California, were conducted to establish
99m
Tc tilmanocept’s clinical safety profile and biodistribution in human subjects.
They demonstrated that tilmanocept exhibited faster injection site clearance
and equivalent primary SLNs compared to filtered 99mTc sulphur colloid. After
peritumoural injections of 12 breast cancer patients with 1.0 nmol of tilmanocept,
a mean injection site clearance half-life of 2.72 ± 1.57 h was measured. This
value was significantly shorter than for filtered 99mTc sulphur colloid, which
exhibited a mean clearance half-life of 49.5 ± 38.5 h (p = 0.0025) [4.40]. There
was no statistical difference (tilmanocept: 0.55% ± 16.8% ID versus filtered
99m
Tc sulphur colloid: 0.65% ± 15.7% ID) in the sentinel node uptake at 3 h
postinjection (p = 0.75). Twelve additional patients were studied at two additional
molar doses (0.2 nmol and 5.0 nmol) [4.37]. No difference in injection site
clearance was observed. The mean amounts of 99mTc tilmanocept in the primary
SLN of the 0.2 nmol, 1.0 nmol and 5.0 nmol groups were 0.11 ± 0.20 pmol, 5.5 ±
4.3 pmol and 11.0 ± 8.5 pmol, respectively.
83
Additional phase 1 trials were performed using a single 0.10 mL intradermal
injection. Figure 4.8 depicts the injection site clearance of radioactivity after a
single dose intradermal administration of tilmanocept and filtered 99mTc sulphur
colloid in two SLN cases [4.41]. In these cases, tilmanocept exhibited a half-life
of 2.62 ± 0.55 h, versus a filtered 99mTc sulphur colloid half-life of 24.1 ± 17.7 h.
The mean SLN uptake of 99mTc tilmanocept, 1.1 ± 0.5 %IA/g (% of injected
activity per gram), was lower than filtered 99mTc sulphur colloid, 2.5 ± 4.9 %IA/g.
These values were higher than the SLN uptake after a peritumoural injection.
A fourth study compared tilmanocept with unfiltered 99mTc sulphur colloid
while specifically using a ‘2 day’ protocol [4.42]. The purpose of this study was to
assess tilmanocept’s effectiveness with delayed lymphoscintigraphy. Imaging and
intraoperative mapping were performed 18–26 h postinjection. Again, tilmanocept
exhibited faster injection site clearance and equivalent SLN accumulation, which
persisted for longer than 24 h [4.43]. This study demonstrated tilmanocept’s high
SLN retention. This property extends the potential time period from injection to
surgery, permitting 2 day surgical procedures and the ability to image on the same
day as injection without the need for additional imaging on the day of surgery.
FIG. 4.8.  Injection site clearance after a single 0.1 mL intradermal administration; 99mTc
tilmanocept (diamonds) exhibited a half-life of 1.16 ± 0.10 h and filtered 99mTc sulphur colloid
(triangles) exhibited a half-life of 36.7 ± 6.5 h.
84
Lastly, a preliminary study was conducted to determine the concordance
between the number of SLNs detected on early and ‘next day’ images, and during
SLNM. Nine women with unilateral breast cancer and one woman with bilateral
breast cancer enrolled in the trial. All patients received 99mTc tilmanocept
(1.0 nmol, 37 MBq) as a single intradermal injection (0.10 mL) the day prior
to surgery. Lymphoscintigraphy was performed at 2 h and 15 h, and SLNB was
performed between 16 h and 24 h postinjection. Lymphoseek administration the
day prior to surgery resulted in the identification of at least one sentinel node in
all cases. A total of 19 lymph nodes were identified by lymphoscintigraphy. The
concordance between the 2 h and 15 h images was 100% (τ = 1.0, p = 0.0013;
(τ = Kendall rank correlation coefficient)). In the operating room, 20 sentinel
nodes were identified using a gamma detector; the concordance between imaging
and SLNM was 93% (τ = 0.93, p = 0.0020). Figure 4.9 shows images acquired at
2 h and 14.5 h after an intradermal injection of 99mTc tilmanocept, demonstrating
sustained uptake by a single lymph node. Only one hot lymph node was found
at surgery 18.9 h postinjection. Although preliminary, this study highlights the
SLN retention of 99mTc tilmanocept. The small molecular diameter provides rapid
SLN uptake, and the high receptor affinity retains the molecules at the SLN for
many hours.
An estimated absorbed radiation dose calculated from these preliminary
studies concluded that the radiation dose for tilmanocept was 25% of that which
would be delivered in an equivalent dose of filtered 99mTc sulphur colloid [4.37].
Furthermore, the remaining radiation dose after tumour excision in melanoma
patients was almost three times higher after filtered 99mTc sulphur colloid injection
FIG. 4.9.  Lymphoscintigraphic images (anterior) acquired at 2 h (left) and 14.5 h (right)
postinjection of 99mTc tilmanocept demonstrated sustained uptake by a single lymph node
(black arrow). The injection site is at the open arrow. Only one hot lymph node was found at
surgery 18.9 h postinjection.
85
compared with tilmanocept. No adverse reactions were reported in any patients,
and no significant alterations in clinical laboratory tests were noted [4.37, 4.44].
In 2006, after safety standards for tilmanocept were established, Navidea

Biopharmaceuticals sponsored a multicentre phase 2 clinical trial [4.45] to
evaluate the effectiveness of tilmanocept in breast cancer and melanoma
patients in five cancer centres across the USA (MD Anderson Cancer Center,
John Wayne Cancer Center, Case Western Reserve University Hospital,
University of California, San Francisco, and the University of Louisville). A total
of 80 patients were enrolled, consisting of 31 patients with breast cancer and
49 patients with melanoma. They received 50 µg (3 nmol) of tilmanocept, which
was injected peritumourally. The primary outcome was to establish and evaluate
the applicability of 99mTc tilmanocept as a radiotracer for SLN identification.
Technetium-99m tilmanocept demonstrated an SLN identification rate of 95%,
and the SLN identification properties were conserved across tumour types.
Concordance between tilmanocept and VBD was assessed: it was 89.5% for
breast cancer patients and 97% for melanoma patients. Again, no adverse events
related to tilmanocept were reported.
Two multicentre phase 3 clinical trials have been completed [4.46, 4.47].
The first, NEO3-05, was completed in December 2009, and involved a total of
136 breast cancer and melanoma patients, who provided a total of 215 lymph
nodes. Fifteen institutions participated. The primary end point of NEO3-05 was
concordance of tilmanocept and VBD in the in vivo detection of excised lymph
nodes, identified as SLNs, and confirmed by pathology. Tilmanocept revealed a
98% overall concordance rate, which was consistent across tumour types. Reverse
concordance was established to determine whether tilmanocept identified lymph
nodes that VBD had not identified, and revealed that tilmanocept was able to
identify 85 additional lymph nodes that had been missed by VBD; 18% of those
missed nodes were found to contain cancer after final pathological evaluation.
This analysis translated into a false negative rate for VBD that was three times
that of tilmanocept.
A second phase 3 clinical trial, NEO3-09, was completed in 2011. This trial,
which used the same entry criteria and end points as NEO3-05, was implemented
to extend the safety database. It involved a total of 229 lymph nodes and
resulted in a concordance rate of 100%, in that all nodes were identified by both
tilmanocept and VBD. Tilmanocept’s failed detection rate in this study was 0%,
86
which confirmed observations from NEO3-05; across the studies, tilmanocept
missed no lymph nodes that contained cancer, and facilitated cancer detection in
6 out of a total of 55 patients (10.9%) with node positive disease whose cancer
would have been entirely misdiagnosed had VBD been used as a single agent.
No serious tilmanocept related adverse events were reported in the
340 patients who participated in NEO3-05 or NEO3-09, whereas VBD caused
anaphylaxis in one patient, which is consistent with reported severe adverse
events associated with VBD and/or other radiolabelled colloids.
An additional phase 3 trial, NEO3-06, has completed enrolment.
This trial studied 83 patients with head and neck cancer. After SLNM with
99m
Tc tilmanocept, a complete lymphadenectomy was performed. The primary
end point of the study was to measure the false negative rate. Results from the
statistical analysis and reporting of the findings will be available upon completion
of full site and data audits planned for later in 2013. An interim evaluation [4.48]
based on 19 disease positive patients from three sites revealed a false negative
rate of 0%.
4.5. RETROSPECTIVE COMPARATIVE STUDIES
Since the completion of the tilmanocept clinical trials, there have been
two studies that addressed tilmanocept’s effectiveness compared to other
sentinel node imaging agents. Both used meta-analysis of data pooled from the
two tilmanocept phase 3 trials, NEO3-05 and NEO3-09, and compared them to
meta-analyses of retrospective reviews of the clinical literature.
4.5.1. Comparison to radiocolloids
Results from one of the retrospective reviews revealed a sulphur colloid

localization rate of 94.13% and a degree of localization of 1.6 after meta-analysis
of the three peer reviewed studies, which, when compared to the tilmanocept data,
showed that tilmanocept was superior in both end points measured. It should be
noted, however, that no distinction between filtered or unfiltered sulphur colloid
was made in this retrospective review.
A second retrospective review compared identical tilmanocept data to a
review of the 99mTc albumin colloid (Nanocoll) literature. Six European trials were
used to derive a 99mTc albumin colloid benchmark to which the tilmanocept end
points could be compared. Meta-analysis [4.49] revealed a Nanocoll localization
rate of 95% and a degree of localization of 1.67, and similarly concluded that
tilmanocept was superior to 99mTc albumin colloid in both end points evaluated.
87
4.6. ADDITIONAL IMAGING MODALITIES
4.6.1. Rationale
One of the design requirements of the present study is the ability to

chemically attach additional imaging reporters. This can be accomplished using
tilmanocept by two independent means. First, the selection of DTPA permits
radiolabelling with other +3 cations such as 68Ga or 111In. The first isotope, which
is generator produced, would permit PET imaging, and the second isotope would
permit imaging at later time points. A second means of adding an additional
imaging reporter would be covalent attachment to one of the aminoterminated
leashes that were not used to couple DTPA or mannose to the dextran backbone.
Approximately five aminoterminated leashes per dextran are available for
conjugation. This mechanism provides an opportunity to attach a fluorescent
reporter [4.50] for optical imaging. The motivation for the fluorescent imaging
reporter would be the ability to combine nuclear and fluorescence imaging.
A dual modality agent composed of both fluorescent and nuclear imaging
reporters would permit whole body or regional imaging, and the fluorescent
reporter would provide images or detection within the surgical field. SLNM is an
excellent clinical setting to utilize this opportunity.
4.6.2. PET imaging
Tilmanocept has been radiolabelled with 68Ga [4.51]. Figure 4.10 is a fused

PET/CT coronal cross-section of a beagle dog acquired 90 min after a transrectal
injection into the prostate. The 1.5 mL dose contained 3.0 nmol of 68Ga tilmanocept
(10 MBq). The right common iliac lymph node had a standardized uptake value
(SUV) of 54 and the SUV of the left common iliac lymph node was 53. SLNM,
conducted 2 h postinjection, detected both lymph nodes; the right common iliac
lymph node had a count rate of 1461 counts/min and accumulated 0.18% ID, and
the left common iliac had a count rate of 2175 counts/min, which represented
1.66% ID. Two other lymph nodes, the right external iliac and periaortic, were
visualized in other cross-sections. Four dogs were studied. There was a high
concordance of PET/CT imaging to intraoperative SLNM, with a sensitivity
approaching 93% for all nodes when the ex vivo count rates exceeded
threefold background.
88
FIG. 4.10.  Coronal fused PET/CT cross-section of a canine pelvis acquired 90 min after a
10 MBq prostate injection of 68Ga tilmanocept. Two lymph nodes (LNs) are visualized (white
arrows); the urinary bladder (blue arrow) and injection site (yellow arrow) are also within the
field of view.
4.6.3. Fluorescence imaging
A near infrared (IR) cyanine (Cy) dye has been covalently attached to
tilmanocept. A series of experiments [4.30] was carried out with 99mTc labelled
Cy7 tilmanocept (Fig. 4.11). The goal was to establish that the attachment of the
fluorescent tag did not alter the receptor binding properties of tilmanocept. In vitro
binding assays to a macrophage cell line demonstrated that the subnanomolar
affinity was maintained and that non-specific binding was not increased. Optical
SLN imaging performed in mice (Fig. 4.12) demonstrated dose dependent SLN
accumulation, which is a hallmark of in vivo receptor mediated binding [4.52].
These studies formed the motivation for development of a fluorescence imaging
version of tilmanocept. Optical imaging will enable a wider dissemination of
the SLN concept to hospitals without access to radiopharmaceuticals. This
is especially true in developing countries, where hospitals are not equipped to
handle radioactivity [4.53].
89
FIG. 4.11.  A fluorescent imaging reporter, Cy7 (blue), was covalently attached to tilmanocept
via an amino terminated leash. Fluorescence spectra demonstrated an emission peak at
780 nm, which is a 7 nm red shift from unattached Cy7.
FIG. 4.12.  An optical image at 150 min after a 0.11 nmol dose of Cy7 tilmanocept produced
an SLN (arrow) with an integrated fluorescence intensity with the region of interest of
780 × 103 counts. The pixel with the highest fluorescence intensity had 64 × 103 counts. A time
intensity curve from the SLN demonstrated sustained accumulation from 140 × 103 counts at
15 min to 800 × 103 counts at 100 min.
ACKNOWLEDGEMENTS
The authors of this chapter acknowledge the following funding sources

