0021-972X/98/$03.00/0
Journal of Clinical Endocrinology and Metabolism
Copyright © 1998 by The Endocrine Society
Vol. 83, No. 12
Printed in U.S.A.
Mutation of the RET Proto-Oncogene Is Correlated with
RET Immunostaining in Subpopulations of Cells in
Sporadic Medullary Thyroid Carcinoma
CHARIS ENG*, GERRY A. THOMAS, DONNA S. NEUBERG, LOIS M. MULLIGAN,
CATHERINE S. HEALEY, CAROL HOUGHTON, ANDREA FRILLING,
FRIEDHELM RAUE, E. DILLWYN WILLIAMS, AND BRUCE A. J. PONDER†
Cancer Research Campaign Human Cancer Genetics Research Group (C.E., C.S.H., C.H., B.A.J.P.)
and Department of Histopathology (G.A.T., E.D.W.), University of Cambridge, Cambridge CB2 2QQ,
United Kingdom; Department of Adult Oncology, Charles A. Dana Human Cancer Genetics Unit
(C.E.), and Department of Biostatistical Science (D.S.N.), Dana-Farber Cancer Institute, Harvard
Medical School (C.E.) and Harvard School of Public Health (D.S.N.), Boston, Massachusetts 021156084; Human Cancer Genetics Program, Comprehensive Cancer Center, Ohio State University, 690
Medical Research Facility, Columbus, Ohio 43210 (C.E.); Departments of Pathology and Paediatrics,
Queen’s University, Kingston, ON K7L 3N6, Canada (L.M.M.); Universitäts-Krankenhaus,
Chirurgische Klinik, Universität Hamburg, 2000 Hamburg 20, Germany (A.F.); and Medizinische
Klinik und Poliklinik, Abteilung Innere Medizin I, Ruprecht-Karls-Universität Heidelberg, 69118
Heidelberg, Germany (F.R.)
ABSTRACT
Mutations in the RET proto-oncogene, which encodes a receptor
tyrosine kinase, are associated with the pathogenesis of medullary
thyroid carcinoma (MTC). Somatic mutations in RET, predominantly
at codon 918, and very rarely at codon 883, have been found in a
proportion of sporadic MTC. We have previously shown that approximately 80% of sporadic MTCs had at least one subpopulation with a
somatic RET mutation. Uneven distribution of somatic mutation
within a single tumor or among metastases from a single individual
was notable. In the present study, we sought to correlate RET expression, as demonstrated by RET immunohistochemistry, with mutation status in sporadic MTC for each tumor. Seventy evaluable
subpopulations, belonging to 28 unrelated sporadic cases, comprising
M
EDULLARY thyroid carcinoma (MTC), a neoplasm of
the calcitonin-secreting thyroid C cells, may occur
sporadically or as a component of the inherited cancer syndrome multiple endocrine neoplasia (MEN) type 2 (1). All
three clinical subtypes of the MEN 2 syndromes are caused
by germline mutations in the RET proto-oncogene (2– 4),
which encodes a receptor tyrosine kinase expressed in tissues
and tumors of neural crest origin (5–9). MEN 2A, which
comprises MTC, pheochromocytoma, and parathyroid hyperplasia, is associated with germline missense mutation in
one of six cysteine codons in the cysteine-rich extracellular
domain of RET (3, 10 –12). Familial MTC (FMTC), characterized by the presence of MTC as the only phenotype in the
Received July 16, 1998. Revised August 13, 1998. Accepted August 17,
1998.
Address all correspondence and requests for reprints to: Charis Eng,
Human Cancer Genetics Program, Ohio State University Comprehensive Cancer Center, 690C Medical Research Facility, 420 West 12th Avenue, Columbus, Ohio 43210. E-mail: eng-1@medctr.osu.edu.
* The Lawrence and Susan Marx Investigator in Human Cancer Genetics and a Barr Investigator.
† A Gibb Fellow of the Cancer Research Campaign.
primary MTC and metastases, were immunostained with two different polyclonal antibodies raised against the C-terminus of RET. The
regional presence of codon 918 or 883 seemed to coincide with increased RET immunopositivity in at least 62 of 70 (89%, P , 0.000001)
tumor subpopulations. The reasons for this concordance are not entirely clear but could be related to either RNA or protein stability.
