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Oncotarget, 2018, Vol. 9, (No. 11), pp: 9875-9884
Research Paper
RET mutation heterogeneity in primary advanced medullary
thyroid cancers and their metastases
Cristina Romei1, Raffaele Ciampi1, Francesca Casella1, Alessia Tacito1, Liborio
Torregrossa2, Clara Ugolini2, Fulvio Basolo2, Gabriele Materazzi2, Paolo Vitti1 and
Rossella Elisei1
1
Endocrine Unit, Department of Clinical and Experimental Medicine, University Hospital of Pisa, Pisa, Italy
2
Department of Surgical, Medical and Molecular Pathology, University Hospital of Pisa, Pisa, Italy
Correspondence to: Rossella Elisei, email: rossella.elisei@med.unipi.it
Keywords: medullary thyroid carcinoma; RET; genetic instability; tumor clonality
Received: August 09, 2017
Accepted: November 16, 2017
Published: January 04, 2018
Copyright: Romei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY
3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT
Purpose: Medullary Thyroid Cancer (MTC) whose pathogenesis is strictly related
to RET proto-oncogene alterations, has been shown to have a heterogenic RET
mutation profile in subpopulations of MTC. The aim of our study was to investigate
the RET somatic mutation profile in primary MTC and in the corresponding metastatic
tissues in a series of advanced metastatic cases.
Results: This study demonstrated that in about 20% of cases a different RET
mutation profile can be found when comparing primary tumor and its corresponding
metastases. Furthermore in 8% of tumors, RET intratumor heterogeneity was
observed We also showed that in some cases an imbalance of RET copy number
was present. We confirmed a high prevalence (90%) of RET somatic mutations in
advanced tumors.
Materials and Methods: Fifty-six MTC patients (50 somatic and 6 hereditary cases)
have been included in the study and a total of 209 specimens have been analysed by
direct sequencing. Multiplex ligation-dependent probe amplification (MLPA) has been
used to investigate amplification/deletion of RET alleles.
Conclusions: In conclusion, this study showed a genetic intra- and intertumor
heterogeneity in MTC, But in only 20% of CASES These results could justify the
relatively moderate level of aggressiveness of the disease with respect to more
aggressive human tumors that are characterized by a high rate of mutation and
heterogeneity.
oncogene have been shown to cause approximately
95–98% of MEN 2 cases [2, 3]. In the other 75% of
cases, MTC is a sporadic tumor, and with exception of
RAS alterations that have been found in approximately
10% of cases [4, 5], somatic mutations in the RET protooncogene appear to be the most common genetic alteration
in MTC tumorigenesis [3]. The most common alterations
in the RET proto-oncogene are missense gain of function
mutations mainly located in the extracellular domain of
RET (exons 10 or 11) and in the RET tyrosine kinase
domain (exons 13, 14, 15 and 16).
These mutations are able to cause the constitutive
activation of the ret onco-protein [6].
INTRODUCTION
Medullary thyroid carcinoma (MTC) is a rare
endocrine tumor originating from parafollicular C cells of
the thyroid. This neoplasia is inherited as an autosomal
dominant trait in 25% of patients [1]. In these cases,
other organs besides the thyroid (e.g., the parathyroid and
adrenal glands) can be involved, thus giving rise to the
multiple endocrine neoplasia type 2 (MEN 2) syndromes,
which are categorized into three different subtypes (e.g.,
MEN 2A, MEN 2B and familial medullary thyroid
carcinoma or FMTC) according to their phenotype [2].
Activating germline point mutations in the RET protowww.impactjournals.com/oncotarget
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Comparison of RET somatic mutations in
different tumoral tissues of the same patient
In particular, we demonstrated that somatic RET
mutation prevalence increases with increasing tumor
size [7], reaching a prevalence of approximately 90% in
advanced cases [8].
The predominant role of a single initiating mutation
in MEN 2 was proposed several years ago [9]. Recently,
studies performed with deep sequencing technologies,
either whole exome sequencing (WES) [10] or targeted
sequencing [11–13], have shown that, with a few
exceptions, RET is the only oncogene altered in MTC.
Over the years, spatial and temporal intratumor
heterogeneity have been demonstrated in several human
cancers such as clear cell renal cancer, glioblastoma,
pancreatic cancer and breast cancer [14–17]. In addition to
a different mutation profile, DNA copy number alterations
represents an additional feature of intratumor heterogeneity
[18]. As far as thyroid cancer genetic heterogeneity is
concerned, little is known about the frequency of different
mutations in metastases of papillary thyroid histotypes
[19–21]. Even less evidence is available on the genetic
heterogeneity of MTC. A single study performed on 28
sporadic MTC cases showed an intratumoral heterogenic
RET mutation profile in 50% of cases [22].
Taking into consideration that RET is almost the
only oncogene altered in MTC, it is conceivable that, if
tumoral heterogeneity exists, different RET mutations
might be present in different tumoral specimens (i.e.,
primary and metastatic tissues). The aim of the present
study was to investigate the RET somatic mutation profiles
in a large series of primary MTC cases and corresponding
synchronous or metachronous metastatic tissues.