that supported the development of tilmanocept at the University of California,
San Diego: the California Breast Cancer Research Program (2RB-0118,
4IB-0051), the Susan G. Komen Breast Cancer Foundation (BASIC99-003204),
the American Cancer Society (RSG-01-249-01-CCE) and the National Cancer
Institute (R01-CA727251, R01-CA82335, R21-CA097643, R21-CA112940,
R21-CA126037, P20-CA128346 and P50-CA114745).
90
[4.1] CABANAS, R.M., An approach for the treatment of penile carcinoma, Cancer. 39
(1977) 491–499.
[4.2] MORTON, D.L., et al., Technical details of intraoperative lymphatic mapping for early
stage melanoma, Arch. Surg. 127 (1992) 392–399.
[4.3] JATOI, I., HILSENBECK, S.G., CLARK, G.M., OSBORNE, C.K., Significance of
axillary lymph node metastasis in primary breast cancer, J. Clin. Oncol. 17 (1999)
2334–2340.
[4.4] SCHULZE, T., MUCKE, J., MARKWARDT, J., SCHLAG, P.M., BEMBENEK, A.,
Long-term morbidity of patients with early breast cancer after sentinel lymph node
biopsy compared to axillary lymph node dissection, J. Surg. Oncol. 93 (2006) 109–119.
[4.5] VERONESI, U., et al., Sentinel lymph node biopsy as an indicator for axillary
dissection in early breast cancer, Eur. J. Cancer 37 (2001) 454–458.
J. Nucl. Med. 42 (2001) 1198–1215.
[4.7] TANGOKU, A., et al., Current status of sentinel lymph node navigation surgery in
breast and gastrointestinal tract, J. Med. Invest. 54 (2007) 1–18.
[4.8] ROSSI, E.C., IVANOVA, A., BOGGESS, J.F., Robotically assisted fluorescence-
guided lymph node mapping with ICG for gynecologic malignancies: a feasibility
study, Gynecol. Oncol. 124 (2011) 78–82.
[4.9] ALEX, J.C., The application of sentinel node radiolocalization to solid tumors of the
head and neck: a 10-year experience, Laryngoscope 114 (2004) 2–19.
[4.10] ALBERTINI, J.J., et al., Intraoperative radiolymphoscintigraphy improves sentinel
lymph node identification for patients with melanoma, Ann. Surg. 223 (1996) 217–224.
[4.11] LARSON, S.M., NELP, W.B., Radiopharmacology of a simplified technetium-99m-
colloid preparation for photoscanning, J. Nucl. Med. 7 (1966) 817–826.
[4.12] GOMMANS, G.M., et al., 99mTc Nanocoll: a radiopharmaceutical for sentinel node
localisation in breast cancer — in vitro and in vivo results, Appl. Radiat. Isot. 67
(2009) 1550–1558.
trisulfide and rhenium sulfide colloids intended for lymphoscintigraphic application,
J. Nucl. Med. 42 (2001) 460–466.
[4.14] CLEMENT, O., LUCIANI, A., Imaging the lymphatic system: possibilities and clinical
applications, Eur. Radiol. 14 (2004) 1498–1507.
[4.15] ALBO, D., et al., Anaphylactic reactions to isosulfan blue dye during sentinel lymph
node biopsy for breast cancer, Am. J. Surg. 182 (2001) 393–398.
[4.16] LAURIE, S.A., KHAN, D.A., GRUCHALLA, R.S., PETERS, G., Anaphylaxis to
isosulfan blue, Ann. Allergy Asthma Immunol. 88 (2002) 64–66.
[4.17] EUROPEAN COMMISION, HEALTH AND CONSUMERS DIRECTORATE-
GENERAL, “Manufacture of Biological Active Substances and Medicinal Products for
Human Use”, EU Guidelines for Good Manufacturing Practice for Medicinal Products
for Human and Veterinary Use, EudraLex: The Rules Governing Medicinal Products
in the European Union, Annex 2, Volume 4, European Commission, Brussels (2012).
91
[4.18] GE HEALTHCARE S.R.L., Summary of Product Characteristics for Nanocoll Kit for
Radiopharmaceutical Preparation, Milan, Italy (2009).
[4.19] CHENG, K.T., et al., “99mTc-diethylenetriaminepentaacetic acid-mannosyl-dextran”,
Molecular Imaging and Contrast Agent Database (MICAD), National Center for
Biotechnology Information (US), Bethesda, MD (2011).
macromolecule for sentinel node detection: [99mTc]DTPA-mannosyl-dextran, J. Nucl.
Med. 42 (2001) 951–959.
[4.21] KREJCAREK, G.E., TUCKER, K.L., Covalent attachment of chelating groups to
macromolecules, Biochem, Biophys. Res. Commun. 77 (1977) 581–583.
[4.22] VERA, D.R., KROHN, K.A., STADALNIK, R.C., SCHEIBE, P.O., Tc-99m galactosyl-
neoglycoalbumin: in vitro characterization of receptor-mediated binding, J. Nucl. Med.
25 (1984) 779–787.
[4.23] SIRLIN, C.B., et al., Gadolinium-DTPA-dextran: a macromolecular MR blood pool
contrast agent, Acad. Radiol. 11 (2004) 1361–1369.
[4.24] VERA, D.R., MATTREY, R.F., A molecular CT blood pool contrast agent, Acad.
Radiol. 9 (2002) 784–792.
[4.25] HUNG, J.C., et al., Filtered technetium-99m-sulfur colloid evaluated for
lymphoscintigraphy, J. Nucl. Med. 36 (1995) 1895–1901.
[4.26] TORIZUKA, K., et al., Phase III multi-center clinical study on 99mTc-GSA, a new agent
for imaging of liver function, Jpn. J. Nucl. Med. 29 (1992) 159–181.
[4.27] SHIRAKAMI, Y., et al., Development of Tc-99m-DTPA-HSA as a new blood pool
agent, Jpn. J. Nucl. Med. 24 (1987) 475–478.
[4.28] VERA, D.R., WALLACE, A.M., HOH, C.K., 99mTc-MAG3-mannosyl-dextran:
a receptor-binding radiopharmaceutical for sentinel node detection, Nucl. Med. Biol.
28 (2001) 493–498.
[4.29] KAWASAKI, T., MIZUNO, Y., MASUDA, T., YAMASHINA, I., Mannan-binding
protein in lymphoid tissues of rats, J. Biochem. (Tokyo) 88 (1980) 1891–1894.
[4.30] EMERSON, D.A., et al., A receptor-targeted fluorescent radiopharmaceutical for
multi-reporter sentinel lymph node imaging, Radiology 265 (2012) 186–193.
[4.31] ECKELMAN, W.C., “Design criteria for targeted molecules: muscarinic
cholinergic systems biology”, Targeted Molecular Imaging (WELCH, M.J.,
ECKELMAN, W.C., Eds), Taylor & Francis Group, Boca Raton, FL (2012) 201–215.
[4.32] VERA, D.R., WISNER, E.R., STADALNIK, R.C., Sentinel node imaging via a
nonparticulate receptor-binding radiotracer, J. Nucl. Med. 38 (1997) 530–535.
[4.33] DE BELDER, D., “Medical application of dextran and its derivatives”,
Polysaccharides in Medicinal Applications (DUMITRIU, S., Eds), Marcel Dekker,
New York (1996) 505–524.
[4.34] MENDEZ, J., WALLACE, A.M., HOH, C.K., VERA, D.R., Detection of gastric and
colonic sentinel nodes via endoscopic administration of Lymphoseek in pigs, J. Nucl.
Med. 44 10 (2003) 1677–1681.
[4.35] ELLNER, S.J., et al., Sentinel lymph node mapping of the colon and stomach using
Lymphoseek in a pig model, Ann. Surg. Oncol. 11 (2004) 674–681.
92
[4.36] SALEM, C.E., WALLACE, A.M., HOH, C.K., VERA, D.R., A preclinical study of
prostate sentinel node mapping with Lymphoseek, J. Urol. 175 (2006) 744–748.
[4.37] ELLNER, S.J., et al., Dose-dependent biodistribution of [99mTc]DTPA-mannosyl-
dextran for breast cancer sentinel node mapping, Nucl. Med. Biol. 30 (2003) 805–810.
[4.38] WOEHNL, A., et al., “Molecular imaging of the sentinel lymph node via
Lymphoseek”, Current Clinical Oncology: From Local Invasion to Metastatic Cancer
(LEONG, S.P.L., Eds), Humana Press, New York (2009) 123–133.
[4.39] HOH, C.K., WALLACE, A.M., VERA, D.R., Preclinical studies of [99mTc]
DTPA -mannosyl-dextran, Nucl. Med. Biol. 30 (2003) 457–464.
[4.40] WALLACE, A.M., HOH, C.K., VERA, D.R., DARRAH, D., SCHULTEIS, G.,
Lymphoseek: a molecular radiopharmaceutical for sentinel node detection, Ann. Surg.
Oncol. 10 (2003) 531–538.
[4.41] WALLACE, A.M., HOH, C.K., DARRAH, D.D., SCHULTEIS, G., VERA, D.R.,
Sentinel lymph node mapping of breast cancer via intradermal administration of
Lymphoseek, Nucl. Med. Biol. 34 (2007) 849–853.
[4.42] YEUNG, H., et al., Lymphoscintigraphy and sentinel node localization in breast
cancer patients: a comparison between 1-day and 2-day protocols, J. Nucl. Med. 42
(2001) 420–423.
[4.43] WALLACE, A.M., et al., Sentinel lymph node accumulation of Lymphoseek and
Tc-99m-sulfur colloid using a “2-day” protocol, Nucl. Med. Biol. 36 (2009) 687–692.
[4.44] WALLACE, A.M., et al., Lymphoseek: a molecular imaging agent for melanoma
sentinel lymph node mapping, Ann. Surg. Oncol. 14 (2007) 913–921.
[4.45] LEONG, S.P.L., et al., A phase 2 study of [99mTc]tilmanocept in the detection of sentinel
lymph nodes in melanoma and breast cancer, Ann. Surg. Oncol. 18 (2011) 961–969.
[4.46] WALLACE, A.M., et al., Comparative evaluation of [99mTc]tilmanocept for sentinel
lymph node mapping in breast cancer patients: result of two phase 3 trials, Ann. Surg.
Oncol. 20 (2013) 2590–2599.
[4.48] MARCINOW, A.M., et al., Use of a novel receptor-targeted (CD206) radiotracer,
99m
Tc-tilmanocept, and SPECT/CT for sentinel lymph node detection in oral cavity
squamous cell carcinoma: initial institutional report in an ongoing phase 3 study,
JAMA Otolaryngolog. Head Neck Surg. 139 (2013) 895–902.
[4.49] TOKIN, C.A., et al., The efficacy of tilmanocept in sentinel lymph node mapping
and identification in breast cancer patients: a comparative review and meta-analysis
of the 99mTc-labeled nanocolloid human serum albumin standard of care, Clin. Exp.
Metastasis 29 (2012) 681–686.
[4.50] VERA, D.R., et al., Cy5.5-DTPA-galactosyl-dextran: a fluorescent probe for in vivo
measurement of receptor biochemistry, Nucl. Med. Biol. 32 (2005) 687–693.
[4.51] STROUP, S.P., et al., Preoperative sentinel lymph node mapping of the prostate using
PET/CT fusion imaging and Ga-68-labeled tilmanocept in a dog model, Clin. Exp.
Metastasis 29 (2012) 673–680.
93
[4.52] VERA, D.R., KROHN, K.A., STADALNIK, R.C., SCHEIBE, P.O., [Tc-99m]
galactosyl-neoglycoalbumin: in vivo characterization of receptor-mediated binding to
hepatocytes, Radiology 151 (1984) 191–196.
[4.53] KESHTGAR, M.R.S., et al., Implementing sentinel lymph node biopsy programmes
in developing countries: challenges and opportunities, World J. Surg. 35 (2011)
1159–1168.
94
Chapter 5
A NEW CLASS OF 99mTc(I) AGENTS FOR SLND:

CHEMICAL DESIGN AND SYNTHESIS
M. MORAIS, J.D.G. CORREIA, I. SANTOS

Unidade de Ciências Químicas e Radiofarmacêuticas, ITN,
Instituto Superior Técnico,
Universidade Técnica de Lisboa,
Sacavém, Portugal
M. PELECANOU, I. PIRMETTIS
Institute of Biosciences & Applications
M. PAPADOPOULOS
Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety,
Demokritos National Center for Scientific Research,

Athens, Greece
5.1. SUMMARY
The unique features of the organometallic technetium and rhenium

tricarbonyl moiety have earned considerable attention in the design of novel
specific radiopharmaceuticals in recent years. As part of the CRP focused on
the Development of 99mTc Radiopharmaceuticals for Sentinel Node Detection
and Cancer Diagnosis, the design of mannosylated dextran derivatives loaded
with pyrazolyl (pz) diamine and cysteine chelating units for coordination to the
organometallic core fac-[M(CO)3]+ (M = Re, 99mTc) are reported in this chapter.
The corresponding rhenium derivatives were prepared as congeners of the
99m
Tc(CO)3 mannosylated dextran and used as non-radioactive alternatives for
large scale synthesis and structural characterization.
5.2. INTRODUCTION
Technetium-99m is the most widely used radioisotope in nuclear medicine,

with an estimated 40 million annual radiodiagnostic procedures carried out
worldwide [5.1]. The preferential use of 99mTc based radiopharmaceuticals
95
reflects the almost ideal nuclear properties of the isotope, as well as their low cost
and on-site availability from commercial 99Mo/99mTc generators. Technetium-99m
emits a 140 keV γ ray with 89% abundance, which is close to optimal for imaging
using commercial gamma cameras. The 6 h half-life is sufficiently long for
preparation of the radiopharmaceutical in situ and for in vivo accumulation in the
target tissue, yet short enough to minimize radiation dose to the patient. Moreover,
this radioisotope presents a wide range of oxidation states, from −I (d8) to + VII
(d0), which enables diverse coordination chemistry and the preparation of a wide
variety of complexes with different biological properties [5.2, 5.3].
Because 99mTc complexes are always obtained in very low concentrations
(10 M−10−7M), their structural characterization is performed indirectly
−10
by synthesizing analogous complexes with the relatively stable 99Tc isomer

(half-life: 2.14 × 105 years, β− decay) or with the non-radioactive VIIB
congener rhenium (Re). The rich coordination chemistry of Tc and Re led to
the design of complexes with improved biological properties towards target
specific SPECT imaging (99mTc) or therapy (186/188Re). The higher oxidation
states (+V, +IV and +III) have been extensively explored for the development
of 99mTc radiopharmaceuticals, with the most studied cores being [Tc=O]3+,
[O=Tc=O]+ and [Tc≡N]2+ at the +V oxidation state. Presently, most of the
perfusion and target specific 99mTc radiopharmaceuticals in routine clinical use
have the [Tc=O]3+ core [5.2–5.5]. Recently, the lower oxidation state +I received
considerable attention when Alberto and co-workers developed a convenient
and fully water based methodology to prepare the organometallic precursors
fac-[M(CO)3(H2O)3]+ directly from permetallated salts [MO4]–(M=Tc,Re)
[5.6–5.9]. Initially, fac-[99mTc(CO)3(H2O)3]+ was synthesized by direct reduction
of Na[99mTcO4], with sodium borohydride in aqueous solution in the presence of
carbon monoxide. After this first important achievement, an even more attractive
method was developed, which involved the use of boranocarbonate, K2[H3BCO2],
to produce CO in situ, and, simultaneously, to reduce Tc(VII) to Tc(I). Currently,
the synthesis of the precursor is carried out using a kit formulation (IsoLink,
Covidien, formerly Mallinckrodt Medical BV).
The tricarbonyl moiety offers a great number of advantages for the design of
novel radiopharmaceuticals. The compact nature of the core fac-[M(CO)3]+ with
an almost spherical shape renders a tight octahedral coordination sphere with the
appropriate ligand system, preventing further ligand attack or reoxidation. The
low spin d6 electronic configuration of the metal centre is, in general, associated
with complexes of high thermodynamic stability and kinetic inertness, which
are key issues for in vivo applications. Moreover, the low MW tricarbonyl
moiety allows the labelling of relevant biomolecules in high yield and with high
specific activity.
96
All three water molecules in the precursor fac-[99mTc(CO)3(H2O)3]+ are
very labile and can be replaced by mono, bi or tridentate chelating ligands to
form stable complexes. Among these, the complexes stabilized by tridentate
ligands display the highest stability, both in vivo and in vitro. Histidine, pyridine,
pyrazole, thioimidazole and cysteine derivatives, together with pure aliphatic
or mixed aromatic aliphatic triamines stabilize the organometallic core quite
efficiently, allowing the preparation of complexes with different biological
properties. Among these, pz diamine and cysteine derivatives have been explored
as efficient tridentate N, N, N and S, N, O donor atom ligands, respectively, for
stabilization of the fac-[M(CO)3]+ moiety. The coordination mode of these model
chelators has been well established by X ray structural analysis (Fig. 5.1) and by
nuclear magnetic resonance (NMR) spectroscopy [5.10−5.13].
Derivatization of such ligands gives bifunctional chelators that allow the
conjugation and labelling of a wide range of biologically active peptides and
small molecules. The relevant biological properties of some of those 99mTc(CO)3
labelled biomolecules in adequate animal models [5.14−5.23], as well as the well
defined coordination capability of pz diamine and cysteine derivatives towards
the fac-[M(CO)3]+ moiety, prompted the conjugation of these bifunctional
FIG. 5.1.  Molecular structures of Re(CO)3 complexes stabilized by pz diamine and

cysteine derivatives.
97
FIG. 5.2.  Schematic representation of mannosylated dextran with pz diamine (DAPM) and
cysteine units (DCM) as chelators for the fac-[M(CO)3]+ moiety (M = Re, 99mTc).
chelators to polymeric nanoparticles such as mannosylated dextran for SLND

(Fig. 5.2).
5.3. RESULTS
The synthesis of mannosylated dextran containing the pz diamine (dextran

aminepyrazolyl diamine mannose; DAPM) or cysteine chelators (dextran
propyl-S-cysteine mannose with MW = 20 000 g/mol; DCM20) was performed
by following a stepwise procedure (Figs 5.3 and 5.4) [5.24, 5.25]. The preparation
of DAPM involved the attachment of amino terminal leashes to the hydroxyl
units of dextran by addition of cysteamine to allyl dextran (1) to yield dextran
amine (2), followed by conjugation of the activated chelator (pz(Boc)COOsucc)
to the free amines. Mannosylation of most of the remaining amino groups in the
final step afforded DAPM (Fig. 5.3).
Depending on the molar ratio pz(Boc)COOsucc/2, the synthetic pathway
allowed the preparation of three mannosylated derivatives that contain 1
(DAPM1), 4 (DAPM4) or 8 (DAPM8) pz units per mole of dextran. In the
case of the cysteine containing dextran derivative DCM20, there are only two
major synthetic steps starting from the allyl dextran derivative (1). The direct
incorporation of the amino acid into the dextran backbone reduced the overall
synthetic steps. Indeed, there is no need to synthesize, activate and conjugate the
bifunctional chelating agent to dextran (Fig. 5.4).
98
FIG. 5.3.  Synthesis of mannosylated dextran derivatives containing 1 (DAPM1), 4 (DAPM4)
or 8 (DAPM8) pz units/mol dextran, respectively: (i) BrC3H5, NaOH (2.5M), H2O; (ii)
NH2(CH2)2SH, (NH4)2S2O8, dimethylsulphoxide; (iii) pz(Boc)COOsucc, borate buffer 0.1M,
pH9; (iv) IME mannose, borate buffer 0.1M, pH9; (v) trifluoroacetic acid (TFA)/H2O.
FIG. 5.4.  Synthesis of DCM20: (i) BrC3H5, NaOH (2.5M), H2O; (ii) L-cysteine, (NH4)2S2O8,
H2O, nitrogen; (iii) 2-imino-2-methoxethyl-1-thio-β-D-mannoside, borate buffer (0.01M, pH9).
99
Moreover, the incorporation of cysteine into the dextran backbone
provides an amino group for conjugation to mannose, whereas the remaining
non-mannosylated cysteine groups serve as S, N, O chelators for stabilization of
the fac-[99mTc(CO)3]+ core.
All compounds were characterized using NMR spectroscopy. The mannose
and the chelator loading in the polymer backbone were determined based on the
1
H NMR data. For the DAPM derivatives, the total numbers of mannose, pz units
and free amines per mole of dextran were calculated based on the intensity
ratios of the peaks corresponding to the mannose anomeric proton (δ, 5.22),
3,5-Me2pz (δ, 2.08) and protons adjacent to free amines (δ, 2.74, He), respectively,
which are easily assigned in the 1H NMR spectra. As an illustrative example,
Fig. 5.5 presents the 1H NMR spectrum obtained for DAPM4, with assignment of
the most relevant peaks.
In an analogous way, the mannose and cysteine loading in DCM20 was
determined based on the relative intensity of the characteristic peaks of each
functionality (Fig. 5.6).
Colorimetric assays (sulphuric acid phenol assay and trinitrobenzene
sulphonate assay) corroborate the NMR results for glucose and amine content per
mole of dextran.
FIG. 5.5.  1H NMR spectrum of DAPM4 in D2O at 25°C.
100
FIG. 5.6.  1H NMR spectrum (range δH, 6.15–1.75) of DCM20 in D2O at 25°C.
FIG. 5.7.  SEC-HPLC trace of DAPM4 (wavelength λ = 200 nm; retention time tR = 15.8 min).
The purity of all the polymeric compounds was checked by analytical