Preliminary studies have suggested that the presence of somatic
codon 918 mutation in MTC has a prognostic significance. If these
preliminary results prove true, then given our data, we can further
explore the feasibility of RET immunocytochemistry as a rapid assessment for the presence of somatic codon 918 for molecular diagnostic and prognostic purposes. (J Clin Endocrinol Metab 83: 4310 –
4313, 1998)
family, is associated with mutations similar to those in MEN
2A and, rarely, with a missense mutation in codon 768 or 804
in the intracellular tyrosine kinase domain (3, 13). MEN 2B
is similar to MEN 2A except for earlier tumor onset, the
presence of developmental abnormalities, and a typical habitus. Clinical hyperparathyroidism is absent, however. MEN
2B is caused by germline mutation in codon 918, M918T, in
more than 95% of cases; and germline mutation, in codon 883,
A883F, in less than 4% (3, 14, 15).
Somatic mutation of RET in the MEN 2B-specific codon 918
occurs in 23– 86% of sporadically occurring MTC (4). Somatic
mutations elsewhere, such as at codon 883, are rare (4). We
have previously shown that in many sporadic MTC, the
presence of somatic M918T and A883F is regionally inhomogenous, occurring in some subpopulations within a single
MTC or occurring in a subset of multiple metastases (16).
These results may be consistent either with clonal evolution
within a tumor or the polyclonal origin of MTC (16), as
suggested by clonality studies using X-linked markers (17).
For the present study, we hypothesized that cells containing a RET mutation would immunostain positively when
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RET MUTATION, IMMUNOSTAINING, AND SPORADIC MTC
exposed to antibodies raised against RET. Hence, uneven
distribution of RET mutation in cell subpopulations in MTC
should be reflected in regional immunostaining patterns, as
well. We, therefore, used immunohistochemical techniques
to examine the relationship between RET expression and
RET mutation status in MTC metastases and subpopulations
within individual tumors from sporadic cases.
Materials and Methods
MTC tumors
A total of 82 MTC populations from 28 sporadic cases were analyzed.
All were obtained as formalin-fixed, paraffin-embedded tissue.
MTC was considered sporadic if the patient did not have multiple
primary tumors and there was no history of a first- or second-degree
relative with MTC or pheochromocytoma.
Mutation analysis
Somatic mutation status in these MTC and their subpopulations have
been reported previously (16).
FIG. 1. RET immunocytochemistry of a
sporadic MTC. A, 103. M918T-positive
and M918T-negative regions are clearly
demarcated and correlate with RET immunostain positive regions. B, 403. Detail of immunostain-positive subpopulation. Individual cells demonstrate
variable immunoreactivity. In general,
immunostaining was cytoplastic, but in
individual cells, submembrane immunopositivity could be detected.
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Immunohistochemistry
Tissue samples were obtained in standard surgical fashion. Samples
were usually obtained from the thyroid (normal tissue or MTC) or as
MTC metastases within lymph nodes. Samples were fixed in 10% buffered formalin, embedded in paraffin, and processed by conventional
hematoxylin and eosin staining method.
Five-micrometer sections were cut from formalin-fixed, paraffin-embedded blocks. After dewaxing and inhibition of endogenous peroxidase, sections were immersed in sodium citrate buffer (pH 6.0) and
pretreated using hydrated autoclaving (in a domestic pressure cooker)
for 2 min (18). Serial sections from each block were incubated overnight
with two polyclonal RET antibodies, G63, a polyclonal rabbit antihuman
TABLE 1. RET mutation status and RET immunostaining in 70
primary sporadic MTC and distinct MTC metastases
RET immuno positive
RET immuno negative
RET mutation
positivea
RET mutation
negative
35
2
6
27
Fisher’s exact test of association, P , 0.000001.
RET 918 mutation1 or 883 mutation1; all subpopulations with
A883F are immunostain positive.
a
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JCE & M • 1998
Vol 83 • No 12
ENG ET AL.
immune serum against the peptide described in Bongarzone et al. (19),
and SC (Santa Cruz Labs, Santa Cruz, CA), whose epitopes correspond
to the C-terminus of the short form of the protein (1:2000 and 1: 100,
respectively). Between successive washes with phosphate-buffered saline, sections were incubated for 35 min each with biotinylated swine
antirabbit antiserum (1:200, Dako Corp., Ely, UK) followed by Strept
ABC (1:500, Dako Corp.). The colorimetric reaction was generated by
incubating the slides in diaminobenzidine for 10 min, and sections were
counterstained in hematoxylin for 1 min. Both antibodies yielded similar
results.