In the whole series of 56 metastatic MTC cases,
we compared the presence of RET somatic mutations
in different tumoral tissues of the same patient. In the
majority of cases (n = 45), the comparison was made
between primary and metastatic tissues, while in the
others (n = 11), the RET genetic profile was compared
in different metastases. The comparison showed that in
45/56 (80.4%) cases, the RET mutation profile, either
positive or negative, was the same in all the specimens
of the same patient. In this group of concordant cases, no
double mutants of RET were observed. Moreover, in 7
cases (Table 1: n. 27, n. 28, n. 34, n. 36, n. 44, n. 53, and n.
55) of which 2 or 3 different sections of the same primary
tissue were available, no differences in the RET genetic
profile, either positive or negative, were observed.
The other 11/56 (19.6%) cases showed a heterogenic
RET mutation profile. As shown in Table 3 (panel A),
in 5 cases (cases n. 3, n. 18, n. 36, n. 53 and n. 54), a
RET heterozygous somatic mutation, either point or
complex, was present in the primary tumor. This subgroup
contained some metastatic lesions that showed the same
RET mutation as found in the primary tumor, while others
were RET negative (n. 3, n. 18, n. 36, n. 53). Moreover,
case n. 53 showed a lymph node metastases with the
same mutation as the primary tumor plus an additional
12 bp deletion of exon 15. Finally, the kidney metastases
of case n. 54 that was characterized by a double somatic
mutation in the primary tumor showed only one of the two
mutations.
Another subgroup of discordant cases (Table 3,
panel B) was characterized by different patterns: a)
different metastases of the same patient were either
positive or negative for a specific RET mutation (n. 6);
b) two different RET mutations, none of them found in
the primary, were present in the same metastatic lesion (n.
10); c) the same RET mutation, not found in the primary,
was present in all lymphnode metastases (n. 23); d) two
different RET mutations or just one of them or even none,
as it happened in the primary, were found in different
metastatic lesions (n 24).
The last two cases (Table 3, panel C) showed
a peculiar RET genetic profile since in some lesions
of the same patient the RET mutation was apparently
homozygous, while in others it was clearly heterozygous
(n. 9 and 14).
In order to verify whether the RET negative tumoral
tissues belonging to cases with other RET positive tissues
were false negative due to a low percentage of cancer cells
in the sample, we evaluated the prevalence of tumoral
cells in the tissue samples. As shown in Table 4, when the
percentage of tumoral cells was at least 20% RET mutation
was detected. Among cases negative for RET mutations,
some cases had a high percentage of tumoral cells and
RESULTS
RET mutations
Among the 56 metastatic MTC cases included
in the study, 7 cases were found to CARRY a RET
germline mutation (Table 1). However case n. 36 was
considered sporadic since it was positive for an A883T
mutation in exon 15 that was previously demonstrated,
both in vitro and in vivo, to have a very low or null
transforming ability [23, 24]. The remaining 49 patients
were found to be negative for the presence of a RET
germline mutation; thus, a total of 50 cases were
considered truly sporadic.
As reported in Table 2, 45/50 (90%) cases were
positive for a RET somatic mutation, while 5 were RET
mutation negative. The most frequent RET somatic
mutation was the M918T mutation in exon 16 that was
found in 34/50 (68%) sporadic cases. Other RET somatic
point mutations or in-frame deletions were found in 5/50
(10%) and 6/50 (12%) cases, respectively. As reported in
Table 2, 4/50 (8%) cases were found to carry 2 different
somatic mutations, either 2 point mutations or 2 deletions,
in the same tissue.
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Table 1: Available tumoral tissues from the 56 patients with metastatic MTC included in the study
1
2
3
4
5
6
7
8
9
10
n.a.
1
1
1
1
n.a.
1
1
1
1
local
recurrence
(LC)/
metastases
2 LNF
1 LNF
1 LC/5 LNF
1 LNF
1 LNF
6 LNF
2 LNF
2 LNF
1 LNF
1 LC
11
1
1 LNF
Neg
39
n.a.
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1
n.a.
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
3 LNF
2 LNF
5 LNF, 2 liver
4 LNF
1 LC/1 LNF
1 LNF
1 LNF
1 LNF
1 LNF
1 LNF
1 LNF
12 LNF
8 LNF
2 LNF
2 LNF
1 LNF
2 LNF
Neg
Neg
Neg
Neg
Neg
Neg
Neg
V804M
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
1
n.a.
1
1
2
n.a.
n.a.
1
1
1
1
1
1
2
1
3
1
num
Primary
tumor
RET
germline
mutation
num
primary
tumor
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
29
30
31
32
33
34
35
36*
37
38
n.a.
1
n.a.
1
1
2
1
3
n.a.