size exclusion high performance liquid chromatography (SEC-HPLC). As an
example, Fig. 5.7 depicts a typical SEC-HPLC trace, showing the formation of a
single major product (>98%) assigned to DAPM4.
101
The rhenium containing mannosylated dextran derivatives were
prepared by reaction of the precursors fac-[Re(CO)3(H2O)]Br or [NEt4]
fac-[ReBr3(CO)3] with DAPM4, DAPM8 or DCM20. The resulting metallated
compounds are surrogates of the 99mTc(CO)3 mannosylated dextran derivatives,
and were used as a non-radioactive alternative for large scale synthesis and
structural characterization.
The synthesis of the rhenium polymeric compounds containing the pz
diamine chelator was monitored by reversed phase high performance liquid
chromatography (RP-HPLC) and 1H NMR spectroscopy. As an example, Fig. 5.8
shows the RP-HPLC traces and 1H NMR spectra for the reaction mixture at 1 h
and 16 h, after appropriate work-up of the collected samples.
FIG. 5.8.  RP-HPLC (254 nm) and 1H NMR data of the reaction mixture at 1 h and 16 h (25°C).
102
After 16 h of reaction, the 1H NMR spectra showed the disappearance of
the singlet (δ, 5.80) corresponding to H(4) of the free pz diamine chelator, and
the appearance of a new singlet (δ, 6.10). At this time point, the reaction was
complete because only one species was detected by RP-HPLC, and the chemical
shifts of the resonances owing to H(4) and 3,5-Me2 groups of the pz ring in
Re(CO)3 DAPM4 and Re(CO)3 DAPM8 compare well with other results
described in the literature for Re analogues [5.21−5.23]. The 13C NMR spectra
for the same compounds allowed identification of the resonances owing to the
carbonyl groups of the fac-[Re(CO)3]+ (δ, 195.2−193). Two intense absorption
bands (2027 cm−1 and 1998 cm−1) in the IR spectra also confirmed the presence
of the organometallic core (data not shown).
Metallation of DCM20 was achieved after 5 h. In the NMR spectra, changes
are evident in the chemical shifts of protons attached to the coordinating S atom
of cysteine (i.e. the βCH2 of cysteine and the OCH2CH2CH2S of the propylene
chain). In addition, the βCH2 protons of cysteine broaden and become barely
visible, hiding in the baseline, and becoming visible only when the temperature
of the sample is raised. This broadening provides a solid sign for the coordination
of the S atom of cysteine to the Re(CO)3+ core (Fig. 5.9) [5.11, 5.12].
FIG. 5.9.  1H spectrum (range δΗ, 5.60–1.70) of Re(CO)3 DCM20 in D2O at 25°C.
103
Moreover, the IR spectra of Re(CO)3 DCM20 show strong bands at
2019 cm−1 and 1895 cm−1, which are attributed to the ν(C≡O) stretching modes,
denoting the presence of the fac-[Re(CO)3]+ core. Furthermore, in the 13C NMR
spectra of Re(CO)3 DCM20, three peaks attributed to the carbonyl groups of the
fac-[Re(CO)3]+ moiety can be clearly seen in the range δC 198–195, which is in
agreement with other S, N, O complexes in the literature [5.11, 5.12]. The purity
of all metallated compounds was checked by SEC-HPLC and RP-HPLC.
The detailed structural characterization of the rhenium congeners allowed
characterization of the 99mTc radioactive nanocompounds. As an example,
Fig. 5.10 displays the RP-HPLC traces obtained for 99mTc(CO)3 DAPM4
(γ detection) and Re(CO)3 DAPM4 (ultraviolet (UV) detection). Similar results
were obtained for all compounds in this study.
DLS and zeta potential measurements allowed physical characterization
(size and charge, respectively) of the mannosylated dextran derivatives, as well
as the metallated analogues. The final compositions of all the dextran derivatives,
their physical parameters and the calculated MWs for each compound are
summarized in Table 5.1.
The DLS data show that mannosylated dextran containing pz diamine
or cysteine units, as well as the rhenium compounds, present similar particle
sizes. Moreover, the hydrodynamic diameter of the nanopolymers increases
with the polymer backbone functionalization, from dextran (4.3 nm) to final
99m
FIG. 5.10.  RP-HPLC traces of M(CO)3 DAPM4 (M = Re, UV/VIS detection; M = Tc,
γ detection).
104
TABLE 5.1.  GROUP DENSITY, HYDRODYNAMIC DIAMETER, ZETA
POTENTIAL AND CALCULATED MWs OF DEXTRAN, DAPM4, DAPM8,
DCM20, Re(CO)3 DAPM4, Re(CO)3 DAPM8 AND ReCO3 DCM20*
Group density Zeta MW

(units/mol dextran) Diameter*
Compound potential* (calculated)
(nm)
Amine pz Mannose Cysteine (mV) (g/mol)
Dextran — — — — 4.3 ± 0.4 −9.9 ± 0.5 10 000

DAPM4 13 4 13 — 7.0 ± 0.3 6.6 ± 0.3 18 820
DAPM8 9 8 13 — 7.0 ± 0.7 7.3 ± 0.6 20 132
DCM20 — — 24 6 6.5 ± 0.5 −6.3 ± 0.1 22 270
Re(CO)3 DAPM4 13 4 13 — 8.4 ± 0.5 7.1 ± 0.7 19 904
Re(CO)3 DAPM8 9 8 13 — 8.7 ± 0.3 7.1 ± 0.1 22 300
ReCO3 DCM20 — — 24 6 8.3 ± 0.5 −6.3 ± 0.1 23 083
Note: —, no data available.

* Results are expressed as mean ± standard deviation.
polymeric compounds (6.5–7 nm). This trend is also observed upon metallation

because the particle size of the derivatives containing pz diamine or cysteine
increased to 8.7 nm or 8.3 nm, respectively. On the other hand, zeta potential
measurements indicated a negative charge for dextran (−9.9 ± 0.5 mV). Dextran
functionalization with amines and pz diamine units led to compounds DAPM4
(6.6 ± 0.3 mV) and DAPM8 (7.3 ± 0.6 mV) with positive surface charges. On
the contrary, DCM2O displayed a negative surface charge (−6.3 ± 0.1 mV).
Coordination of fac-[Re(CO)3]+ moiety to the previous compounds produced no
effects on their surface charges.
ACKNOWLEDGEMENTS
M. Morais thanks the Portuguese Science Foundation (FCT) for a PhD grant
(SFRH/BD/48066/2008). Covidien is also acknowledged for providing the
tricarbonyl kits.
105
[5.1] WORLD NUCLEAR ASSOCIATION, Radioisotopes in Medicine (2014),

http://www.world-nuclear.org/info/inf55.html
[5.2] ARANO, Y., Recent advances in 99mTc radiopharmaceuticals, Ann. Nucl. Med. 16
(2002) 79–93.
[5.3] BANERJEE, S.R., et al., New directions in the coordination chemistry of 99mTc:
a reflection on technetium core structures and a strategy for new chelate design, Nucl.
Med. Biol. 32 (2005) 1–20.
[5.4] LIU, S., The role of coordination chemistry in the development of target-specific
radiopharmaceuticals, Chem. Soc. Rev. 33 (2004) 445–461.
[5.5] LIU, S., Bifunctional coupling agents for radiolabeling of biomolecules and target-
specific delivery of metallic radionuclides, Adv. Drug Deliv. Rev. 60 (2008) 1347–1370.
[5.6] ALBERTO, R., et al., Metal carbonyl syntheses XXII. Low pressure carbonylation of
[MOCl4]− and [MO4]: the technetium(I) and rhenium(I) complexes [NEt4]2[MCl3(CO)3],
J. Organomet. Chem. 493 (1995) 119–127.
[5.7] ALBERTO, R., SCHIBLI, R., EGLI, A., SCHUBIGER, A.P., A novel organometallic
aqua complex of technetium for the labeling of biomolecules: synthesis of
fac-[99mTc(H2O)3(CO)3]+ from [99mTcO4] in aqueous solution and its reaction with a
bifunctional ligand, J. Am. Chem. Soc. 120 (1998) 7987–7988.
[5.8] ALBERTO, R., SCHIBLI, R., WAIBEL, R., ABRAM, U., SCHUBIGER, A.P.,
Basic aqueous chemistry of [M(OH2)3(CO)3]+ (M = Re, Tc) directed towards
radiopharmaceutical application, Coord. Chem. Rev. 190–192 (1999) 901–919.
[5.9] ALBERTO, R., ORTNER, K., WHEATLEY, N., SCHIBLI, R., SCHUBIGER, A.P.,
Synthesis and properties of boranocarbonate: a convenient in situ CO source for the
aqueous preparation of [99mTc(OH)2(CO)3]+, J. Am. Chem. Soc. 123 (2001) 3135–3136.
[5.10] ALVES, S., PAULO, A., CORREIA, J.D.G., DOMINGOS, A., SANTOS, I.,
Coordination capabilities of pyrazolyl containing ligands towards the fac-[Re(CO)3]+
moiety, J. Chem. Soc. Dalton Trans. 24 (2002) 4714–4719.
[5.11] STAVEREN, D., et al., S-functionalized cysteine: powerful ligands for the
labelling of bioactive molecules with triaquatricarbonyltechnetium-99m(1+)
([99mTc(OH2)3(CO)3]+), Helv. Chim. Acta 88 (2005) 447–460.
[5.12] HE, H., et al., Re(CO)3 complexes synthesized via an improved preparation of
aqueous fac-[Re(CO)3(H2O)3]+ as an aid in assessing 99mTc imaging agents. Structural
characterization and solution behavior of complexes with thioether-bearing amino
acids as tridentate ligands, Inorg. Chem. 44 (2005) 5437–5446.
[5.13] HE, H., LIPOWSKA, M., CHRISTOFOROU, A.M., MARZILLI, L.G., TAYLOR, A.T.,
Initial evaluation of new 99mTc(CO)3 renal imaging agents having carboxyl-rich
thioether ligands and chemical characterization of Re(CO)3 analogues, Nucl. Med.
Biol. 34 (2007) 709–716.
[5.14] ALVES, S., et al., Pyrazolyl derivatives as bifunctional chelators for labeling
tumor-seeking peptides with the fac-[M(CO)3]+ moiety (M = 99mTc, Re), Bioconjug.
Chem. 16 (2005) 438–449.
106
[5.15] ALVES, S., et al., Pyrazolyl conjugates of bombesin: a new tridentate ligand framework
for the stabilization of fac-[M(CO)3]+ moiety, Nucl. Med. Biol. 33 (2006) 625–634.
[5.16] ALVES, S., et al., In vitro and in vivo evaluation of a novel 99mTc(CO)3-pyrazolyl
conjugate of cyclo-(Arg-Gly-Asp-D-Tyr-Lys), Bioconjug. Chem. 18 (2007) 530–537.
[5.17] PALMA, E., et al., A new biphosphonate-containing 99mTc(I) tricarbonyl complex
potentially useful as a boneseeking agent: synthesis and biological evaluation, J. Biol.
Inorg. Chem. 12 (2007) 667–679.
[5.18] VITOR, R.F., et al., Pyrazolyl–diamine ligands that bear anthracenyl moieties and
their rhenium(I) tricarbonyl complexes: synthesis, characterisation and DNA-binding
properties, ChemBioChem. 9 (2008) 131–142.
[5.19] MORAIS, M., RAPOSINHO, P.D., OLIVEIRA, M.C., CORREIA, J.D.G.,
SANTOS, I., Evaluation of novel (99m)Tc(I)-labeled homobivalent α-melanocyte-
stimulating hormone analogs for melanocortin-1 receptor targeting, J. Biol. Inorg.
Chem. 17 (2012) 491–505.
[5.20] CHOI, K.-H., HONG, Y.-D., CHOI, O.-J., CHOI, S.-J., Synthesis and physical
evaluation of 99mTc(CO)3-labeled cysteine–arylpiperazines for a neuroreceptor(5-HT1A)
imaging, Bull. Korean Chem. Soc. 27 (2006) 1689–1692.
[5.21] ALBERTO, R., PAK, J.K., STAVEREN, D., MUNDWILER, S., BENNY, P., Mono-,
bi-, or tridentate ligands? The labeling of peptides with 99mTc-carbonyls, Biopolymers
76 (2004) 324–333.
[5.22] KARAGIORGOU, O., et al., (S)-(2-(2’-pyridyl)ethyl)cysteamine and
(S)-(2-(2’-pyridyl)ethyl)-D,L-homocysteine as ligands for the “fac-[M(CO)3]+”
(M = Re, 99mTc) core, Inorg. Chem. Comm. 44 (2005) 4118–4120.
[5.23] PARK, S.H., JANG, B.S., PARK, K.B., Synthesis and biological characterization of
99mTc(I) tricarbonyl cysteine complex, a potential diagnostic for assessment of renal
function, J. Lab. Compd. Radiopharm. 48 (2005) 63–73.
[5.24] MORAIS, M., et al., Mannosylated dextran derivatives labeled with fac-[M(CO)3]+
(M = 99mTc, Re) for specific targeting of sentinel lymph node, Mol. Pharm. 8
(2011) 609–620.
[5.25] PIRMETTIS, I., et al., New 99mTc(CO)3 mannosylated dextran bearing S-derivatized
cysteine chelator for sentinel lymph node detection, Mol. Pharm. 9 (2012) 1681–1692.
107
Chapter 6
A NEW CLASS OF 99mTc(I) AGENTS FOR SLND:

LABELLING AND QUALITY CONTROL
M. MORAIS, J.D.G. CORREIA, I. SANTOS

Unidade de Ciências Químicas e Radiofarmacêuticas, ITN,
Instituto Superior Técnico,
Universidade Técnica de Lisboa,
Sacavém, Portugal
M. PELECANOU, I. PIRMETTIS
Institute of Biosciences & Applications
M. PAPADOPOULOS
Institute of Nuclear & Radiological Sciences & Technology, Energy & Safety,
Demokritos National Center for Scientific Research,

Athens, Greece
6.1. SUMMARY
In an attempt to prepare a new class of tracers for SLND, the radiolabelling

of mannosylated dextran derivatives containing the tridentate pz diamine and
cysteine chelators for stabilization of the organometallic core fac-[99mTc(CO)3]+
are reported. This chapter demonstrates the optimization of the labelling
conditions to achieve high radiochemical purity and specific activity. These
results are of paramount importance for the biological properties of the
compounds, namely their mannose receptor targeting properties.
6.2. INTRODUCTION
The successful introduction of a stable, water soluble Tc(I) organometallic

precursor by Alberto et al. opened a new avenue in radiopharmaceutical
sciences, mainly when a fully aqueous based preparation of the precursor
fac-[99mTc(CO)3(H2O)3]+ by direct reduction of Na[99mTcO4] with sodium
borohydride in the presence of carbon monoxide was reported [6.1, 6.2]. The
potential of this preparation was enhanced by a remarkable and innovative
109
procedure that used potassium boranocarbonate, K2[H3BCO2], to reduce Tc(VII)
to Tc(I) and simultaneously generate CO in situ [6.2]. Currently, the synthesis
of the precursor can be carried out using a kit formulation (IsoLink, Covidien,
formerly Mallinckrodt Medical BV).
Using the tricarbonyl core, a wide variety of new complexes with
relevant biological properties have been designed and evaluated in the past few
years [6.3−6.14].
The easy availability of this Tc(I) precursor has contributed towards
significant progress in radiopharmaceutical chemistry, with the major advantages
including: (a) applicability to a wide range of biologically relevant molecules,
(b) high thermodynamic and kinetic stability of the resultant 99mTc complexes, (c)
high specific activity of the labelled biomolecules, often without any purification
step, (d) relatively low MW of the metal moiety and (e) easy fine tuning of the
pharmacokinetics of the final complexes owing to the wide range of bifunctional
chelators available for the metal core.
In this chapter, the labelling of mannosylated dextran derivatives (DAPM1,
DAPM4, DAPM8 and DCM20) containing the tridentate pz diamine and
cysteine chelators with the organometallic moiety fac-[99mTc(CO3)]+ is described.
Chromatographic characterization of the resulting radiolabelled dextran
derivatives, as well as the assessment of their stability in vitro, is also reported.
6.3. RESULTS
6.3.1. Radiolabelling with [99mTc(CO)3]+
The mannosylated dextran derivatives bearing the pz diamine (DAPM1,

DAPM4 and DAPM8) or S-derivatized cysteine (DCM20) chelating units have
been labelled with the fac-[99mTc(CO)3]+ moiety under different experimental
conditions [6.15, 6.16]. The dextran derivatives DAPM1, DAPM4 and DAPM8
(final concentration: 2.5 × 10−5M−5 × 10−5M) reacted with the precursor
fac-[99mTc(CO)3(H2O)3]+ in a N2 flushed capped vial at 100°C for 30 min,
yielding quantitatively 99mTc(CO)3 labelled mannosylated dextran derivatives
of the type 99mTc(CO)3 L (L = DAPM1, DAPM4 and DAPM8) (Fig. 6.1)
[6.15]. The dextran derivative DCM20 (final concentration: 2.2 × 10−6M)
reacted with the precursor fac-[99mTc(CO)3(H2O)3]+ in a capped vial at 75°C for
25 min, yielding quantitatively the 99mTc(CO)3 labelled mannosylated dextran
derivative 99mTc(CO)3 DCM20 (Fig 6.2) [6.16]. The tricarbonyl precursor
fac-[99mTc(CO)3(OH2)3]+ was prepared using a kit formulation (IsoLink).
Briefly, addition of freshly eluted Na[99mTcO4] solution (1.5−2.0 mL) to an
IsoLink kit, followed by heating for 30 min at 100°C, afforded the radioactive
110
FIG. 6.1.  Synthesis of 99mTc(CO)3 DAPM1, 99mTc(CO)3 DAPM4 and 99mTc(CO)3 DAPM8.
99m
FIG. 6.2.  Synthesis of Tc(CO)3 DCM20.
synthon fac-[99mTc(CO)3(OH2)3]+. The reaction vial was cooled and the solution
neutralized with 1M HCl (160–170 μL) to destroy the remaining boranocarbonate.
The product was controlled by RP-HPLC.
DCM20 was also quantitatively labelled at low ligand concentration (final
concentration: 1.5 × 10−6M) through an efficient one pot reaction, by dissolving
the compound (50 μg) in a Na[99mTcO4] solution (1.5 mL) and transferring
the resulting solution into the IsoLink kit. The vial was then kept at 100°C for
30 min [6.16].
6.3.2. Quality control
The radiochemical control of all radiolabelled mannosylated dextran

derivatives of the type 99mTc(CO)3 − L (L = DAPM1, DAPM4, DAPM8, and
DCM20) was performed by ITLC, RP-HPLC and SEC-HPLC [6.15, 6.16].
111
6.3.2.1. ITLC
Analysis was performed using PALL Life Sciences (product 61886) or

Gelman Sciences Inc. (product 51432) strips and three different eluent systems
(A, B, C):
—— System A: Methyl ethyl ketone. The [99mTcO4]− migrates with the solvent
front (Rf = 1; Rf = retention factor), whereas fac-[99mTc(CO)3(H2O)3]+,
radioactive nanoconjugates and colloidal species stay at the origin (Rf = 0).
—— System B: 5% HCl 6N/CH3OH. fac-[99mTc(CO)3(H2O)3]+ and [99mTcO4]−
migrate with the solvent front (Rf = 1), whereas radioactive nanoconjugates
and colloidal species stay at the origin (Rf = 0).
—— System C: C5H5N/CH3COOH/H2O (3:5:1). Colloidal species stay at
the origin (Rf = 0), whereas fac-[99mTc(CO)3(H2O)3]+, [99mTcO4]− and
radioactive nanoconjugates migrate with the solvent front (Rf = 1).
Radioactivity detection was performed on a radiochromatographer

(Berthold LB 2723) equipped with a 20 mm diameter NaI(Tl) scintillation crystal.
6.3.2.2. HPLC
Technetium-99m(CO)3 DAPM1, 99mTc(CO)3 DAPM4 and 99mTc(CO)3