As negative controls for each tumor or normal thyroid, additional
serial sections from each corresponding block underwent the same procedure and were incubated with the same dilutions of normal rabbit
serum. All these were immunonegative.
Subpopulations were scored as immunostain positive if at least 20%
of the cells in that section stained with the antibody. In addition, the
staining must have been 21 or stronger (where 11 is weak staining, and
41 is very intense staining). Immunostain negative was only scored
when the intensity was no more than background (i.e. graded 0). Note
should be made that normal C cells either do not stain or stain at 0.5–11,
at best.
Results
Seventy subpopulations comprising primary MTC and
distinct metastases from sporadic cases could be scored for
both mutation status and RET immunostaining (Table 1).
The remaining 12 subpopulations either could not be assessed for mutation status (e.g. repeated PCR failure) or
could not be assessed for immunostaining (for technical
reasons), or both. Assessment of RET mutation status and
immunostaining were performed independently in a parallel fashion and scored blindly by 2 independent individuals on 3 occasions. Thirty-seven subpopulations were
RET mutation positive: 34/37 M918T and 3/37 A883F (see
also Table 1 and Ref. 16). Of the RET mutation-positive
subpopulations, 35 (95%) were RET immunopositive (with
intensity of staining graded at 31 and 41) and 2 were
immunonegative. Thirty-three subpopulations were RET
mutation negative and of those, 27 were RET immunonegative (82%); but 6 were RET immunopositive (with intensity of staining ranging from 21 to 41). In sum, therefore, there was overall concordance between the presence
of codon 918 or 883 mutation and positive RET immunostaining, and between the absence of the mutation and
negative immunostaining in a total of 62 (89%) tumors (P ,
0.000001, Fisher’s exact test). Of note, the correlation was
observed at the level of subpopulations within the tumors,
as well [e.g. Fig. 1 compared with Fig. 2, lanes 14 –17, of
Eng et al., 1996 (16)]. Interestingly, there was a higher
discordance rate in the RET mutation negative category
(Table 1).
Discussion
Regional localization of RET codon 918 mutation or codon
883 mutation seems to be correlated with the presence of RET
immunopositivity. Positive immunoreactivity is usually associated with increased synthesis of the target protein, in this
case, RET, or increased stability of the protein. This is analogous to the situation for p53. In general, the presence of p53
mutation is correlated with increased p53 immunoreactivity,
although some discordance is also noted (e.g. Ref. 20). It is
unclear whether the presence of somatic RET mutation per se
would cause increased synthesis, but it is possible that the
codon 918 and codon 883 mutations could cause increased
stability of RET transcript and/or the protein. Alternatively,
cells with somatic RET 918 (or 883) mutation are those which
are actively dividing and/or not differentiated.
For RET mutation negative cases, concordance rates were
slightly lower. A technical explanation is possible, however.
This category included sections that were largely normal
thyroid, with a miniscule MTC focus that showed RET immunoreactivity or normal tissue with a thin sliver of tumor
at one edge, which may have been lost before DNA
extraction.
The apparent concordance between RET immunostaining and mutation status may prove clinically useful if these
data can be confirmed with larger studies. A few small
studies have shown that the presence of a somatic M918T
predicted for a poor clinical outcome (21, 22), although one
other study did not (23). If indeed the presence of somatic
M918T in sporadic MTC is correlated with a poor prognosis, and if our immunostaining-M918T association holds
true, then RET immunostaining of all sporadic MTC can be
included as part of the molecular diagnostic armamentarium, the results of which could serve as an adjunct to the
classic prognostic indicators.
Acknowledgments
We are grateful to the clinicians who obtained MTC specimens. We
thank Drs. Patricia L. M. Dahia and Oliver Gimm for critical review of
the manuscript.
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26th Annual Meeting of the European
Thyroid Association
August 29 –September 1, 1999
Milan, Italy
For information, please contact: Professor P. Beck-Peccoz, ICH, Via Manzoni 56 20089 Rozzano, Milan, Italy.
Telephone: 139-02-822.44515; fax: 139.02.822.44590; E-mail: paolo.beck-peccoz@humanitas.it.
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