1
local
recurrence
(LC)/
metastases
7 liver
1 LNM
3 LNF
4 LNF
1 LNF CC
2 LNF
1 LNF
11 LNF 1
2 LNF
1 LNF
3 LNF, 2 brain,
2 liver, 1 kidney,
1 adrenal
1 LNF
2 LNF
1 liver
1 LNF
1 LNF
2 LNF
5 LNF
2
1 LNF
4 LNF, 2 PHEO
1 trachea
1 LNF
1 LNF
7 LNF
1 kidney
3 LNF
2 LNF
RET
germline
mutation
Neg
Neg
Neg
Neg
Neg
C634S
Neg
A883T
Neg
Neg
C634R
Neg
Neg
Neg
Neg
Neg
Neg
Neg
M918T
Neg
M918T
Neg
M918T
Neg
Neg
Neg
Neg
Neg
n.a. not available; * this patient was considered as sporadic since A883T is not transforming (ref 24) and had the somatic
deletion p.D898_E901del, c.2694_2705del12 reported in Table 2. PHEO: pheochromocitoma.
could be considered real negative instead some cases had
low percentage of tumoral cells (10–15%) and they could
be potentially false negative. Nevertheless, if we exclude
these latter cases, the number of heterogenic cases would
not change and affect the final results of the present study.
encompassing codons 632–634 (Figure 1, panel A). Very
interestingly, the same deletion was found as homozygous
in other metastatic lesions (Figure 1, panel B). No RET
alterations were found at the germline level (Figure 1,
panel C).
MLPA was performed on all tumoral tissues of case n.
14 to determine if the “homozygosity” of the deletion was
caused by a RET copy number variation. As shown in Figure
1, panel D, no copy number variation was found within
the RET gene in the tumor that was characterized by the
heterozygous somatic 6 bp deletion of exon 11. However, one
Multiplex ligation-dependent probe
amplification (MLPA) assay
As previously stated, some tumoral tissues of patient
n. 14 showed a heterozygous somatic deletion in exon 11
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Table 2: RET somatic mutations in sporadic cases
Sporadic cases (n = 50)
RET somatic mutations (45/50 = 90%)
mutation
M918T*
C620A
C634G
C634R
A883F
p.D899_E902del, c.2694_2705
p.D898_E901del, c.2692_2703del12
p.Glu632fs c.1894_1904del11
p.Ile638fs c.1912_1918del7**
n. of cases/50 (%)
34 (70.4)
2 (4)
1 (2)
1 (2)
1 (2)
2 (4)
1 (2)
1 (2)
p.E632_L633del, c.1894_1899del6 + p.D898_E901del, c.2692_2703del12 **
1 (2)
p.E632_L633del, c.1894_1899del6
1 (2)
NM
5 (11.5)
*
in 2 cases M918T mutation was associated with an additional point mutation in the same tissue (S891A or C620F);
cases two different deletions in exon 11 and in both exon 11 and 15, respectively, were found in the same tissue.
**
in 2
Figure 1: Sanger sequencing pherograms and MLPA graphics of the case n 14. Heterozygous deletion in exon 11 encompassing
codons 632–634 (panel A) is revealed by the presence of double peaks in the pherogram starting from codon 634 (see red arrow); homo/
hemizygous RET somatic deletion in exon 11 encompassing codons 632–634 is shown in (panel B) and revealed by the absence of
both codons 632 and 633 (see black arrow). No RET deletion was found at the germline level (panel C) as demonstrated by the wild
type sequence of RET oncogene. MLPA showed that no copy number variation was found within the RET gene in the tissues with the
heterozygous somatic 6 bp deletion of exon 11 (panel D) suggesting a balance between mutated and not mutated alleles. At variance, an
amplification of one RET allele was observed in the tissue with apparent homozygous 6 bp deletion of exon 11 (panel E) suggesting that
the amplified allele should be the mutated one.
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Table 3: Cases with a different RET mutation profile in different samples
PANEL A
LNF
M918T (2/5)
NM (3/5)
NM (1/1)
p.D898_E901del,
c.2694_2705del12 (10/11)
NM (1/11)
M918T (1/7)
p.D898_E901del,
c.2694_2705del12 + M918T (1/7)
NM (5/7)
Not present
PANEL B
M918T (5/6)
NM (1/6)
patient
Primary
3
M918T (1/1)
18
M918T (1/1)
36
p.D898_E901del,
c.2694_2705del12 (3/3)
53
M918T (2/2)
54
M918T/S891A (1/1)
6
n.a.
10
NM (1/1)
23
NM (1/1)
24
NM (1/1)
9
M918T homo/hemizygous
(1/1)
M918T heterozygous (1/1)
n.a.