DAPM8: HPLC analysis was performed on Perkin Elmer equipment coupled to a
gamma detector (Berthold Lb 509) and a UV/VIS detector (Shimadzu SPD-10 AV
or Perkin Elmer Lc 290). Analysis was carried out either by RP-HPLC or by
SEC-HPLC. RP-HPLC: Supelco Discovery Bio Wide Pore C18 25 cm × 4.6 mm,
5 μm analytical column; flow, 1 mL/min; eluents, A, trifluoroacetic acid (TFA)
0.1% in H2O; B, TFA 0.1% in CH3CN. SEC-HPLC: Shodex OHpack SB-803 HQ
analytical column; flow, 0.5 mL/min; eluent, ammonium acetate 0.5M. Resolution
of the column was calculated to be 0.76 ± 0.29 min. The wavelengths for
UV detection were 220 nm and 254 nm for RP-HPLC and SEC-HPLC, respectively.
Technetium-99m DCM20: HPLC analysis was performed by two systems.
System I: A Waters 600 chromatography system was coupled to a Waters 991
photodiode array detector and a GABI gamma detector (Raytest). HPLC solvents
consisted of water containing 0.1% TFA (solvent A) and methanol containing
0.1% TFA (solvent B). For the radiochemical analysis, a Nucleosil C18 reversed
phase column (10 μm, 250 × 4 mm) was used. The HPLC system started
with 100% of A from 0 min to 1 min followed by a linear gradient to 30% A
(70% B) in 9 min; this composition was held for another 10 min. The flow rate
112
was 1 mL/min. System II: A Waters 600E chromatography system was coupled
to a Waters 486 UV detector and a GABI gamma detector (Raytest) (γ trace
for 99mTc). Separations were achieved on a size exclusion column (Shodex
SB-803HQ) (8 mm × 300 mm) eluted with water at a flow rate of 1 L/min.
All radioactive probes were obtained with high radiochemical purity
and specific activity, as illustrated by RP-HPLC radiochromatograms of
99m
Tc(CO)3 DAPM4 and 99mTc (CO)3−DCM20 (Fig. 6.3).
6.3.3. Stability
The in vitro stabilities of 99mTc(CO)3 DAPM1, 99mTc(CO)3 DAPM4,

99m
Tc(CO)3 DAPM8 and 99mTc (CO)3− DCM20 towards transchelation were
studied in the presence of a large excess of cysteine or histidine in PBS (~24 h at
37°C). These amino acids have been selected because they are present in several
proteins in vivo and contain a set of donor atoms with high affinity for Tc(I).
Table 6.1 summarizes the radiochemical purities of the radiolabelled dextran
derivatives at different time points.
The results in Table 6.1 show that, despite being obtained in almost
quantitative yield, 99mTc(CO)3 DAPM1 displays relatively low stability in
the presence of cysteine and histidine. On the contrary, 99mTc(CO)3 DAPM4,
99m
Tc(CO)3 DAPM8 and 99mTc(CO)3 DCM20 are highly stable under the test
conditions, presenting, in both cases, high radiochemical purity (≥90%), even
after long incubation times. Therefore, 99mTc(CO)3 DAPM4, 99mTc(CO)3 DAPM8
and 99mTc(CO)3 DCM20 were selected as the most promising radioactive
compounds to pursue animal studies.
FIG. 6.3.  RP-HPLC radiochromatograms of (A) 99mTc(CO)3 DAPM4 and (B) 99mTc (CO)3−DCM20.
cpm: counts per minute.
113
TABLE 6.1.  IN VITRO STABILITIES OF RADIOLABELLED
MANNOSYLATED DEXTRAN DERIVATIVES 99mTc(CO)3 L (L = DAPM1,
DAPM4, DAPM8, DCM20) IN THE PRESENCE OF CYSTEINE AND
HISTIDINE AT DIFFERENT TIME POINTS
Activity bound to dextran (%)
Compound Cysteine Histidine
0 h 2 h 4 h 6 h 2 h 4 h 6 h
99m
Tc(CO)3 DAPM1 98 85 75 ND 89 85 ND
99m
Tc(CO)3 DAPM4 98 96 94 90 99 98 97
99m
Tc(CO)3 DAPM8 98 95 94 90 99 98 97
99m
Tc (CO)3−DCM20 98 95 94 90 98 96 97
Note: ND: not determined.
ACKNOWLEDGEMENTS
M. Morais thanks the FCT for a PhD grant (SFRH/BD/48066/2008).
[6.1] ALBERTO, R., et al., A novel organometallic aqua complex of technetium for the
labeling of biomolecules: synthesis of fac-[99mTc(H2O)3(CO)3]+ from [99mTcO4]− in
aqueous solution and its reaction with a bifunctional ligand, J. Am. Chem. Soc. 120
(1998) 7987–7988.
[6.2] Alberto, R., Ortner, K., Wheatley, N., Schibli, R., Schubiger, A.P., Synthesis and
properties of boranocarbonate: a convenient in situ CO source for the aqueous
preparation of [99mTc(OH)2(CO)3]+, J. Am. Chem. Soc. 123 (2001) 3135–3136.
[6.3] Alberto, R., Schibli, R., Waibel, R., Abram, U., Schubiger, A.P., Basic aqueous
chemistry of [M(OH2)3(CO)3]+ (M = Re, Tc) directed towards radiopharmaceutical
application, Coord. Chem. Ver. 190–192 (1999) 901–919.
[6.4] SANTOS, I., PAULO, A., CORREIA, J.D.G., Rhenium and technetium complexes
anchored by phosphines and scorpionates for radiopharmaceutical applications, Top.
Curr. Chem. 252 (2005) 45–84.
114
[6.5] ALBERTO, R., The particular role of radiopharmacy within bioorganometallic
chemistry, J. Organomet. Chem. 692 (2007) 1179–1186.
[6.6] GARCIA, R., PAULO, A., SANTOS, I., Rhenium and technetium complexes
with anionic or neutral scorpionates: an overview of their relevance in biomedical
applications, Inorg. Chim. Acta 362 (2009) 4315–4327.
[6.7] Alberto, R., The chemistry of technetium-water complexes within the manganese triad:
challenges and perspectives, Eur. J. Inorg. Chem. 2009 (2009) 21–31.
[6.8] Alberto, R., Organometallic radiopharmaceuticals, Top. Organomet. Chem. 32 (2010)
219–246.
[6.9] LIPOWSKA, M., et al., First evaluation of a 99mTc-tricarbonyl complex,
99m
Tc(CO)3(LAN), as a new renal radiopharmaceutical in humans, J. Nucl. Med. 47
(2006) 1032–1040.
[6.10] LIU, S., Ether and crown ether-containing cationic 99mTc complexes useful as

radiopharmaceuticals for heart imaging, Dalton Trans. 2007 (2007) 1183–1193.
[6.11] LIU, Z.L., et al., Kinetic characterization of a novel cationic 99mTc(I)-tricarbonyl

complex, 99mTc-15C5-PNP, for myocardial perfusion imaging, J. Nucl. Card. 17 (2010)
858–867.
[6.12] MARIA, L., et al., Tris(pyrazolyl)methane 99mTc tricarbonyl complexes for myocardial
imaging, Dalton Trans. 2009 (2009) 603–606.
[6.13] GOETHALS, L.R., et al., Rapid hepatic clearance of 99mTc-TMEOP: a new candidate
for myocardial perfusion imaging, Cont. Med. Mol. Imag. 6 (2011) 178–188.
[6.14] MORAIS, M., RAPOSINHO, P.D., CORREIA, J.D.G., SANTOS, I., Technical

University of Lisbon, Portugal, unpublished data.
[6.15] Morais, M., et al., Mannosylated dextran derivatives labeled with fac-[M(CO)3]+

(M = 99mTc, Re) for specific targeting of sentinel lymph node, Mol. Pharm. 8 (2011)
609–620.
[6.16] Pirmettis, I., et al., New 99mTc(CO)3 mannosylated dextran bearing S-derivatized

115
Chapter 7
4 + 1 Tc(III) COMPLEXES IN THE DESIGN AND

DEVELOPMENT OF 99mTc LABELLED
DEXTRAN MANNOSE DERIVATIVES AS
POTENTIAL RADIOPHARMACEUTICALS FOR SLND
A. REY
Cátedra de Radioquímica, Facultad de Química,
Universidad de la República,
Montevideo, Uruguay
H.-J. PIETZSCH
Helmholtz-Zentrum,
Dresden-Rossendorf,
Germany
7.1. SUMMARY
With the objective to develop a potential 99mTc radiopharmaceutical for

SLND, it was decided to focus on the formation of 4 + 1 Tc(III) complexes. This
system is based on an oxofree core with the metal at a low oxidation state, and
provides a non-polar building block that is stable against ligand exchange in vivo.
DC25 and DCM30 were derivatized to incorporate the isocyanide necessary to
coordinate 99mTc. The Tc complexes were formed by simultaneous coordination
of the tetradentate and tripodal ligand 2,2', 2'' nitrilotris ethanethiol and
isocyanide dextrans as the coligand. Labelling was achieved by substitution using
99m
Tc ethylenediaminetetraacetic acid (EDTA) as the precursor. Radiochemical
purity, determined by SEC-HPLC and thin layer chromatography (TLC), was
above 90%. The biodistribution of the derivative containing mannose as the
pharmacophore (DCM30-iso) in Wistar rats showed a high uptake in popliteal
lymph nodes and a very low uptake in external lumbar nodes and other organs.
This result was corroborated by dynamic and static imaging. The same derivative
without mannose (DC25-iso), on the other hand, showed negligible uptake in all
lymph nodes. Results suggest that uptake in lymph nodes can be attributed to
mannose. With the objective to assess the influence of the labelling method on the
biological behaviour of labelled dextran mannose derivatives, the biodistribution
of 99mTc DCM30 was also studied under the same conditions. A high uptake in the
first lymph node (popliteal) was observed at all time points, while the activity in
117
the second node (external lumbar) was significantly lower. However, the uptake
in popliteal lymph nodes was significantly lower compared to 99m Tc DCM30-iso
at 15 min and 30 min postinjection. The uptake in other organs was negligible,
except for in the blood and liver, which demonstrated low uptakes.
7.2. INTRODUCTION
The 4 + 1 Tc(III) complexes are a class of Tc complexes bearing the Tc(III)

core proposed by Pietzsch et al. [7.1] as a new non-polar Tc chelate system for
the design of neutral and lipophilic complexes. Figure 7.1 depicts the general
structure of this type of complex.
Formed by combination of a tetradentate tripodal 2-[bis-(2-mercapto-
ethyl)-amino]-ethanethiol (NS3) ligand and a monodentate coligand (isocyanide
or tertiary phosphine), these complexes have the advantage of high versatility
in conjugating biomolecules because both monodentate and tetradentate ligands
are, at least in principle, able to bear functional groups, although the introduction
of the biomolecule in the tetradentate ligand might generate a chiral carbon [7.2].
The donors can also be varied, but phosphines have the problem of sometimes
being rather bulky cosubstituents [7.3].
FIG. 7.1.  General structure of 4 + 1 Tc(III) complexes.
118
A very important feature of these complexes is that the metal is well
shielded by the 4 + 1 donor atom arrangement, thus avoiding interference
with biological activity [7.4–7.6]. Furthermore, complexes are very stable,
both in labelling milieu and in human plasma. In vitro challenge experiments
with glutathione clearly indicated that no transchelation reactions occur. There
were also no indications for in vivo reoxidation of Tc(III) to Tc(V) species or
pertechnetate [7.7, 7.8].
The tripodal molecule 2,2', 2''-nitrilotris(ethanethiol) is the most widely
used NS3 tetradentate ligand. Derivatization of this ligand to introduce
hydrophilic groups such as hydroxy groups, carboxyl groups or carbohydrates
increases the hydrophilicity of the overall complex and is useful for fine tuning
pharmacokinetics [7.9−7.11]. Figure 7.2 depicts the application of this concept to
the development of a 99mTc labelled arginylglycylaspartic acid (RGD) peptide for
imaging tumoural neoangiogenesis [7.12].
This labelling concept has also been applied to the development of 99mTc
dextran mannose derivatives as potential radiopharmaceuticals for SLND. For
this purpose, a dextran derivative bearing mannose as the active moiety and an
isocyanopropyl group as the monodentate ligand for Tc(III) complexation were
necessary. Figure 7.3 depicts the structure.
FIG. 7.2.  RGD peptide labelled by means of 4 + 1 Tc(III) complexes.
119
FIG. 7.3.  Structure of a dextran derivative bearing an isocyanide moiety as the monodentate
ligand for Tc(III) complexation.
FIG. 7.4.  Conjugation of isocyanide moiety to the biomolecule though an amino group.
The amino group of cysteine was used as the point for attaching both the
mannose and the electron donor group. Introduction of the latter is achieved
by reaction of the amine groups of the cysteine with 4-isocyano-butyric acid
2,5-dioxo-pyrrolidin-1-yl ester, as shown in Fig. 7.4.
The procedure can be performed as a batch, and kept for at least 2 weeks
at 5°C−10°C. Derivatization in small quantities prior to each labelling procedure
was also performed. On average, dextran derivative bears 45 cysteine branches:
120
1 cysteine is derivatized with the isocyanopropyl group, 36 cysteine branches are
mannosylated and 8 remain free.
7.3. MATERIALS AND METHODS
7.3.1. General
All laboratory chemicals were reagent grade and used without further
purification. Solvents for chromatographic analysis were HPLC grade.
Technetium-99m NaTcO4 was obtained from a commercial generator. The dextran
derivative containing S-derivatized cysteine (DC25, MW ~ 23 kg/mol) and the
mannosylated dextran derivative containing S-derivatized cysteine (DCM30)
were provided by I. Pirmettis (Institute of Nuclear & Radiological Sciences
& Technology, Energy & Safety, Demokritos National Centre for Scientific
Research, Greece). Radiochemical purity was checked by TLC in silica gel using
water as the mobile phase and by SEC-HPLC on a Superdex Peptide 10/300
GL column eluted with 50mM, pH7.0 PBS and 0.15M NaCl, at 0.5 mL/min
flow rate. Radioactivity was detected with a homemade γ ray NaI(Tl) detector.
NMR spectra were recorded in D2O at 25°C on a Bruker 500 MHz Avance DRX
spectrometer using 2,2,3,3-tetradeutero-3-trimethylsilyl-propionic acid sodium
salt as the internal reference. Assignment of the spectra was based on a series of
1
H–1H and 1H–13C correlation experiments. The 2,2', 2''-nitrilotris(ethanethiol)
was prepared by reaction of tris(2-chloroethyl)amine hydrochloride with
potassium thioacetate, followed by reduction with LiAlH4. The final product
was precipitated as oxalate salt and used as such in further reactions [7.2].
The coupling agent 4-isocyanobutyric acid 2,5-dioxopyrrolidin-1-yl ester was
synthesized according to previously published methods [7.1, 7.8, 7.11].
7.3.2. Synthesis of dextran derivative with isocyanopropyl arms
7.3.2.1. Dextran cysteine derivative with isocyanide arms (DC25-iso)
The dextran cysteine derivative DC25 (30 mg, 0.1 µmol) and triethylamine

(100 µL) were dissolved in water (1 mL) in a vessel protected from light.
N-hydroxysuccinimidyl 4-(isocyano)butanoate (12.3 mg, 58 µmol) was added.
The reaction was stirred at room temperature until the reagent was completely
consumed. The solution was evaporated in vacuo, and the white solid was washed
with methanol and diethyl ether. The yield was 73% [7.13].
121
7.3.2.2. Dextran cysteine mannose derivative with isocyanide arms (DCM30-iso)
The dextran cysteine mannose derivative DCM30 (12 mg, 0.3 µmol) and

triethylamine (100 µL) were dissolved in water (1 mL) in a vessel protected
from light. N-hydroxysuccinimidyl 4-(isocyano)butanoate (4.5 mg, 21 µmol)
was added. The reaction was stirred at room temperature until the reagent was
completely consumed. The solution was evaporated in vacuo, and the white solid
was washed with methanol and diethyl ether. The yield was 75% [7.13].
7.3.3. Radiolabelling and control of radiochemical purity
As shown in Fig. 7.5, labelling was performed by a two step substitution

procedure using 99mTc EDTA/mannitol as the precursor. This precursor was
obtained by direct reduction of pertechnetate using stannous chloride as the
reducing agent, as follows. Technetium-99m [TcO4] Na (300−1000 MBq, 1 mL)
was added to a vial containing EDTA sodium salt (5 mg), mannitol (5 mg) and
stannous chloride dihydrate (0.08 mg), and the mixture was incubated at room
temperature for 5 min. The radiochemical purity was checked by TLC on silica
gel using acetone and water as the mobile phases. The radiochemical purity was
higher than 90%.
FIG. 7.5.  Labelling procedure.
122
Substitution was achieved by the simultaneous action of the dextran
derivative and the tetradentate NS3 coligand 2,2', 2''-nitrilotris(ethanethiol).
The experimental procedure used for substitution was as follows.
A solution of derivatized dextran mannose derivative in saline solution (800 μg
in 250−500 μL) and 2,2',2''-nitrilotris(ethanethiol) oxalate (1 mg) was mixed
with 99mTc EDTA and incubated at 70°C for 30 min. The in vitro stability of
the 99mTc labelled compounds incubated at 37°C for up to 4 h postlabelling was
evaluated using SEC-HPLC.
The radiochemical purity could be assessed by a single TLC run using
water as the mobile phase because the labelled dextran remained at the origin,
while 99mTc EDTA and 99mTcO4− migrated with the solvent front.
To confirm that coordination of the dextran derivative with 99mTc took
place through the isonitrile, the labelling procedure was also performed using a
derivative without the isocyanide group. The radiochemical purity was assessed
using TLC and HPLC.
7.3.4. Biological evaluation
7.3.4.1. Biodistribution studies
Experimental animal studies were approved by the Ethics Committee of

the Faculty of Chemistry at the University of the Republic, Uruguay. Normal
Wistar rats (female, 250–300 g, three animals per group) were anaesthetized
with sodium thiopental (50 mg/kg) administered in the peritoneal cavity. Then,
99m
Tc DCM30-iso derivatives (50 µL, 0.37 MBq, 0.2 nmol) were injected
subcutaneously in the rear foot-pad, and 10 min before sacrifice, a volume of
0.05 mL of patent blue was also injected for visual identification of lymph nodes.
Animals were sacrificed at different time intervals postinjection (15 min, 30 min,
60 min, 180 min and 24 h). Popliteal and external lumbar lymph nodes, as well
organs and samples of blood and muscle, were collected, weighed and assayed
for radioactivity. The injected foot was also removed for determination of
activity remaining at the injection site. To establish if lymph node uptake can be
attributed to the presence of the mannose moiety, biodistribution of labelled 99mTc
DC25-iso was also performed under the same conditions. Popliteal extraction (E)
was calculated according to:
%ID POPLITEAL −%ID INGUINAL

E (%) = ×100 (7.1)
%ID POPLITEAL
123
7.3.4.2. Imaging studies
Imaging studies were performed in normal Wistar rats (female, 250–300 g).