14
p.E632_L633del,
c.1894_1899del6 heterozygous
(1/1)
p.E632_L633del, c.1894_1899del6
heterozygous (4/5)
p.E632_L633del, c.1894_1899del6
homo/hemizygous(1/5)
p.E632_L633del, c.1894_1899del6
heterozygous (1/2)
p.E632_L633del, c.1894_1899del6
homo/hemizygous (1/2)
n.a.
p.D898_E901del,
c.2692_2703del12 (12/12)
C620F+M918T (2/8)
M918T (5/8)
NM (1/8)
PANEL C
Distant met
n.a.
n.a.
n.a
n.a
only M918T (1/1)
n.a.
p.E632_L633del, c.1894_1899del6
p.D898_E901del,
c.2692_2703del12 (1/1)
n.a.
n.a.
n.a. not available
confirmed by the results of the present study, since the
prevalence of somatic RET mutations was 90% in this
advanced metastatic MTC series, with M918T mutations
being the most frequent. Among the RET mutations
other than M918T, we found several complex somatic
alterations, almost exclusively deletions, affecting
exons 11 and/or 15. The presence of these type of RET
mutations, although more rare than point mutations, has
been previously reported in other series, and they have
been demonstrated to have high transforming capabilities
[28, 29].
Only few cases of sporadic MTC with multiple RET
mutations in the same tumoral tissue have been reported so
far [30]. In our series, we found 4 cases with intratumoral
heterogeneity characterized by the presence of 2 RET
alterations, either point mutations or deletions, in the
same tumoral specimen. Although the prevalence of these
mixed cases (8% of our series) is rather low, this finding is
something new with respect to the results of Eng et al. [22],
of the two RET alleles was highly amplified with respect to
the other in the tissue with the apparent homozygous somatic
6 bp deletion of exon 11 (Figure 1, panel E). Based on these
MLPA results, we can reasonably assume that the amplified
allele was the mutated one, thus leading to the apparently
homozygous pattern observed with the sequencing analysis.
DISCUSSION
Several studies have demonstrated that RET is the
most prevalent oncogene involved in MTC tumorigenesis.
From the study of the International RET consortium [25],
the prevalence of RET mutations in sporadic MTC cases
was found to be approximately 40%, with a high rate
of M918T mutations. Over the years, the overall RET
mutation prevalence was essentially confirmed, but RET
mutation levels were demonstrated to be significantly
lower in small size tumors and much higher in advanced
metastatic cases [7, 8, 26, 27]. This latter finding has been
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Table 4: Comparison between RET mutation status and percentage of tumoral cells in the analysed
tumoral tissue
patient
3
Type of tissue
LNF
LNF1
LNF3
primary tumor
% of tumoral cells
n.a.
80
70
20
RET mutation
NM
NM
NM
M918T
53
LNF1
LNF2
LNF3
LNF4
LNF5
LNF6
5
5
20
60
30
n.a.
NM
NM
p.D898_E901del,c.2694_2705del12
NM
NM
NM
6
24
LNF4
LNF6
LNF7
LNF8
primary tumor
20
30
10
10
20
NM
M918T/C620F
NM
NM
NM
53
LNF1
LNF2
LNF3
LNF4
LNF5
LNF6
LNF LC SIN
5
5
20
60
30
n.a.
50
NM
NM
p.D898_E901del,c.2694_2705del12
NM
NM
NM
M918T
The first pattern was characterized by the presence of a
RET heterozygous somatic mutation in the primary tumor
that was not necessarily found in all the corresponding
metastases or an additional unique mutation (from
that found in the primary tumor) that was found in one
metastatic lymph node. To understand this finding, we
might hypothesize that the primary tumor is composed
of RET-positive and RET-negative cell subpopulations.
Although we cannot be sure that different cells within a
tumor can carry different mutations, the study of Eng et al
[22] clearly demonstrated that it was possible. As far as the
case with the additional mutation is concerned, the most
plausible explanation is the evolutionary theory of cancer
according to which new mutations can occur in already
mutated, developing tumors [31, 32].
In the second pattern, the primary tumor was RET
negative, and different metastases of the same patient were
either positive or negative for a specific RET mutation. It
is conceivable that a few cells in the primary tumor were
positive for the mutations found in the lymphnodes which
received a growth advantage during the selective pressures
of the metastatic process and produced RET-positive
metastases [32]. This explanation and the possibility that
during the growth of mutated cells other mutations can
be added could also justify those cases in which different
metastases are genetically different.
The third pattern of heterogeneity showed a peculiar
RET genetic profile: In some lesions, the RET mutation
who found a high prevalence of mixed subpopulations, but
only mixed in regards to RET positive or negative mutation
status. However, it must be noted that the methodology used
in the Eng et al study was rather restrictive and did not allow
for the identification of all RET mutations and in particular
could not detect complex mutations. The pathogenic role
of a double mutant in the same tissue is still not clear, but
Nowell’s evolutionary theory of cancer, which describes
how mutations can accumulate in cell subpopulations,
remains the most likely explanation [31, 32].
Recently, some studies have described the genetic
heterogeneity in different human cancers [14–17]. These
findings are rather expected in those human tumors
(i.e., lung and melanoma) in which multiple oncogenes
(up to 163 and 147 different oncogenes, respectively)
have been found to be altered in the same tumor [33–
35]. Differentiated thyroid cancers have been shown
to have a low number of altered oncogenes, which are
usually mutually exclusive [36]. This is particularly
true for sporadic MTC, which has a high prevalence of
RET mutations and a low prevalence of RAS mutations,
with no other driver oncogenes found so far [3, 5]. For
this reason, we concentrated our attention on the RET
oncogene alone and we showed that 20% of MTC cases
were characterized by a different RET mutation profile
in primary and metastatic tissues. As reported in Table 2,
three different patterns of heterogeneity were found, and
different hypotheses can be proposed to explain them.