After peritoneal anaesthesia with sodium thiopental, animals were injected in the
rear foot-pad with 99mTc labelled dextran derivatives DCM30-iso and DC25-iso
(0.05 mL, 100 MBq, 2 nmol). Imaging was performed using a rectangular field
(53.8 cm × 39.9 cm) gamma/camera (Sophy Camera DSX) equipped with a low
energy, high resolution, parallel hole collimator. Static images were obtained at
15 min, 30 min, 45 min and 60 min postinjection.
7.4. RESULTS AND DISCUSSION

with isocyano propyl arms
Dextran isocyanide derivatives DC25-iso and DCM30-iso were synthesized

in high yields from their respective dextran precursors (~75% yields). Detailed
NMR experiments allowed for complete assignment of signals to their respective
nuclei. Two dimensional 1H–1H (correlation spectroscopy and nuclear Overhauser
effect spectroscopy) and 1H–13C correlation techniques were able to confirm the
expected structure.
7.4.2. Radiochemical purity and stability of 99mTc dextran derivatives
Mannosylated and non-mannosylated dextran isocyanide derivatives

(DCM30-iso and DC25-iso, respectively) gave the 4 + 1 complex with the
NS3 ligand at a high yield (~90%). Figure 7.6 depicts a typical chromatographic
profile of the precursor (99mTc EDTA) and of the final product, clearly
demonstrating that substitution was complete and that 99mTc labelled dextran was
obtained as the only reaction product.
When dextran derivative without the isocyanide group was used in the
4 + 1 labelling process, both chromatographic methods showed that the final
product was 99mTc EDTA, indicating that the isocyanide group is crucial for
achieving an adequate labelling yield. The radiochemical purity remained
over 90% for 4 h, demonstrating that no decomposition occurred within the
studied period.
124
FIG 7.6. Determination of radiochemical purity of 99mTc labelled dextran mannose derivative
using SEC-HPLC.
Table 7.1 summarizes the results obtained at different time points between

15 min and 24 h after subcutaneous administration in the right foot-pad of
Wistar rats of 99mTc DCM30-iso. Table 7.2 shows the results obtained after
administration of the non-mannosylated isocyanide dextran derivative analogue
(99mTc DC25-iso).
125
TABLE 7.1. BIODISTRIBUTION OF 99mTc DCM30-ISO IN RATS AFTER SUBCUTANEOUS ADMINISTRATION* (cont.)
126
% ID/organ
Organs
15 min 30 min 60 min 180 min 24 h
Blood 3.9 ± 1.6 2.38 ± 0.94 2.26 ± 0.67 8.51 ± 0.71 0.64 ± 0.11
Liver 2.8 ± 1.9 4.33 ± 0.66 5.17 ± 0.30 11.26 ± 0.76 6.28 ± 0.41
Lungs 0.09 ± 0.09 0.07 ± 0.01 0.07 ± 0.01 0.39 ± 0.18 0.05 ± 0.01
Spleen 0.10 ± 0.09 0.05 ± 0.02 0.08 ± 0.02 0.049 ± 0.25 0.11 ± 0.01
Kidneys 0.38 ± 0.36 0.34 ± 0.12 0.22 ± 0.05 1.072 ± 0.092 0.70 ± 0.18
Thyroid 0.03 ± 0.03 0.01 ± 0.01 0.010 ± 0.005 0.014 ± 0.013 0.010 ± 0.005
Muscle 0.40 ± 0.40 1.13 ± 0.57 0.84 ± 0.39 1.70 ± 0.75 0.46 ± 0.14
Stomach 0.14 ± 0.10 0.08 ± 0.02 0.20 ± 0.11 0.244 ± 0.036 0.18 ± 0.15
Intestine 0.40 ± 0.29 0.87 ± 0.18 0.98 ± 0.20 6.5 ± 1.3 1.36 ± 0.02
Bladder + urine 0.20 ± 0.27 1.30 ± 0.70 3.18 ± 1.24 15.9 ± 8.3 49.4 ± 9.4
Popliteal lymph node 9.4 ± 1.2 5.90 ± 0.82 3.55 ± 1.40 5.42 ± 0.88 7.0 ± 1.0
TABLE 7.1. BIODISTRIBUTION OF 99mTc DCM30-ISO IN RATS AFTER SUBCUTANEOUS ADMINISTRATION* (cont.)
% ID/organ
Organs
15 min 30 min 60 min 180 min 24 h
External lumbar lymph node 1.18 ± 0.52 1.33 ± 0.91 1.42 ± 0.99 0.79 ± 0.54 3.40 ± 0.43
Popliteal extraction 87.8 ± 4.0 78.6 ± 12.6 76.4 ± 12.3 86.3 ± 7.8 51.3 ± 0.80
Injected foot 78.0 ± 6.4 77.1 ± 2.6 79.8 ± 3.0 45.7 ± 3.1 19.8 ± 1.5
* Results are expressed as % ID per organ and whole blood (mean ± standard deviation on three animals per time point).
127
TABLE 7.2.  BIODISTRIBUTION OF 99mTc DC25-ISO IN RATS AFTER
SUBCUTANEOUS ADMINISTRATION*
% ID/organ
Organs
15 min 30 min 60 min 24 h
Blood 9.0 ± 2.5 5.8 ± 1.6 5.7 ± 0.4 0.54 ± 0.10
Liver 10.4 ± 1.8 11.0 ± 2.0 10.8 ± 0.3 1.62 ± 0.51
Lungs 0.53 ± 0.12 0.27 ± 0.08 0.30 ± 0.02 0.04 ± 0.01
Spleen 0.12 ± 0.02 0.06 ± 0.02 0.08 ± 0.01 0.04 ± 0.02
Kidneys 4.8 ± 2.4 2.9 ± 0.02 4.6 ± 0.9 8.5 ± 1.8
Thyroid 0.054 ± 0.004 0.03 ± 0.02 0.05 ± 0.02 0.010 ± 0.005
Muscle 8.9 ± 1.4 6.0 ± 1.2 4.7 ± 0.2 0.40 ± 0.08
Stomach 0.47 ± 0.06 0.25 ± 0.01 0.50 ± 0.21 0.52 ± 0.14
Intestine 5.7 ± 2.5 8.1 ± 1.3 14.4 ± 0.9 2.28 ± 0.20
Bladder + urine 3.4 ± 1.7 15.3 ± 2.8 25.6 ± 2.9 76.8 ± 6.5
Popliteal lymph node 0.25 ± 0.04 0.20 ± 0.12 0.20 ± 0.07 0.55 ± 0.07
External lumbar lymph node 0.06 ± 0.03 0.11 ± 0.06 0.10 ± 0.04 0.25 ± 0.02
Injected foot 41.1 ± 5.4 25.8 ± 6.3 15.6 ± 1.2 2.23 ± 0.37
* Results are expressed as % ID per organ and whole blood (mean ± standard deviation on
three animals per time point)
Lymph node uptake of 99mTc DCM30-iso (dextran with mannose pending

arms) was much higher than that of the corresponding dextran conjugate
without mannose (99mTc DC25-iso). These findings support the hypothesis that
radioactivity localization in lymph nodes has occurred by a receptor mediated
mechanism to macrophages present in these organs.
128
7.4.3.2. Imaging studies
Dynamic images up to 30 min after subcutaneous administration of

99m
Tc DCM30-iso and 99mTc DC25-iso in rats are shown in Fig. 7.7.
Biodistribution profiles of both compounds are consistent with the results
obtained by dissection. Lymph nodes are clearly visible for 99mTc DCM30-iso.
Liver and bladder can also be visualized. On the other hand, the uptake in lymph
nodes is significantly lower for 99mTc DC25-iso. In this case, liver and bladder
are the organs that show a higher uptake.
Static images acquired 30 min postadministration confirm the above
findings, as shown in Fig. 7.8.
7.5. CONCLUSIONS
With the aim of developing new radiopharmaceuticals for SLND, evaluation

and biological evaluation of mannosylated and non-mannosylated isocyanide
dextran derivatives labelled with Tc(III) in a 4+1 linking arrangement have been
studied. Labelling with high yields and radiochemical purities was achieved with
both derivatives. Biodistribution results demonstrated high uptakes in the first
lymph node and low uptakes in the following node, as well as low activities in the
rest of the body when mannose moieties were present. On the other hand, lack of
mannose resulted in negligible uptakes in all lymph nodes. Imaging experiments
corroborated the biodistribution data. These results are promising, and further
biological evaluation is required to confirm the findings.
99m 99m
Tc DCM30-iso Tc DC25-iso
FIG. 7.7.  Dynamic acquisitions (0–30 min) after subcutaneous administration of

99m
Tc DCM30-iso (left) and 99mTc DC25-iso (right) in rats.
129
FIG. 7.8.  Static acquisitions (30 min) after subcutaneous administration of
99m
Tc DCM30-iso (left) and 99mTc DC25-iso (right).
[7.1] PIETZSCH, H.-J., et al., Mixed-ligand technetium(III) complexes with tetradendate/

monodendate NS3/isocyanide coordination: a new nonpolar technetium chelate system
for the design of neutral and lipophilic complexes stable in vivo, Bioconjug. Chem. 12
(2001) 538–544.
[7.2] SPIES, H., GLASER, M., Synthesis and reactions of trigonal-bipyramidal rhenium and
technetium complexes with a tripodal, tetradentate NS3 ligand, Inorg. Chim. Acta 240
(1995) 465–478.
[7.3] ALBERTO, R., “Technetium”, Comprehensive Coordination Chemistry II
(McCLEVERTY, J.A., MEER, T.S., Eds), Vol. 5, Elsevier Science, Amsterdam
(2003) 127–271.
[7.4] DREWS, A., et al., Synthesis and biological evaluation of technetium(III) mixed-ligand
complexes with high affinity for the cerebral 5-HT1A receptor and the alpha1-
adrenergic receptor, Nucl. Med. Biol. 29 (2002) 389–398.
[7.5] WALTHER, M., et al., Synthesis and biological evaluation of a new type of (99m)
technetium-labeled fatty acid for myocardial metabolism imaging, Bioconjug. Chem.
18 (2007) 216–230.
[7.6] KÜNSTLER, J.U., et al., Organometallic 99mTc(III) ‘4 + 1’ bombesin(7-14) conjugates:
synthesis, radiolabeling, and in vitro/in vivo studies, Bioconjug. Chem. 18 (2007)
1651–1661.
130
[7.7] SCHILLER, E., et al., Mixed-ligand rhenium-188 complexes with tetradentate/
monodentate NS3/P (‘4 + 1’) coordination: relation of structure with antioxidation
stability, Bioconjug. Chem. 16 (2005) 634–643.
[7.8] SEIFERT, S., et al., Novel procedures for preparing Tc-99m(III) complexes with
tetradentate/monodentate coordination of varying lipophilicity and adaptation to
Re-188 analogues, Bioconjug. Chem. 15 (2004) 856–863.
[7.9] KÜNSTLER, J.U., et al., Novel 99mTc ‘4 + 1’ peptide conjugates: tuning the
biodistribution by variation of coligands, Eur. J. Med. Chem. 45 (2010) 3645–3655.
[7.10] KÜNSTLER, J.U., et al., Efficient preparation of 99mTc(III) ‘4 + 1’ mixed-ligand
complexes for peptide labeling with high specific activity, Appl. Radiat. Isot. 68
(2010) 1728–1733.
[7.11] KÜNSTLER, J.U., et al., Impact of functionalized coligands on the pharmacokinetics
of 99mTc(III) ‘4 + 1’ mixed-ligand complexes conjugated to bombesin, J. Inorg.
Biochem. 105 (2011) 1383–1390.
[7.12] GIGLIO, J., et al., “Design and evaluation of potential 99mTc-radiopharmaceuticals
based on the Tc-carbonyl, 4 + 1 mixed-ligand chelate system and Tc-nitrido
approaches”, Labelling of Small Biomolecules Using Novel Technetium-99m Cores,
Technical Reports Series No. 459, IAEA, Vienna (2007) 261–285.
[7.13] GIGLIO, J., et al., Design and development of (99m)Tc-‘4 + 1’-labeled dextran-mannose
derivatives as potential radiopharmaceuticals for sentinel lymph node detection, Cancer
Biother. Radiopharm. 28 (2013) 541–551.
131
Chapter 8
BIOLOGICAL EVALUATION OF
LOW VALENCE 99mTc DEXTRAN DERIVATIVES
FOR SLND: RESULTS OF A MULTILABORATORY STUDY
R. PASQUALINI
CIS bio international/IBA, Saclay, RP Innovative,
Clamart, France
8.1. SUMMARY
SLNB allows accurate staging of regional lymph node basins by surgical

removal and histological examination of those lymph nodes that receive direct
lymph drainage from the tumour site. This technique has become the standard
of care in breast cancer and melanoma, and is increasingly being applied to
other solid cancers with high metastatic potential in lymph nodes. The SLNM
can be performed using blue dyes, radiotracers or both. Radiopharmaceuticals
based on 99mTc colloids (either inorganic or of albumin origin) are the currently
used radiotracers, although a newly approved dextran based, receptor targeted
agent, 99mTc tilmanocept, is expected to be gradually adopted by the nuclear
medicine community.
Recognizing the importance of the receptor targeted approach for SLNM,
the IAEA initiated a CRP in 2007 to evaluate the ability of newer low valence
99m
Tc dextran derivatives to selectively localize in the SLN. This chapter is based
on work carried out at participating institutions in several Member States.
Low MW dextran (12 kg/mol and 20 kg/mol), to which mannose residues
were added for specificity to mannose receptors expressed by SLN macrophages,
was derivatized to incorporate either cysteine/cysteamine or pz moiety for
facilitating radiolabelling with a Tc(I) carbonyl core. Isocyanide groups were
also added to mannosylated dextran for the formation of 4 + 1 Tc(III) complexes.
The new dextran derivatives (20 kg/mol and 30 kg/mol MW) were
quantitatively labelled with 99mTc (>90% radiochemical purity). Both Tc(I) and
Tc(III) complexes were inert to ligand exchange with cysteine and histidine, and
remained stable after 6 h at room temperature.
The biological properties of the radiolabelled dextran derivatives were
tested in vivo (gamma imaging) and ex vivo (biodistribution) after intradermal/
subcutaneous administration to mice, rats and rabbits. Approved 99mTc labelled
colloids (rhenium sulphide, sulphur colloids and calcium phytate) were used
133
to standardize the protocol for identifying the sentinel node and the secondary
nodes in animals. Animal experiments have shown rapid and high accumulation
in the popliteal lymph node that remained almost stable for a long period of time.
Uptake in the SLN was similar to that displayed by 99mTc tilmanocept, showing a
saturable receptor mediate mechanism.
Ranking the 99mTc dextran derivatives according to their biological
properties led to [99mTc(CO)3 DCM20] and [99mTc(CO)3 DAPM8] being selected
as the most promising agents for potential use in humans.
8.2. INTRODUCTION
The SLN is the hypothetical first lymph node, or group of nodes, that
receives lymph collected from a primary tumour mass. If cancerous dissemination
occurs, it is postulated that the SLN will trap any metastasizing cells leaving
the tumour. If the SLN is negative for tumour metastasis, the presence of
cancerous cells in all other lymph nodes in the same lymphatic chain is highly
improbable [8.1–8.5].
This technique has become the standard of care in breast cancer [8.6, 8.7]
and melanoma [8.8, 8.9], and is increasingly being applied to other solid cancers
with high metastatic potential in lymph nodes, such as oral and oropharyngeal
squamous cell carcinoma [8.10]. SLNM can be performed using a dye (patent
blue or isosulphan blue), using radiotracers or both. Radiopharmaceuticals
based on 99mTc colloids (either inorganic or of albumin origin) are the currently
used radiotracers [8.11]. Despite their long standing use, large variations exist
in the nature and size of 99mTc labelled colloids currently used for the detection
of SLNs [8.2, 8.12]. Studies in the USA were initiated with sulphur colloid,
which is a radiopharmaceutical that was initially approved for liver and spleen
scintigraphy [8.13]. This radiocolloid (filtered or unfiltered) is still used. In
Europe, albumin colloids and a preformed rhenium sulphide colloid have been
approved for SLND. Of the two, albumin colloid is the most frequently used
tracer in Europe. Technetium-99m labelled antimony trisulphide is the colloid
of choice in Australia, whereas 99mTc calcium phytate is the tracer used in Japan.
Ideally, an SLN imaging agent would rapidly clear from the injection site to
allow easy detection of nodes that are located close to the injection site. Residence
of activity in the SLN should be selective and long enough to allow different
protocols for detection (external acquisition or radioguided surgery). The
radiotracer should be obtained with high specific activity to avoid saturation of
the phagocytic process, with consequent leakage of radioactivity to the higher
echelon nodes of the region. Finally, its preparation should be easy to perform,
and the administered product should be safe. Radiocolloids hardly match all the
134
above mentioned requirements. In particular, clearance from the injection site
may be slow, especially for large sized colloids. On the other hand, colloids based
on HSA, although displaying the appropriate size range, may raise potential
concerns about the quality of the donated blood, which has to be monitored for
viral contamination. Some of these drawbacks have been overcame by the
introduction, by Vera et al., of a totally synthetic 99mTc DTPA mannosyl dextran
derivative that accumulates in the lymphatic tissues of rats and rabbits by binding
to mannose receptors present on the surface of macrophage cells [8.14]. The
compound consists of a dextran backbone with an average MW of 9500 g/mol, to
which mannose glycosides and DTPA moiety are attached through cysteamine
arms. The final amino, mannose and DTPA densities were 23, 55 and 8 moles per
dextran, respectively. The MW of the dextran derivative was approximately
36 000 g/mol. This receptor based agent has shown excellent results in
animals [8.14], and has been investigated in a multicentre, open label,
comparative study against VBD for the detection of SLNs in melanoma and
breast tumours [8.15]. The clinical study demonstrated the concordance of in
vivo detection rates of this new agent and VBD in lymph nodes. The compound,
named 99mTc labelled tilmanocept, was approved by the FDA in March 2013 for
use in patients with breast cancer or melanoma who are undergoing surgery to
remove tumour draining lymph nodes. The drug, marketed under the name of
Lymphoseek, has been approved by the European Medicines Agency
(an extensive discussion of this new tracer is given by Vera et al. in Chapter 4).
Recognizing the importance of the receptor targeted approach for SLNM,
the IAEA initiated a CRP in 2007 aimed at synthesizing newer low valence
99m
Tc dextran derivatives and evaluating their potentiality in mapping SLNs in
rodents. This chapter is based on the biological work carried out at institutions
participating in the CRP Development of 99mTc Radiopharmaceuticals for
Sentinel Node Detection and Cancer Diagnosis (see the list of contributors to
drafting and review at the end of this publication).
In an effort to explore new mannosylated dextran derivatives that
could produce a superior 99mTc labelled SLNM agent, it was decided to
investigate the potential of the fac-[99mTc(CO)3]+ core and the [99mTc(NS3)]
fragment for 99mTc labelling. The former core has recently attracted growing
interest, particularly after Alberto et al. reported an aqueous preparation of
the [99mTc(CO)3(H2O)3]+ precursor [8.16] and the availability of the IsoLink
boronocarbonate kit by Mallinckrodt Medical BV [8.17]. As a tridentate ligand
is required for the replacement of the aquo ligands and the formation of a stable
complex with a 99mTc tricarbonyl core, mannosylated dextran molecules were
equipped with S-derivatized cysteine arms (providing the S, N, O set of ligating
atoms) [8.18–8.20] or with S-derivatized pz diamine arms (providing the N, N,
N set of ligating atoms) [8.21, 8.22]. Isocyanide groups were also appended to
135
mannosylated dextran for ligation of the [99mTc(NS3)] fragment in the so called
4 + 1 mixed ligand arrangement [8.23–8.26]. The chemical structures of dextran
derivatives evaluated in this study are reported in Fig. 8.1.
136
FIG. 8.1.  Chemical structure of dextran derivatives under study.
8.3. MATERIALS AND METHODS
8.3.1. Synthesis of dextran derivatives
Detailed synthesis of the different dextran derivatives is discussed in

Chapter 5. Briefly, dextran bearing S-derivatized cysteine chelator (DC15) and
mannosylated dextran (DCM20) were obtained through a two and three step
synthesis, respectively. Dextran (MW = 11 800 g/mol) was reacted with allyl
bromide to yield allyl dextran with about 40% coupling. Addition of cysteine
to allyl dextran in the presence of ammonium persulphate resulted in the
S-derivatized cysteine, compound DC15. The mannosylated product DCM20
was obtained in good yield after in situ hydrolysis and activation of cyanomethyl
2,3,4,6-tetra-O-acetyl-1-thio-β-D-mannoside (CNM thiomannose) and coupling
to DC15. All intermediates, as well as the final products, were purified by
ultrafiltration using a membrane with an MW cut-off of 3000 g/mol, then
lyophilized [8.27].
137
Mannosylated dextran bearing pz diamine chelators (DAPM1–4–8) were
obtained using a synthetic strategy similar to that used for the preparation of
DC15/DCM20 compounds. Dextran (MW = 9500–10 500 g/mol) was reacted
with allyl bromide to yield allyl dextran with about 50% coupling. The addition
of aminoethanthiol and ammonium persulphate resulted in the S-derivatized
amine. This intermediate was reacted in dry CH2Cl2 with the carboxylic acid
of the Boc protected pz diamine chelator using N,N'-dicyclohexylcarbodiimide
and N-hydroxysuccinimide as activators. CNM thiomannose was deacetylated
with sodium methoxide. After the removal of methanol, the CNM mannose was
reacted with dextran bearing the Boc protected pz diamine arms. The protecting
group was removed with TFA/H2O, giving the final conjugates, as TFA salt,
in quantitative yields. All intermediates and the final product were purified by
dialysis, then lyophilized [8.28].
Mannosylated dextran derivatives bearing isocyanide groups for ligation
with 99mTc(III) (DCM-iso) were prepared following a four step synthesis.
Dextran (MW = 18 000 g/mol) was reacted with allyl bromide to yield allyl
dextran (40% of the glucose units were allylated). Addition of cysteine to allyl
dextran in the presence of ammonium persulphate resulted in the S-derivatized
cysteine, compound DC25. Mannose residues were introduced by reacting DC25
with CNM thiomannose as previously described (DCM30). The isocyanide
group was introduced by reacting DCM30 with 4-isocyano-butyric acid
2,5-dioxo-pyrrolidin-1-yl ester under basic conditions. All intermediates, as well
as the final products, were purified by ultrafiltration using a membrane with a
MW cut-off of 3000 g/mol [8.29]. The procedure can be performed as a batch
and kept for at least 2 weeks at 5°C–10°C, or performed in small quantities prior
to each labelling procedure.
Proton and 13C NMR spectroscopy (300–500 MHz) was used to establish
the chemical identity of intermediates and final products. Colorimetric tests
were used to determine the density of amine residues per mole of dextran
(2,4,6-trinitrobenzene sulphonic acid assay) and to obtain the number of sugar
units per dextran molecule (sulphuric acid/phenol colorimetric assay).
8.3.2. Physical characterization of dextran derivatives
The hydrodynamic diameters of DCM and DAPM derivatives and the

starting dextran were determined by DLS using a ZetaSizer Nano ZS-90 from
Malvern Instruments (UK). Measurements were carried out at 25°C with a
173° scattering angle. All the compounds were dissolved in saline (2 mL),
resulting in different concentrations because different quantities of compounds
were used for labelling (50 µg DCM20, 400 µg DAPM).
138
8.3.3. Radiolabelling
Technetium-99m, as 99mTc [TcO4] Na, was obtained from commercial

generators available in each Member State.
8.3.3.1. Dextran derivative formulation for 99mTc labelling
DC15/DCM20: 1 mL of 0.22 µm Millipore filtered aqueous solutions of

DC15 or DCM20 (50 µg/mL or 400 µg/mL) was dispensed in 10 mL penicillin
vials and lyophilized at −4°C for 24 h. At the end of the lyophilization cycle, the
vials were stoppered and sealed under vacuum.
DAPM1–4–8: Vials containing DAPM derivatives (50–500 µg) were
prepared either extemporaneously, by dispensing the appropriate volume of a
water solution, or by lyophilization of aqueous solutions of the derivatives.
DC25-iso/DCM30-iso: Vials containing DC25-iso or DCM30-iso (500 µg)
were prepared extemporaneously before labelling.
Dextran derivatives were labelled either with a [99mTc(CO)3]+ core (dextran
propyl-S-cysteine/DCM/DPAM derivatives) or with a [99mTc(NS3)] fragment
(DC25-iso/DCM30-iso derivatives). Common labelling and quality control
procedures were established to obtain comparable results within laboratories.
8.3.3.2. Labelling with [99mTc(CO)3]+ core: formation of

[99mTc(CO)3(H2O)3]+ precursor
Radiolabelling with [99mTc(CO)3]+ core was performed by a two step

procedure using [99mTc(CO)3(H2O)3]+ as a precursor. The precursor was
obtained using the IsoLink kit (Mallinckrodt Medical BV) or by a homemade
kit (10 mL vial containing 4.0 mg Na2CO3, 5.5 mg NaBH4, 15–20 mg Na/K
tartrate and 1 atm. carbon monoxide headspace volume). Typically, 1 mL of
99m
TcO4 (37–1850 MBq/mL, 1–50 mCi/mL) was added to the IsoLink vial and
heated for 25 min at 100°C in a water bath. The solution was cooled, and the pH
was adjusted to 7–8.5 with the addition of 0.1M HCl or PBS/HCl solution.
8.3.3.3. Labelling with [99mTc(CO)3]+ core: formation of

[99mTc(CO)3 DC15/DCM20] and [99mTc(CO)3 DAPM]
Typically, the vials containing solid dextran derivatives were reconstituted

with 200 µL of saline and flushed with nitrogen before radiolabelling.
Approximately 500 µL of the pH adjusted [99mTc(CO)3(H2O)3]+ intermediate
was added to the individual vials. DC15 and DC25 were labelled at 70°C–80°C
139
for 25–30 min, whereas labelling of DAPM derivatives occurred at 100°C for
30 min.
8.3.3.4. Labelling with [99mTc(NS3)] fragment: formation of

99m
Tc EDTA precursor
The 99mTc EDTA precursor was prepared by adding 99mTcO4− (300 MBq,

1 mL) to a vial containing 5 mg EDTA, 5 mg mannitol and 0.08 mg SnCl2.
Formation of the precursor took place in 5 min at room temperature.
8.3.3.5. Labelling with [99mTc(NS3)] fragment: formation of

[99mTc (DC25-iso + NS3)] and [99mTc (DCM30-iso + NS3)]
A solution of DC25-iso or DCM30-iso in saline (800 μg in 250–500 μL)

and NS3 (1 mg) was mixed with 99mTc EDTA and incubated at 70°C for 30 min.
8.3.4. Control of radiochemical purity
8.3.4.1. Radiochemical purity of [99mTc(CO)3 DC15/DCM20] and

[99mTc(CO)3 DAPM]
Correct formation of the [99mTc(CO)3(H2O)3]+ precursor, as well as the

radiochemical purity of the final products, was ascertained using a reversed
phase C18 column. The chromatographic conditions varied slightly from one
laboratory to another. The following is an example of the analytical conditions for
determination of the radiochemical purity. Solvents consisted of water containing
0.1% TFA (solvent A) and methanol containing 0.1% TFA (solvent B). The
column was a Nucleosil C18 reversed phase column (10 µm, 250 mm × 4 mm).
Separation started with 100% of A from 0 min to 1 min, followed by a linear
gradient to 30% A (70% B) in 9 min; this composition was held for another
10 min. The flow rate was 1 mL/min. Peak elution was monitored using an
appropriate radioactivity detector. Radioactive preparations were also tested by
SEC-HPLC (e.g. Shodex SB-803HQ, 8 mm × 300 mm) eluted with water at a
flow rate of 1 mL/min. In this system, retention times were as follows: 99mTcO4−,
tR = 3.5 min; fac-[99mTc(OH2)3(CO)3]+ precursor, tR = 5.0 min; 99mTc dextrans,
tR = 11–13 min.
For a rapid evaluation of radiochemical purity, samples were also
analysed by ITLC using methanol:HCl (99:1) as the mobile phase, or paper
chromatography (Whatman 1) using acetone as the mobile phase.
140
The radiochemical stability of the [99mTc(CO)3] dextran derivatives in the
final preparation was evaluated at room temperature. The chemical inertness of
the complexes was also investigated by challenging each 99mTc complex with a
large molar excess (1:100) of cysteine or histidine solutions in 1 × 10−3M PBS at
pH7.4. The samples were incubated at 37°C and analysed by RP-HPLC.
8.3.4.2. Radiochemical purity of [99mTc EDTA] precursor
Correct formation of the [99mTc EDTA] precursor was checked by TLC in

silica gel using acetone and water as the mobile phase.
8.3.4.3. Radiochemical purity of [99mTc (DC25-iso + NS3)] and

[99mTc (DCM30-iso + NS3)]
Radiochemical purity was checked by TLC in silica gel using water as

the mobile phase and by molecular exclusion HPLC on a Superdex Peptide
10/300 GL column, using 50mM PBS, pH7.0, in 0.15M NaCl at 0.5 mL/min
flow rate.
8.3.5. Determination of partition coefficient of some

99m
Tc(CO)3 dextran derivatives
The octanol water partition coefficient was determined at room

temperature for the following labelled dextran derivatives: [99mTc(CO)3 DC15],
[99mTc(CO)3 DCM20] and [99mTc(CO)3 DAPM1].
Experimental animal studies were approved by each respective local ethics

committee. Commercially approved 99mTc labelled colloids (rhenium sulphide,
sulphur colloids or calcium phytate) were used to standardize the protocol for
identifying the sentinel node and the secondary nodes in animals.
Biodistribution studies were performed in female (Wistar or Sprague

Dawley) rats. Some participating institutions were also able to perform
biodistribution studies in mice and in rabbits.
Rats weighing 200–250 g were used in experiments. Each animal was
first anaesthetized with a mixture of xylazine/ketamine (or acepromazine/
ketamine) administered in the peritoneal cavity. Then, approximately 50 μL of
141
the 99mTc labelled preparation (~1.8 MBq) was injected subcutaneously in the
foot-pad region (in some experiments, the intradermal route was used). This was
followed by gentle massage of the area of injection for up to 2 min with a strip
of gauze (animals were rejected if more than 1% of the administered activity was
recovered in the gauze). The animals (at least three per time point) were then kept
for the respective incubation periods (60 min and 180–240 min) under normal
conditions, with water provided ad libitum. A volume of 50 µL of 1% patent blue
solution was injected into the same site approximately 10 min before sacrifice
for visual detection of lymph nodes. The popliteal and inguinal lymph nodes,
injection site and tissue and organs of interest were removed and weighed
before radioactivity measurement. Results were expressed as a percentage of the
administered activity found in sampled tissues and organs. The popliteal lymph
node extraction (E) was calculated as:
%ID POPLITEAL −%ID INGUINAL

E(%) = ×100 (8.1)
%ID POPLITEAL
A similar protocol was followed for experiments in male BALB/c mice

(20 ± 2 g). In these animals, a smaller volume (5–25 µL) and a reduced activity
(90–180 kBq) were used.
In rabbits, intramuscular injection of 0.2 mL of ketamine was used as
anaesthesia. An activity of 111 MBq in 0.2 mL dextran derivative was injected
intradermally in both rear foot-pads. After injection, both foot-pads were
massaged for 3 min. At 50 min postinjection, 0.1 mL of a vital dye (patent blue
V) was injected intradermally in both foot-pads. Surgical dissection was started
10 min later to measure the uptake in popliteal and iliac nodes.
The influence of administered mass and volume on tracer localization was
also studied in rats and mice.
8.3.6.2. Gamma camera imaging
Anaesthetized animals (rats/rabbits) were horizontally placed under the

collimator of a gamma camera, equipped with a low energy, high resolution
collimator, using a 256 × 256 matrix size, zoom 2 (1 min/frame) for dynamic
studies and a 512 × 512 matrix size, zoom 2 for static studies. For optimal
image resolution, the distance between the collimator and animal was about
10 cm. SPECT/CT images were also obtained in rats and mice for some
selected compounds.
142
8.4. RESULTS
All dextran derivatives depicted in Fig. 8.1 were synthesized in high

yields (~90% yields per synthetic step). Detailed NMR experiments allowed for
the complete assignment of signals to their respective nuclei. Two dimensional
1
H–1H (correlation spectroscopy and nuclear Overhauser effect spectroscopy) and
1
H–13C (heteronuclear single quantum coherence spectroscopy and heteronuclear
multiple bond) correlation techniques were able to confirm the expected structure
as depicted in Fig. 8.1. Detailed information on the characterization of dextran
derivatives are reported in Chapter 5.
8.4.2. Physical characterization of dextran derivatives
The size distribution measurements revealed the formation of aggregates as

early as 30 min after preparation, increasing the size of particles from 7–10 nm
to 230–310 nm and even 700 nm. That was the case for all preparations. In the
case of DCM20, a rapid switch to 314 nm and a small number of aggregates of
sizes greater than 5000 nm (240 nm after sonication) were observed. In the case
of DAPM, the initial distribution showed particles and aggregates of size 11 nm
(close to the expected value, corresponding with analyses after synthesis), 84 nm
and 277 nm, which was switched in time (24 h) to 266 nm. Even aggregates of
sizes greater than 5000 nm were formed (but only with 1.3% intensity).
8.4.3. Radiochemical purity and stability of 99mTc dextran derivatives
8.4.3.1. Complexes [99mTc(CO)3 DC15/DCM20]
The mannosylated compound DCM20, as well as its non-mannosylated

derivative DC15, were successfully labelled with 99mTc(CO)3 core
(95% radiochemical yields), even at low ligand concentration (1.5 × 10−6M) using
the fac-[99mTc(OH2)3(CO)3]+ precursor. Figure 8.2 shows radioactive profiles
of DCM20 preparations, as well as the precursor fac-[99mTc(OH2)3(CO)3]+ and
pertechnetate obtained using RP-HPLC.
143
FIG. 8.2.  RP-HPLC profiles of 99mTcO4− (left), fac-[99mTc(OH2)3(CO)3]+(middle) and
99m
Tc(CO)3(DCM20) (right). cpm: counts per minute.
The [99mTc(CO)3 DC15] and [99mTc(CO)3 DCM20] complexes showed

high inertness towards histidine challenge (<3% of the total radioactivity was
transchelated after 6 h incubation).
8.4.3.2. Complexes [99mTc(CO)3 DAPM]
The mannosylated dextran pz diamine derivatives, DAPM1–4, were

successfully labelled with 99mTc(CO)3 core (radiochemical yield of 98% at the
time of labelling). However, up to 25% of the radioactivity bound to the DAPM
derivative bearing only one pz diamine group underwent transchelation after
histidine and cysteine challenges. No appreciable 99mTc activity was lost from
DAPM4 and DAPM8 complexes after 6 h of chemical challenge.
8.4.3.3. Complexes [99mTc (DC25-iso + NS3)] and [99mTc (DCM30-iso + NS3)]
The isocyanide derivatives of dextran and mannosylated dextran gave the

4 + 1 complex with the NS3 ligand with high yield (~90%).
Typical SEC-HPLC profiles of 99mTc [EDTA] and [99mTc (DCM30-iso + NS3)]
are shown in Fig. 8.3, clearly demonstrating that substitution was complete and
that 99mTc labelled dextran was obtained as the only product of the reaction.
The stability of the labelled dextran for at least 4 h was also established by
both previously described chromatographic methods.
8.4.4. Octanol water partition coefficients
The octanol water partition coefficients, obtained at room temperature, of

some selected 99mTc(CO)3 dextran derivatives are reported in Table 8.1.
144
FIG. 8.3.  SEC-HPLC profiles of 99mTc [EDTA] (top) and [99mTc (DCM30-iso + NS3)] (bottom).
TABLE 8.1.  OCTANOL WATER PARTITION COEFFICIENTS OF 99mTc(CO)3

DEXTRAN DERIVATIVES
Ligand Octanol water Log P
DC15 0.003 −2.495
DCM20 0.007 −2.133
DAPM1 0.060 −1.225
All products were highly hydrophilic, with values of log P (P = octanol/water

partition coefficient) in the negative range.
145
8.4.5. Biodistribution studies
The results of biodistribution studies in mice, rats and rabbits on

99m
Tc dextran derivatives synthesized for the purposes of this study are presented
in Table 8.2.
TABLE 8.2.  BIODISTRIBUTION OF 99mTc(CO)3 DEXTRAN DERIVATIVES

IN MICE AT 1 H AND 4 H AFTER INTRADERMAL ADMINISTRATION*
DC15 DCM20 DAPM4

Organ
2 µg/25 µL 2 µg/25 µL 2.3 µg/25 µL
Liver 1 h p.i. 13.41 ± 0.63 23.34 ± 3.50 2.97 ± 0.40
Liver 4 h p.i. 2.94 ± 0.36 21.60 ± 3.29 3.76 ± 0.43
Spleen 1 h p.i. 0.90 ± 0.05 13.82 ± 3.62 1.03 ± 0.11
Spleen 4 h p.i. 0.91 ± 0.21 15.41 ± 3.48 1.52 ± 0.53
Blood 1 h p.i. 1.29 ± 0.12 2.93 ± 0.44 0.87 ± 0.14
Blood 4 h p.i. 0.68 ± 0.11 1.27 ± 0.07 0.76 ± 0.06
IS 1 h p.i. 3.13 ± 0.92 30.68 ± 8.33 68.28 ± 11.00
IS 4 h p.i 1.77 ± 0.21 36.09 ± 3.09 67.30 ± 1.45
SLN 1 h p.i. 0.24 ± 0.02 3.18 ± 0.18 4.77 ± 1.01
SLN 4 h p.i. 0.13 ± 0.01 3.47 ± 1.02 4.12 ± 1.14
2LN 1 h p.i. 0.07 ± 0.01 2.16 ± 0.41 1.40 ± 0.49
2LN 4 h p.i. 0.08 ± 0.01 2.07 ± 0.47 1.19 ± 0.36
PE 1 h p.i. 70.8 32.1 70.6
PE 4 h p.i. 70.8 32.1 70.6
Note: IS: injection site; PE: popliteal extraction; p.i.: postinjection; SLN: sentinel lymph node;
2LN: second lymph node.