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was apparently homozygous, while in others, it was clearly
heterozygous. In these cases, a loss of heterozygosity
(LOH) (as reported also by Dvorakova et al in their series
[30, 37]) or a copy number alteration should be responsible
for the observed pattern. The MLPA experiment suggested
that an increased number of RET mutated alleles were
present in our sample. This result agrees with our previous
findings, which showed that RET gene amplifications were
present in RET-positive tumors [38].
A limitation of the present study can be due the
sensitivity of the Sanger method that has been estimated
to be between 10–20% [7, 39, 40]. This implies the
possibility to have a technical bias due to the presence of
micro metastases, with a low number of metastatic tumoral
cells surrounded by normal cells and for these reason not
detectable by Sanger sequencing. We tried to rule out this
problem and effectively we found that when the tumoral cells
were > 20% RET mutation was detectable but we also had
cases with > 20% of tumoral cells that were RET negative.
These latter can be considered as true negative cases. For
those cases with < 20% of tumoral cells we cannot exclude
the possibility they were false negative cases. Nevertheless,
the number of heterogenic cases would not change if we
exclude these potential false negative cases.
In conclusion, this study showed that a different
RET mutation profile between primary and metastatic
tissues of the same MTC was present in 20% of cases,
and in 8% of these, RET intratumor heterogeneity was
observed. These results, together with the evidence
that MTC has a low rate of mutations other than RET,
explains the relatively moderate level of aggressiveness
of the disease with respect to other, more aggressive
tumors (lung adenocarcinoma or melanoma) that
are characterized by a high rate of mutation and
heterogeneity.
from paraffin-embedded tissue blocks. A peripheral blood
sample was available for both sporadic and familial cases.
All patients provided written informed consent. This
investigation was approved by our institutional review
board and by the local Ethic Committee (protocol number
469, approved 29/1/2015).
MATERIALS AND METHODS
Multiplex ligation-dependent probe
amplification (MLPA) assay
Methods
Pathologic diagnosis
Patients underwent total or subtotal thyroidectomy
at the Department of Surgery of the University of Pisa,
Italy. The presence of typical histological (i.e., tumoral
cells arranged in trabecular, insular or sheet-like growth
patterns) and immunohistochemical (cells positive for
calcitonin and chromogranin) features defined an MTC
histological diagnosis. In some cases, the percentage of
tumoral cells in the sample was recorded. High levels of
serum calcitonin were confirmatory of the C cell origin of
the cases.
Genotyping
DNA was prepared from blood, fresh and paraffinembedded tumoral tissues according to a previously
described protocol [8]. RET exons 10, 11, 13, 14, 15 and
16 were analyzed by direct sequencing [41]. To exclude
false negative cases, primary tumors and metastases found
to be discordant for the presence/absence of the M918T
RET mutation were further analyzed using a more sensitive
TaqMan SNP genotyping assay (ThermoFisher Scientific,
Waltham, Massachusetts, USA). The experiments were
run according to the manufacturer’s guidelines.
Patients
MLPA was used to detect potential large deletions
in RET gene. Experiments were performed on primary
and metastatic tumor samples using the SALSA MLPA
P169 HIRSCHSPRUNG PROBEMIX (MRC-Holland,
Amsterdam, the Netherlands). Coffalyser.Net software
(MRC-Holland, Amsterdam, the Netherlands) was used
to identify copy number variations. Experiments were
repeated at least twice. Three reference DNAs from the
blood of healthy subjects and a negative control (a sample
without DNA) were included in all experiments.
We studied the tumoral tissues of 56 patients (24
females and 32 males) affected with metastatic sporadic or
familial MTC. The histological diagnosis and classification
of tumoral tissues were performed by an experienced team
of local pathologists. MTC cases were included in the
study only if at least two tumoral tissues obtained from
different lesions were available, either from primary and
metastatic tissues or from several metastases. A total of
209 tumoral specimens were analyzed. In particular, 54
primary tumoral tissues, 2 local recurrences, 132 lymph
node metastases and 21 distant metastases (i.e., lung,
liver, kidney, adrenal gland, brain) were included in the
study. The details of the analyzed tumoral tissues and their
corresponding patients’ statistics are reported in Table 1.
Tissues were either collected at surgery, immediately
frozen in liquid nitrogen and kept at -80°C, or recovered
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CONFLICTS OF INTEREST
The authors declare that there is no conflicts of
interest that could affect the impartiality of the reported
research.
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medullary thyroid cancer reveals dominant and mutually
exclusive oncogenic mutations in RET and RAS. J
Clin Endocrinol Metab. 2013; 98:E364-9. https://doi.
org/10.1210/jc.2012-2703.