* Results are expressed as % ID per gram of organ and blood, except for the radioactivity
found in lymph nodes and remaining at the injection site, for which the recoveries are
expressed as % ID (mean ± standard deviation are calculated on five animals per time point).
146
8.4.5.1. Biodistribution in mice
Table 8.2 summarizes the results obtained 1 h and 4 h after intradermal
administration in the right foot-pad of BALB/c mice of 2 µg/50 µL of 99mTc(CO)3
dextran derivatives.
The SLN uptake of 99mTc[(CO)3 DCM20] (dextran with mannose pending
arms) was much higher than that of the corresponding dextran conjugate without
mannose ([99mTc(CO)3 DC15]). These findings support the hypothesis that
radioactivity localization in lymph nodes has occurred by a receptor mediated
mechanism to macrophages present in these organs.
8.4.5.2. Biodistribution in rats
Table 8.3 summarizes the results obtained 1 h and 3 h after

subcutaneous administration in the right foot-pad of Wistar rats of 99mTc(CO)3
99m
TABLE 8.3.  BIODISTRIBUTION OF Tc(CO)3 DEXTRAN
DERIVATIVES IN RATS AT 1 h AND 3 h AFTER SUBCUTANEOUS
ADMINISTRATION* (cont.)
DCM20 DAPM1 DAPM4 DAPM8

Organ
~4 µg/50 µL ~50 µg/50 µL 50 µg/50 µL 50 µg/50 µL
Liver 1 h p.i. 0.97 ± 0.05 1.01 ± 0.07 4.80 ± 1.03 5.44 ± 1.06
Liver 3 h p.i. 1.16 ± 0.12 1.66 ± 0.13 6.49 ± 0.09 6.84 ± 0.01
Spleen 1 h p.i. 0.03 ± 0.00 0.02 ± 0.01 0.32 ± 0.10 0.25 ± 0.11
Spleen 3 h p.i. 0.07 ± 0.04 0.09 ± 0.01 0.31 ± 0.04 0.45 ± 0.04
Blood 1 h p.i. 0.64 ± 0.13 0.02 ± 0.03 0.91 ± 0.02 1.72 ± 0.81
Blood 3 h p.i. 0.73 ± 0.48 0.10 ± 0.02 0.96 ± 0.37 1.55 ± 0.65
IS 1 h p.i. 43.53 ± 2.50 92.58 ± 0.94 83.85 ± 1.37 81.58 ± 0.35
IS 3 h p.i. 48.51 ± 5.41 79.48 ± 4.27 79.50 ± 5.02 81.13 ± 0.01
SLN 1 h p.i. 4.40 ± 0.01 4.91 ± 0.06 6.71 ± 2.35 7.53 ± 069
147
99m
TABLE 8.3.  BIODISTRIBUTION OF Tc(CO)3 DEXTRAN
DERIVATIVES IN RATS AT 1 h AND 3 h AFTER SUBCUTANEOUS
ADMINISTRATION* (cont.)
DCM20 DAPM1 DAPM4 DAPM8

Organ
~4 µg/50 µL ~50 µg/50 µL 50 µg/50 µL 50 µg/50 µL
SLN 3 h p.i. 0.02 ± 0.01 4.45 ± 0.86 5.98 ± 1.68 5.21 ± 0.78
2LN 1 h p.i. 0.05 ± 0.08 0.39 ± 0.08 2.59 ± 1.06 0.41 ± 0.15
2LN 3 h p.i. 0.08 ± 0.01 0.42 ± 0.06 1.41 ± 0.50 0.59 ± 0.14
PE 1 h p.i. 99.6 ± 0.2 92.6 ± 0.9 61.8 ± 2.4 94.5 ± 2.5
PE 3 h p.i. 98.0 ± 3.1 79.5 ± 4.3 76.7 ± 1.7 87.8 ± 3.8
Note: IS: injection site; PE: popliteal extraction; SLN: sentinel lymph node; 2LN: second
lymph node.
* Results are expressed as % ID per whole organ and for total blood (mean ± standard deviation
are calculated on four animals per time point).
Table 8.4 summarizes the results obtained 1 h and 3 h after subcutaneous
administration in the right foot-pad of Wistar rats of [99mTc (DCM30-iso + NS3)]
and [99mTc (DC25-iso + NS3)] derivatives.
The SLN uptake of [99mTc (DCM30-iso + NS3)] (dextran with mannose
pending arms) was significantly higher than that of the corresponding dextran
conjugate without mannose ([99mTc (DC25-iso + NS3)]). Again, these findings are
in favour of the hypothesis of radioactivity localization by a receptor mediated
mechanism to macrophages present in the lymph nodes.
8.4.5.3. Biodistribution in rabbits
A planar gamma camera study of rabbits showed that only popliteal nodes
were clearly imaged, whereas signals from iliac lymph nodes (second echelon
lymph nodes) were overlapped by radioactivity accumulated in the bladder.
Therefore, to obtain information about the biodistribution patterns of these new
99m
Tc dextran derivatives in rabbits, ex vivo experiments were carried out.
148
TABLE 8.4.  BIODISTRIBUTION OF [99mTc (DCM30-ISO + NS3)] AND
[99mTc (DC25-ISO + NS3)] IN RATS 1 h AND 3 h AFTER SUBCUATNEOUS
ADMINISTRATION*
DCM30-iso DC25-iso
Organ
~25 µg/50 µL ~25 µg/50 µL
Liver 1 h p.i. 5.17 ± 0.30 10.80 ± 0.30
Liver 3 h p.i. 11.26 ± 0.76 NP
Spleen 1 h p.i. 0.08 ± 0.02 0.02 ± 0.01
Spleen 3 h p.i. 0.05 ± 0.25 NP
Blood 1 h p.i. 2.26 ± 0.67 0.08 ± 0.01
Blood 3 h p.i. 8.51 ± 0.71 NP
IS 1 h p.i. 79.80 ± 3.00 15.6 ± 1.2
IS 3 h p.i. 45.70 ± 3.10 NP
SLN 1 h p.i. 3.55 ± 1.40 0.20 ± 0.07
SLN 3 h p.i. 5.42 ± 0.88 NP
2LN 1 h p.i. 1.42 ± 0.99 0.10 ± 0.04
2LN 3 h p.i. 0.79 ± 0.54 NP
PE 1 h p.i. 60.0 ± 1.3 50.0 ± 0.1
PE 3 h p.i. 85.4 ± 0.7 NP
Note: IS: injection site; NP: not performed; PE: popliteal extraction; p.i.: postinjection;
SLN: sentinel lymph node; 2LN: second lymph node.
* Results are expressed as % ID per whole organ and total blood (mean ± standard deviation
are calculated on three animals per time point).
Table 8.5 summarizes the results obtained 1 h after subcutaneous

administration in the right foot-pad of rabbits of some 99mTc(CO)3
149
TABLE 8.5.  BIODISTRIBUTION OF SOME 99mTc(CO)3 DEXTRAN
DERIVATIVES IN RABBITS AT 1 h AFTER SUBCUTANEOUS
ADMINISTRATION*
DCM20 DAPM4 DAPM8

Organ
~60 µg/0.2 mL ~60 µg/0.2 mL ~60 µg/0.2 mL
Liver 1 h p.i. 0.75 ± 0.01 2.11 ± 0.03 1.50 ± 0.12
Spleen 1 h p.i. 0.44 ± 0.14 0.49 ± 0.14 0.40 ± 0.14
Blood 1 h p.i. 0.45 ± 0.01 0.19 ± 0.01 0.48 ± 0.01
IS 1 h p.i. 36.44 ± 2.12 42.88 ± 3.99 41.98 ± 2.79
SLN 1 h p.i. 4.11 ± 1.90 5.56 ± 1.55 6.79 ± 1.89
2LN 1 h p.i. 0.27 ± 0.06 0.78 ± 0.03 0.34± 0.07
PE 1 h p.i. 91.9 ± 0.1 85.8 ± 2.2 94.8 ± 1.76
lymph node.
* Results are expressed as % ID per gram, except for the radioactivity found in lymph nodes
and at the injection site, for which the recoveries are expressed as % ID (mean ± standard
deviation are calculated on three animals per time point).
8.4.5.4. Effect of the mass of administered tracer on biodistribution
To investigate whether uptake of mannosylated dextran derivatives is a

saturable process, the influence of the mass of administered tracer on SLN uptake
was explored. Experiments were performed in mice and rats. Table 8.6 shows the
results of a study in which the effect of the amount of [99mTc(CO)3 DCM20] on
biodistribution was tested.
The results on [99mTc(CO)3 DCM20] showed that the biological behaviour
of this complex is very sensitive to the injected dosage. When the injected mass
in mice decreased from 2 µg to 0.005 µg, the uptake of SLN doubled, and the
uptake in the second lymph node decreased by a factor of four (but it should be
150
TABLE 8.6.  BIODISTRIBUTION OF [99mTc(CO)3 DCM20] IN MICE AND
RATS AT 1 h AFTER INTRADERMAL ADMINISTRATION*
2 µg/25 µL 0.005 µg/5 µL 0.05 µg/30 µL 0.05 µg/5 µL

Organ
mice mice rats rats
Blood (whole) 2.93 ± 0.44 1.09 ± 0.13 3.98 ± 0.11 1.07 ± 0.14
IS 30.68 ± 8.33 76.65 ± 4.14 55.02 ± 2.16 49.10 ± 2.64
SLN 3.18 ± 0.18 6.37 ± 0.86 17.76 ± 1.89 32.10 ± 8.21
2LN 2.16 ± 0.41 0.54 ± 0.09 0.70 ± 0.19 0.74 ± 0.13
PE 32.1 ± 0.2 91.5 ± 0.8 96.1 ± 1.9 97.7 ± 8.1
lymph node.
* Results are expressed as % ID (mean ± standard deviation are calculated on four to five
animals per time point).
noted that the injected volume also decreased from 25 µL to 5 µL). This induced
a sharp increase of popliteal extraction from 30%–40% to 90%. In this study, the
SLN uptake in rats was very high, and the activity remaining at the injection site
1 h after the application of the tracer was relatively low.
8.4.6. Gamma camera imaging
Examples of dynamic acquisitions in rats after subcutaneous administration

of 9–18 MBq of 99mTc dextran derivatives in 0.1 mL are shown in Figs 8.4
and 8.5. The images of the earliest acquisitions (0–1 min) are in the upper left
corners, whereas the latest acquisitions (14–15 min) are in the lower right corners
of Figs 8.4 and 8.5.
Figure 8.5 shows anterior views of the animals positioned upside down. The
injection site (hottest spot), SLN, bladder, kidneys and liver are easily discernible
on the images.
The radioactivity pattern of 99mTc dextran derivatives under study was also
clearly delineated after static acquisition, as shown in Fig. 8.6 for three different
compounds administered to rats.
151
FIG. 8.4.  Dynamic acquisitions (0–15 min) after intradermal administration of
[99mTc(CO)3 DCM20].
FIG. 8.5.  Dynamic acquisitions (0–15 min) after intradermal administration of

[99mTc(CO)3 DAPM8].
152
FIG. 8.6.  Static acquisitions in rats 30 min postinjection of 0.5 µg of [99mTc(CO)3 DCM20]
(left), [99mTc(CO)3 DAPM8] (middle) and [99mTc (DCM30-iso + NS3)] (right).
FIG. 8.7.  SPECT (left) and SPECT/CT fusion (right) images of rats receiving [99mTc(CO)3
DCM20] from foot-pads subcutaneously. The images were taken at: (A) 30 min, (B) 1 h (B)
and (C) 3 h postinjection.
The ability of these tracers to visualize SLNs was also demonstrated by

SPECT images. Figure 8.7 shows the images obtained in rats after subcutaneous
administration of [99mTc(CO)3 DCM20] in the rear foot-pad. The radioactivity
levels at the popliteal lymph node were found to be significantly higher
than those in the inguinal lymph node (the second lymph node in this animal
153
mode), although high radioactivity levels were also detected at the injection
site. The SPECT images remained almost unchanged from 15 min to 180 min
postinjection, indicating that this agent satisfied another important criterion:
sustained accumulation in the SLN.
8.5. DISCUSSION
The dextran derivatives under study have been successfully synthesized

and thoroughly characterized using chemical and NMR techniques. The synthetic
pathways used are robust and flexible enough to allow the preparation of
derivatives in which the number of grafted chelator and mannosylated arms can
be adjusted predictably and accurately. The chemical structure of the synthesized
compounds is in agreement with the hydrodynamic diameter determined by DLS
in saline. In freshly prepared samples, DCM and DAPM show hydrodynamic
sizes ranging from 7 nm to 10 nm. These values are similar to those found for
DTPA mannosyl dextran (7.1 ± 0.9 nm) [8.14] and MAG3 mannosyl dextran
(5.5 ± 2.4 nm) [8.30], and have been confirmed in two recently published articles
dealing with DCM [8.27] and DAPM [8.28]. In this work, size distribution
measurements revealed the formation of aggregates as early as 30 min after
preparation, increasing the sizes of particles from 7–10 nm to 230–310 nm and
even 700 nm. These findings, not reported in the two articles cited above, need to
be confirmed by additional experiments.
The mannosylated dextran derivatives, as well as their non-mannosylated
analogues, were successfully labelled with a 99mTc carbonyl core and 99mTc(NS3)
fragments in high radiochemical yields. Of particular interest are dextran
derivatives bearing cysteine arms (providing the S, N, O set of ligating atoms
to bind to a 99mTc carbonyl core) that may be labelled, even at a very low ligand
concentration (1.5 × 10−6M) [8.27]. Mannosylated dextran bearing cysteine arms
are stable compounds that can be stored in the form of lyophilized powder for
long periods of time. In contrast, owing to the propensity of isocyanide groups
to polymerize, mannosylated dextran bearing isocyanide arms are less stable
compounds and should be prepared shortly before the radiolabelling process.
The 99mTc labelled dextran derivatives are inert complexes, as demonstrated by
the results obtained after challenging tests with cysteine and histidine, and may
undergo high dilutions without significant degradation.
Biological evaluation in mice, rats and rabbits has shown rapid and high
accumulation in the popliteal lymph node (the SLN in our animal model) that
remained almost stable for long periods of time. Uptake characteristics are
similar to those displayed by 99mTc tilmanocept [8.31]. The results of this work
have clearly confirmed that, as for 99mTc tilmanocept, uptake in lymph nodes is
154
the result of a receptor mediated mechanism. Indeed, the uptake by lymph nodes
of mannosylated dextran derivatives, either labelled with a 99mTc carbonyl core or
with a Tc(NS3) fragment, was much higher than that of the corresponding dextran
derivative without mannose. Additionally, experiments in mice and rats were
able to demonstrate that the biological behaviour of 99mTc mannosylated dextran
complexes was very sensitive to injected mass. It is likely that this characteristic
has contributed to induce some degree of heterogeneity that has been observed
between results obtained in different laboratories.
Results from biodistribution can be used to classify the tracers under study.
There may be different criteria for establishing a rank order of imaging tracers on
the basis of their best biological properties. For SLNM agents, it was decided to
assign a rank on the basis of qualitative scores driven by the assessment of the
following properties:
—— Extent of uptake in the SLN (at 30 min postinjection);