FUNDING
This work was supported by the Associazione
Italiana Ricerca sul Cancro (AIRC, Investigator grant
2014, project code 15431).
11. Simbolo M, Mian C, Barollo S, Fassan M, Mafficini A,
Neves D, Scardoni M, Pennelli G, Rugge M, Pelizzo MR,
Cavedon E, Fugazzola L, Scarpa A. High-throughput
mutation profiling improves diagnostic stratification of
sporadic medullary thyroid carcinomas. Virchows Arch.
2014; 465:73–8. https://doi.org/10.1007/s00428-0141589-3.
REFERENCES
1. Raue F, Frank-Raue K. Epidemiology and Clinical
Presentation of Medullary Thyroid Carcinoma. Recent
Results Cancer Res. 2015; 204:61–90. https://doi.
org/10.1007/978-3-319-22542-5_3.
12. Sherman SI, Clary DO, Elisei R, Schlumberger MJ,
Cohen EEW, Schoffski P, Wirth LJ, Mangeshkar M, Aftab
DT, Brose MS. Correlative analyses of RET and RAS
mutations in a phase 3 trial of cabozantinib in patients with
progressive, metastatic medullary thyroid cancer. Cancer.
2016; 122:3856–64. https://doi.org/10.1002/cncr.30252.
2. Frank-Raue K, Raue F. Hereditary Medullary Thyroid
Cancer Genotype-Phenotype Correlation. Recent Results
Cancer Res. 2015; 204:139–56. https://doi.org/10.1007/9783-319-22542-5_6.
3. Romei C, Ciampi R, Elisei R. A comprehensive overview of
the role of the RET proto-oncogene in thyroid carcinoma.
Nat Rev Endocrinol. 2016; 12:192–202. Available from
http://www.ncbi.nlm.nih.gov/pubmed/26868437.
13. Wei S, LiVolsi VA, Montone KT, Morrissette JJD, Baloch
ZW. Detection of Molecular Alterations in Medullary
Thyroid Carcinoma Using Next-Generation Sequencing: an
Institutional Experience. Endocr Pathol. 2016; 27:359–62.
https://doi.org/10.1007/s12022-016-9446-3.
4. Ciampi R, Mian C, Fugazzola L, Cosci B, Romei C,
Barollo S, Cirello V, Bottici V, Marconcini G, Rosa PM,
Borrello MG, Basolo F, Ugolini C, et al. Evidence of a
low prevalence of RAS mutations in a large medullary
thyroid cancer series. Thyroid. 2013; 23:50–7. https://doi.
org/10.1089/thy.2012.0207.
14. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder
D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey
P, Varela I, Phillimore B, Begum S, et al. Intratumor
heterogeneity and branched evolution revealed by
multiregion sequencing. N Engl J Med. 2012; 366:883–92.
https://doi.org/10.1056/NEJMoa1113205.
5. Moura MM, Cavaco BM, Leite V. RAS proto-oncogene in
medullary thyroid carcinoma. Endocr Relat Cancer. 2015;
22:R235-52. https://doi.org/10.1530/ERC-15-0070.
15. Sottoriva A, Spiteri I, Piccirillo SGM, Touloumis A, Collins
VP, Marioni JC, Curtis C, Watts C, Tavare S. Intratumor
heterogeneity in human glioblastoma reflects cancer
evolutionary dynamics. Proc Natl Acad Sci U S A. 2013;
110:4009–14. https://doi.org/10.1073/pnas.1219747110.
6. Hedayati M, Zarif Yeganeh M, Sheikholeslami S, Afsari F.
Diversity of mutations in the RET proto-oncogene and its
oncogenic mechanism in medullary thyroid cancer. Crit Rev
Clin Lab Sci. 2016; 53:217–27. https://doi.org/10.3109/104
08363.2015.1129529.
16. Yachida S, Jones S, Bozic I, Antal T, Leary R, Fu B,
Kamiyama M, Hruban RH, Eshleman JR, Nowak MA,
Velculescu VE, Kinzler KW, Vogelstein B, et al. Distant
metastasis occurs late during the genetic evolution of
pancreatic cancer. Nature. 2010; 467:1114–7. https://doi.
org/10.1038/nature09515.
7. Romei C, Ugolini C, Cosci B, Torregrossa L, Vivaldi
A, Ciampi R, Tacito A, Basolo F, Materazzi G, Miccoli
P, Vitti P, Pinchera A, Elisei R. Low prevalence of the
somatic M918T RET mutation in micro-medullary thyroid
cancer. Thyroid. 2012; 22:476–81. https://doi.org/10.1089/
thy.2011.0358.
17. Campbell PJ, Yachida S, Mudie LJ, Stephens PJ, Pleasance
ED, Stebbings LA, Morsberger LA, Latimer C, McLaren S,
Lin ML, McBride DJ, Varela I, Nik-Zainal SA, et al. The
patterns and dynamics of genomic instability in metastatic
pancreatic cancer. Nature. 2010; 467:1109–13. https://doi.
org/10.1038/nature09460.
8. Romei C, Casella F, Tacito A, Bottici V, Valerio L, Viola
D, Cappagli V, Matrone A, Ciampi R, Piaggi P, Ugolini
C, Torregrossa L, Basolo F, et al. New insights in the
molecular signature of advanced medullary thyroid cancer:
evidence of a bad outcome of cases with double RET
mutations. J Med Genet. 2016. https://doi.org/10.1136/
jmedgenet-2016-103833.
18. Martinez P, Birkbak NJ, Gerlinger M, McGranahan N,
Burrell RA, Rowan AJ, Joshi T, Fisher R, Larkin J, Szallasi
Z, Swanton C. Parallel evolution of tumour subclones
mimics diversity between tumours. J Pathol. 2013;
230:356–64. https://doi.org/10.1002/path.4214.
9. Mulligan LM, Gardner E, Smith BA, Mathew CG, Ponder
BA. Genetic events in tumour initiation and progression in
multiple endocrine neoplasia type 2. Genes Chromosomes
Cancer. 1993; 6:166–77.
19. Yang X, Li J, Li X, Liang Z, Gao W, Liang J, Cheng S,
Lin Y. TERT Promoter Mutation Predicts RadioiodineRefractory Character in Distant Metastatic Differentiated
Thyroid Cancer. J Nucl Med. 2017; 58:258–65. https://doi.
org/10.2967/jnumed.116.180240.
10. Agrawal N, Jiao Y, Sausen M, Leary R, Bettegowda C,
Roberts NJ, Bhan S, Ho AS, Khan Z, Bishop J, Westra
WH, Wood LD, Hruban RH, et al. Exomic sequencing of
www.impactjournals.com/oncotarget
9882
Oncotarget
20. Ricarte-Filho JC, Ryder M, Chitale DA, Rivera M,
Heguy A, Ladanyi M, Janakiraman M, Solit D, Knauf JA,
Tuttle RM, Ghossein RA, Fagin JA. Mutational profile
of advanced primary and metastatic radioactive iodinerefractory thyroid cancers reveals distinct pathogenetic
roles for BRAF, PIK3CA, and AKT1. Cancer Res. 2009;
69:4885–93. https://doi.org/10.1158/0008-5472.CAN-090727.
different spectrum of mutations in sporadic type from
hereditary type. Jpn J Cancer Res. 1999; 90:1231–7.
29. Ceccherini I, Pasini B, Pacini F, Gullo M, Bongarzone I,
Romei C, Santamaria G, Matera I, Mondellini P, Scopsi
L, Pinchera A, Pierotti MA, Romeo G. Somatic in frame
deletions not involving juxtamembranous cysteine residues
strongly activate the RET proto-oncogene. Oncogene. 1997;
14:2609–12. https://doi.org/10.1038/sj.onc.1201079.
21. Melo M, Gaspar da Rocha A, Batista R, Vinagre J,
Martins MJ, Costa G, Ribeiro C, Carrilho F, Leite V,
Lobo C, Cameselle-Teijeiro JM, Cavadas B, Pereira L, et
al. TERT, BRAF, and NRAS in Primary Thyroid Cancer
and Metastatic Disease. J Clin Endocrinol Metab. 2017;
102:1898–907. https://doi.org/10.1210/jc.2016-2785.
30. Dvorakova S, Vaclavikova E, Sykorova V, Duskova J,
Vlcek P, Ryska A, Novak Z, Bendlova B. New multiple
somatic mutations in the RET proto-oncogene associated
with a sporadic medullary thyroid carcinoma. Thyroid.
2006; 16:311–6. https://doi.org/10.1089/thy.2006.16.311.
31. Nowell PC. The clonal evolution of tumor cell populations.
Science. 1976; 194:23–8.
22. Eng C, Mulligan LM, Healey CS, Houghton C, Frilling A,
Raue F, Thomas GA, Ponder BA. Heterogeneous mutation
of the RET proto-oncogene in subpopulations of medullary
thyroid carcinoma. Cancer Res. 1996; 56:2167–70.
32. Greaves M, Maley CC. Clonal evolution in cancer. Nature.
2012; 481:306–13. https://doi.org/10.1038/nature10762.
33. Hong SM, Park JY, Hruban RH, Goggins M. Molecular
signatures of pancreatic cancer. Arch Pathol Lab Med. 2011;
135:716–27. https://doi.org/10.1043/2010-0566-RA.1.
23. Cosci B, Vivaldi A, Romei C, Gemignani F, Landi S, Ciampi
R, Tacito A, Molinaro E, Agate L, Bottici V, Cappagli V,
Viola D, Piaggi P, et al. In silico and in vitro analysis of
rare germline allelic variants of RET oncogene associated
with medullary thyroid cancer. Endocr Relat Cancer. 2011;
18:603–12. https://doi.org/10.1530/ERC-11-0117.
34. Brugarolas J. Molecular genetics of clear-cell renal cell
carcinoma. J Clin Oncol. 2014; 32:1968–76. https://doi.
org/10.1200/JCO.2012.45.2003.