—— First to second lymph node ratio;
—— Residence of radioactivity in the first lymph node;
—— Activity remaining at the injection site;
—— Activity in organs (liver/kidneys) and blood.
The activity remaining at the site of injection was considered to have less
impact on the overall quality of the tracer than the ‘specificity’ for the first lymph
node. Thus, this characteristic was not used as a primary selection criterion
(activity remaining at the site of injection is of little or no concern for SLND
in melanoma, and is not a big issue in the case of breast tumours). Potential for
kit pharmaceutical development was not included in the ranking score. Because
the biological results obtained during this multilaboratory project were obtained
using different animal models and following slightly different protocols,
experimental data could not be simply mixed together to increase the statistical
power of the decision tree process. However, although with several limitations,
the biodistribution results allowed selection of [99mTc(CO)3 DCM20] and
[99mTc(CO)3 DAPM8] as the most promising agents for potential use in humans.
8.6. CONCLUSIONS
Results in mice, rats and rabbits showed that low valence 99mTc mannosylated
dextran derivatives are rapidly localized in SLNs following a saturable receptor
mediate mechanism. These radioconjugates, in particular [99mTc(CO)3 DCM20]
and [99mTc(CO)3 DAPM8], retained a high binding capacity to SLNs. Imaging
in rats and rabbits revealed a rapid uptake into SLNs. In addition to the uptake
155
in SLNs, the clearance from the injection site was also found to be inversely
related to the mass of the administered compound. Further evaluation in a well
controlled protocol and in a large number of animals will be needed to explore
the real potential of these compounds as SLNM agents in humans.
ACKNOWLEDGEMENTS
The contributions of all research laboratories who actively conducted

biological characterization of the novel class of dextran based 99mTc imaging
agents for SLND during the course of the CRP on Development of 99mTc
Radiopharmaceuticals for Sentinel Node Detection and Cancer Diagnosis are
gratefully acknowledged. Names and institutions are listed in the contributors to
drafting and review.
[8.1] CABANAS, R.M., An approach for the treatment of penile carcinoma, Cancer 39
(1977) 456–466.
Semin. Nucl. Med. 27 (1997) 55–67.
cancer with clinically negative lymph-nodes, Lancet 349 (1997) 1864–1867.
[8.4] KESHTGAR, M.R.S., ELL, P.J., Sentinel lymph node detection and imaging, Eur. J.
Nucl. Med. 26 (1999) 57–67.
J. Nucl. Med. 42 (2001) 1198–1215.
[8.7] GIAMMARILE, F., et al., The EANM and SNMMI practice guideline for
lymphoscintigraphy and sentinel node localization in breast cancer, Eur. J. Nucl. Med.
Mol. Imaging. 40 (2013) 1932–1947.
[8.8] ALAZRAKI, N., GLASS, E.C., CASTRONOVO, F., OLMOS, R.A., PODOLOFF, D.,
Society of Nuclear Medicine Procedure guideline for lymphoscintigraphy and the use
of intraoperative gamma probe for sentinel lymph node localization in melanoma of
intermediate thickness 1.0, J. Nucl. Med. 43 (2002) 1414–1418.
[8.9] WONG, S.L., et al., Sentinel lymph node biopsy for melanoma: American Society of
Clinical Oncology and Society of Surgical Oncology joint clinical practice guideline,
J. Clin. Oncol. 30 (2012) 2912–2918.
156
[8.10] ALKUREISHI, L.W., et al., Joint practice guidelines for radionuclide
lymphoscintigraphy for sentinel node localization in oral/oropharyngeal squamous cell
carcinoma, Eur. J. Nucl. Med. Mol. Imaging 36 (2009) 1915–1936.
[8.11] WILHELM, A.J., MIJNHOUT, G.S., FRANSSEN, E.J., Radiopharmaceuticals in
sentinel lymph-node detection: an overview, Eur. J. Nucl. Med. 26 (1999) S36–S42.
trisulfide and rhenium sulfide colloids intended for lymphoscintigraphic application,
J. Nucl. Med. 42 (2001) 460–466.
[8.13] ALAZRAKI, N.P., et al., Sentinel node staging of early breast cancer using
lymphoscintigraphy and the intraoperative gamma-detecting probe, Semin. Nucl. Med.
30 (2000) 56–64.
macromolecule for sentinel node detection: (99m)Tc-DTPA-mannosyl-dextran, J. Nucl.
Med. 42 (2001) 951–959.
[8.16] ALBERTO, R., SCHLIBI, R., EGLI, A., SCHUBIGER, P.A., A novel organometallic
aqua complex of technetium for the labeling of biomolecules: synthesis of
[99mTc(H2O)3(CO)3]+ from [99mTcO4]− in aqueous solution and its reaction with a
bifunctional ligand, J. Am. Chem. Soc. 120 (1998) 7987–7988.
[8.17] ALBERTO, R., ORTNER, K., WHEATLEY, N., SCHIBLI, R., SCHUBIGER, A.,
Synthesis and properties of boranocarbonate: a convenient in situ CO source for
aqueous preparation of fac-[99mTc(H2O)3(CO)3]+, J. Am. Chem. Soc. 123 (2001)
3135–3136.
[8.18] VAN STAVEREN, D.R., et al., Functionalized cysteine: powerful ligands for
the labelling of bioactive molecules with triaquatricarbonyltechnetium-99m(1+)
([99mTc(OH2)3(CO)3]+), Helv. Chim. Acta 88 (2005) 447–460.
[8.19] KARAGIORGOU, O., et al., (S)-(2-(2'-pyridyl)ethyl)cysteamine and
(S)(2-(2'-pyridyl)ethyl)-d,l-homocysteine as ligands for the “fac-[M(CO)3]+'
(M = Re, 99mTc) core, Inorg. Chem. 44 (2005) 4118–4120.
[8.20] HE, H., et al., Re(CO)3 complexes synthesized via an improved preparation of
aqueous fac-[Re(CO)3(H2O)3]+ as an aid in assessing 99mTc imaging agents. Structural
characterization and solution behavior of complexes with thioether-bearing amino
acids as tridentate ligands, Inorg. Chem. 44 (2005) 5437–5446.
[8.21] XAVIER, C., PAK, J.-K., SANTOS, I., ALBERTO, R., Evaluation of two chelators for
labeling a PNA monomer with the fac-[99mTc(CO)3]+ moiety, J. Organomet. Chem. 692
(2007) 1332–1339.
[8.22] ALVES, S., et al., Pyrazolyl derivatives as bifunctional chelators for labeling
tumor-seeking peptides with the fac-[M(CO)3]+ moiety (M) = 99mTc, Re): synthesis,
characterization, and biological behaviour, Bioconjug. Chem. 16 (2005) 438–449.
[8.23] CORREIA, J.D.G., PAULO, A., SANTOS, I., Re and Tc complexes with pyrazolyl-
containing chelators: from coordination chemistry to target specific delivery of
radioactivity, Curr. Radiopharm. 2 (2009) 277–294.
157
[8.24] SPIES, H., GLASER, M., PIETZSCH, H.-J., HAHN, F.E., LÜGGER, T., Synthesis and
reactions of trigonal-bipyramidal rhenium and technetium complexes with tripodal,
tetradentate NS3 ligand, Inorg. Chim. Acta 240 (1995) 465–478.
[8.25] PIETZSCH, H.-J., GUPTA, A., SYHRE, R., LEIBNITZ, P., SPIES, H., Mixed-ligand
technetium(III) complexes with tetradentate/monodentate NS3/isocyanide coordination:
a new nonpolar technetium chelate system for the design of neutral and lipophilic
complexes stable in vivo, Bioconjug. Chem. 12 (2001) 538–544.
[8.26] SEIFERT, S., et al., Novel procedures for preparing 99mTc(III) complexes with
tetradentate/monodentate coordination of varying lipophilicity and adaptation to 188Re
analogues, Bioconjug. Chem. 15 (2004) 856–863.
[8.27] KÜNSTLER, J.-U., et al., Impact of functionalized coligands on the pharmacokinetics
of 99mTc(III) ‘4 + 1’ mixed-ligand complexes conjugated to bombesin, J. Inorg.
Biochem. 105 (2011) 1383–1390.
[8.28] PIRMETTIS, I., et al., (99m)Tc(CO)3 New mannosylated dextran bearing S-derivatized
[8.29] MORAIS, M., et al., Mannosylated dextran derivatives labeled with
fac-[M(CO)3]+ (M = (99m)Tc, Re) for specific targeting of sentinel lymph node, Mol.
Pharm. 8 (2011) 609–620.
[8.30] GIGLIO, J., et al., Design and development of (99m)Tc-‘4 + 1’-labeled dextran-mannose
derivatives as potential radiopharmaceuticals for sentinel lymph node detection, Cancer
Biother. Radiopharm. 28 (2013) 541–551.
[8.31] VERA, D.R., WALLACE, A.M., HOH, C.K., [99mTc]MAG3-mannosyl-dextran: a
receptor-binding radiopharmaceutical for sentinel node detection, Nucl. Med. Biol. 28
(2001) 493–498.
158
CONTRIBUTORS TO DRAFTING AND REVIEW
Arano, Y. Chiba University, Japan
Arteaga de Murphy, C. Salvador Zubirán National Institute of

Medical Sciences and Nutrition, Mexico
Boschi, A. University of Ferrara, Italy
Chen, B.J. China Institute of Atomic Energy, China
Colombo, L. A. Roffo Institute of Oncology, Argentina
Correia, J.D.G. Universidade Técnica de Lisboa, Portugal
Dar, U-e-K. Institute of Nuclear Medicine and Oncology, Pakistan
De León Rodriguez, L.M. University of Guanajuato, Mexico
De Oliveira, E.A. Nuclear and Energy Research Institute,

National Nuclear Energy Commission, Brazil
De Oliveira Filho, R.S. Federal University, Brazil
D’Orio, E.G. A. Roffo Institute of Oncology, Argentina
Duatti, A. University of Ferrara, Italy, and International Atomic

Energy Agency
Erzsébet, S. Institute of Isotopes, Hungarian Academy of Sciences,

Hungary
Faintuch, B.L. Nuclear and Energy Research Institute, National

Nuclear Energy Commission, Brazil
Fernández, S. Universidad de la República, Uruguay
Ferro-Flores, G. National Nuclear Research Institute, Mexico
Giglio, J. Universidad de la República, Uruguay
Gomez, S.I. Tecnonuclear S.A., Argentina
Guerrini, R. University of Ferrara, Italy
Hall, D.J. UCSD Moores Cancer Center, University of

California, San Diego, United States of America
159
Hernandez, M. Tecnonuclear S.A., Argentina
Hoh, C.K. UCSD Moores Cancer Center, University of

Horváth, V. Institute of Isotopes, Hungarian Academy of Sciences,

Hungary
Hyder, S.W. Institute of Nuclear Medicine & Oncology, Pakistan
Janevik-Ivanovska, E. Goce Delcev University, Stip,

The Former Yugoslav Republic of Macedonia
Javed, M. Institute of Nuclear Medicine and Oncology, Pakistan
Jentschel, C. Forschungszentrum Dresden-Rossendorf, Germany
Khan, I.U. Institute of Nuclear Medicine and Oncology, Pakistan
Környei, J. Institute of Isotopes, Hungarian Academy of Sciences,

Hungary
Li, H.Y. China Institute of Atomic Energy, China
Liang, J.X. China Institute of Atomic Energy, China
Lu, J. China Institute of Atomic Energy, China
Luo, H.Y. China Institute of Atomic Energy, China
Luo, Z.F. China Institute of Atomic Energy, China
Mallia, M. Bhabha Atomic Research Centre, India
Mariani, G. Regional Center of Nuclear Medicine,

University of Pisa Medical School, Italy
Medina, L.A. UNAM e Instituto Nacional de Cancerología, Mexico
Morais, M. Universidade Técnica de Lisboa, Portugal
Morales-Avila, E. National Nuclear Research Institute, Mexico
Mustaciosu, C. Horia Hulubei National Institute for Physics and

Nuclear Engineering, Romania
Niculae, D. Horia Hulubei National Institute for Physics and

160
Nunez, E.G.F. Nuclear and Energy Research Institute,
Ocampo-García, B.E. National Nuclear Research Institute, Mexico
Orsini, F. Regional Center of Nuclear Medicine,

University of Pisa Medical School, Italy
Pandey, U. Bhabha Atomic Research Centre, India
Paolino, A. Universidad de la República, Uruguay
Papadopoulos, M. Demokritos National Center for Scientific Research,

Greece
Pasquali, M. University of Ferrara, Italy
Pasqualini, R. CIS bio international/IBA, Saclay, RP Innovative,

France
Patrascu, I. Horia Hulubei National Institute for Physics and

Pedraza-Lopez, M. Salvador Zubirán National Institute of

Medical Sciences and Nutrition, Mexico
Pelecanou, M. Demokritos National Center for Scientific Research,

Greece
Pietzsch, H.-J. Forschungszentrum Dresden-Rossendorf, Germany
Pirmettis, I. Demokritos National Center for Scientific Research,

Greece
Ramirez, F.M. National Nuclear Research Institute, Mexico
Rey, A. Universidad de la República, Uruguay
Salvadori, S. University of Ferrara, Italy
Samuel, G. Bhabha Atomic Research Centre, India
Santos, I. Universidade Técnica de Lisboa, Portugal
Santo-Cuevas, C.L. National Nuclear Research Institute, Mexico
Silva, N.G. Nuclear and Energy Research Institute,

161
Subramanian, S. Bhabha Atomic Research Centre, India
Tokin, C.A. UCSD Moores Cancer Center, University of

Tóth, G. Biological Center of the Hungarian Academy of

Sciences, Hungary
Trapella, C. University of Ferrara, Italy
Tsotakos, T. Demokritos National Center for Scientific Research,

Greece
Tuta, C. Horia Hulubei National Institute for Physics and

Uccelli, L. University of Ferrara, Italy
Venkatesh, M. Bhabha Atomic Research Centre, India
Vera, D.R. UCSD Moores Cancer Center, University of

Wallace, A.M. UCSD Moores Cancer Center, University of

Yang, C.H. China Institute of Atomic Energy, China
Zheng, D.Q. China Institute of Atomic Energy, China
Technical Meetings
Vienna, Austria: 2–7 September 2012
Research Coordination Meetings
Vienna, Austria: 12–16 November 2007

Athens, Greece: 18–22 May 2009
Vienna, Austria: 22–26 November 2010
162
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This publication summarizes the current status of and future
trends in the development of radiopharmaceuticals for sentinel
lymph node detection (SLND), a widely recognized, essential
diagnostic tool for the effective treatment of superficial
cancers such as breast tumours and melanoma. The first part
of the publication covers all current aspects of this diagnostic
methodology, including the production of nanocolloidal
particles, their biological mechanism of action and relevant
clinical applications. In the second part, recent outstanding
developments in the field fuelled by the introduction of molecular
imaging agents for SLND and of multimodality optical and
radioactive agents are discussed. A portion of the results on the
novel generation of SLND radiopharmaceuticals presented here
were obtained through an IAEA co-ordinated research project.
INTERNATIONAL ATOMIC ENERGY AGENCY

VIENNA
ISBN 978–92–0–109714–9
ISSN 2077–6462

Radiopharmaceuticals For Sentinel Lymph Node Detection - Status and Trends

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IAEA RADIOISOTOPES AND RADIOPHARMACEUTICALS SERIES No.

IAEA RADIOISOTOPES AND RADIOPHARMACEUTICALS SERIES

IAEA RADIOISOTOPES AND RADIOPHARMACEUTICALS REPORTS

Further information is available from:

Marketing and Sales Unit

AFGHANISTAN GHANA OMAN

INTERNATIONAL ATOMIC ENERGY AGENCY

Marketing and Sales Unit, Publishing Section

Printed by the IAEA in Austria

IAEA Library Cataloguing in Publication Data

In a continuous effort to support and promote scientific research in cancer

CHAPTER 2. BASIC DESCRIPTION OF

CHAPTER 4. TECHNETIUM-99m TILMANOCEPT:

CHAPTER 5. A NEW CLASS OF 99mTc(I) AGENTS FOR SLND:

CHAPTER 6. A NEW CLASS OF 99mTc(I) AGENTS FOR SLND:

6.1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

CHAPTER 7. 4 + 1 Tc(III) COMPLEXES IN THE DESIGN AND

CHAPTER 8. BIOLOGICAL EVALUATION OF LOW VALENCE

8.1. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

CONTRIBUTORS TO DRAFTING AND REVIEW . . . . . . . . . . . . . . . . . . 159

The scientific objectives of the CRP can be outlined as follows:

—— Design and synthesis of dextran multifunctional ligands with pendant

In Chapter 2, an overview of the biology and physiology of the lymphatic

BASIC DESCRIPTION OF THE LYMPHATIC SYSTEM

The lymphatic system is part of the circulatory system. It is closely

The purpose of this chapter is to provide readers with a basic description

2.3. THE LYMPHATIC SYSTEM: A GENERAL VIEW

The lymphatic system is closely associated with the cardiovascular system.

2.4. STRUCTURE OF THE LYMPHATIC NETWORK

The lymphatic network begins as lymphatic capillaries merge to form

2.4.1. Lymphatic capillaries

2.4.2. Lymphatic vessels

Lymphatic vessels form as lymph capillaries merge. The walls of lymphatic

2.4.3. Lymphatic trunks and collecting ducts

2.5. LYMPH PRODUCTION AND MOVEMENT

2.5.1. Role of the interstitial space

—— An interstitial fluid containing salts and plasma proteins;

Collagen fibres provide much of the structural framework of the tissue.

2.5.2. Formation of lymph

Tissue fluid is composed of water and dissolved substances that migrate

Lymph nodes are located along the lymphatic pathways, particularly

2.6.1. Structure of a lymph node

FIG. 2.4. Section of a lymph node.

Lymph nodes have two primary functions:

2.6.2.1. Filtering function of lymph nodes

2.6.2.2. Receptor based uptake of foreign matters

In the lymph node, phagocytosis is the main process of macrophages.

—— The C type lectin-like domain binds glycoproteins with exposed mannose,

In recent years, macrophage specific delivery systems have gained

2.7.1. The SLN concept

2.7.2. Physicochemical aspects of lymph node delivery of

Basically, to be taken up by the SLN, large molecules or colloids

Size is the major factor determining the behaviour of particulate materials

Colloid composition Size range Comments References

Human serum albumin 7–23a Registered in different countries [2.59, 2.60]

Human serum albumin 100–600b Registered in different countries Refer to

Stannous/stannic hydroxide 30–200a Approved in some European [2.60]

Rhenium sulphide 8–68a Registered in different countries [2.61]

Sulphur colloid 50–1000c Registered in USA [2.50, 2.61]

FIG. 2.4.  Section of a lymph node.

FIG. 4.2.  Production of tilmanocept starts with the attachment of aminoterminated leashes,