35. Riesco-Eizaguirre G, Santisteban P. Endocrine Tumours:
Advances in the molecular pathogenesis of thyroid cancer:
lessons from the cancer genome. Eur J Endocrinol. 2016;
175:R203–17. https://doi.org/10.1530/EJE-16-0202.
24. Elisei R, Cosci B, Romei C, Agate L, Piampiani P, Miccoli
P, Berti P, Basolo F, Ugolini C, Ciampi R, Nikiforov Y,
Pinchera A. Identification of a novel point mutation in the
RET gene (Ala883Thr), which is associated with medullary
thyroid carcinoma phenotype only in homozygous
condition. J Clin Endocrinol Metab. 2004; 89:5823–7.
https://doi.org/10.1210/jc.2004-0312.
36. Cancer Genome Atlas Research Network. Integrated
genomic characterization of papillary thyroid carcinoma.
Cell. 2014; 159:676–90. https://doi.org/10.1016/j.
cell.2014.09.050.
25. Eng C, Clayton D, Schuffenecker I, Lenoir G, Cote G, Gagel
RF, van Amstel HK, Lips CJ, Nishisho I, Takai SI, Marsh
DJ, Robinson BG, Frank-Raue K, et al. The relationship
between specific RET proto-oncogene mutations and
disease phenotype in multiple endocrine neoplasia type 2.
International RET mutation consortium analysis. JAMA.
1996; 276:1575–9.
37. Dvorakova S, Vaclavikova E, Sykorova V, Vcelak J,
Novak Z, Duskova J, Ryska A, Laco J, Cap J, Kodetova
D, Kodet R, Krskova L, Vlcek P, et al. Somatic mutations
in the RET proto-oncogene in sporadic medullary thyroid
carcinomas. Mol Cell Endocrinol. 2008; 284:21–7. https://
doi.org/10.1016/j.mce.2007.12.016.
26. Wells SAJ, Asa SL, Dralle H, Elisei R, Evans DB, Gagel
RF, Lee N, Machens A, Moley JF, Pacini F, Raue F, FrankRaue K, Robinson B, et al. Revised American Thyroid
Association guidelines for the management of medullary
thyroid carcinoma. Thyroid. 2015; 25:567–610. https://doi.
org/10.1089/thy.2014.0335.
38. Ciampi R, Romei C, Cosci B, Vivaldi A, Bottici V, Renzini
G, Ugolini C, Tacito A, Basolo F, Pinchera A, Elisei R.
Chromosome 10 and RET gene copy number alterations
in hereditary and sporadic Medullary Thyroid Carcinoma.
Mol Cell Endocrinol. 2012; 348:176–82. https://doi.
org/10.1016/j.mce.2011.08.004.
27. Schlumberger M, Jarzab B, Cabanillas ME, Robinson B,
Pacini F, Ball DW, McCaffrey J, Newbold K, Allison R,
Martins RG, Licitra LF, Shah MH, Bodenner D, et al. A
Phase II Trial of the Multitargeted Tyrosine Kinase Inhibitor
Lenvatinib (E7080) in Advanced Medullary Thyroid
Cancer. Clin Cancer Res. 2016; 22:44–53. https://doi.
org/10.1158/1078-0432.CCR-15-1127.
39. Monzon FA, Ogino S, Hammond MEH, Halling KC, Bloom
KJ, Nikiforova MN. The role of KRAS mutation testing
in the management of patients with metastatic colorectal
cancer. Arch Pathol Lab Med. 2009; 133:1600–6. https://
doi.org/10.1043/1543-2165-133.10.1600.
40. Ihle MA, Fassunke J, Konig K, Grunewald I, Schlaak
M, Kreuzberg N, Tietze L, Schildhaus HU, Buttner R,
Merkelbach-Bruse S. Comparison of high resolution
melting analysis, pyrosequencing, next generation
sequencing and immunohistochemistry to conventional
Sanger sequencing for the detection of p.V600E and non-p.
28. Uchino S, Noguchi S, Yamashita H, Sato M, Adachi M,
Yamashita H, Watanabe S, Ohshima A, Mitsuyama S,
Iwashita T, Takahashi M. Somatic mutations in RET exons
12 and 15 in sporadic medullary thyroid carcinomas:
www.impactjournals.com/oncotarget
9883
Oncotarget
V600E BRAF mutations. BMC Cancer. 2014; 14:13.
https://doi.org/10.1186/1471-2407-14-13.
of sporadic medullary thyroid cancer (MTC) allows the
preclinical diagnosis of unsuspected gene carriers and the
identification of a relevant percentage of hidden familial
MTC (FMTC). Clin Endocrinol (Oxf). 2011; 74:241–7.
https://doi.org/10.1111/j.1365-2265.2010.03900.x.
41. Romei C, Cosci B, Renzini G, Bottici V, Molinaro E, Agate
L, Passannanti P, Viola D, Biagini A, Basolo F, Ugolini
C, Materazzi G, Pinchera A, et al. RET genetic screening
www.impactjournals.com/oncotarget
9884
Oncotarget