AJCP / Review Article
Molecular Pathology of Hereditary and Sporadic Medullary
Thyroid Carcinomas
Rebecca D. Chernock, MD,1,2 and Ian S. Hagemann, MD, PhD1
From the Departments of 1Pathology and Immunology and 2Otolaryngology Head and Neck Surgery, Washington University School of Medicine,
St Louis, MO.
CME/SAM
Key Words: Medullary thyroid carcinoma; RET; RAS; Tyrosine kinase; Multiple endocrine neoplasia; Familial medullary thyroid carcinoma
Am J Clin Pathol June 2015;143:768-777
DOI: 10.1309/AJCPHWACTTUYJ7DD
ABSTRACT
Objectives: Medullary thyroid carcinoma (MTC) is a
relatively uncommon type of thyroid malignancy, with unique
histologic features and molecular pathology. It is important
to recognize, because its management, which is in part
driven by the genetic basis of this disease, is different from
follicular-derived thyroid tumors. The aim of this article is
to briefly review the histopathologic features of MTC and
then explore its molecular pathology, including the role of
molecular diagnostic testing and the use of targeted therapy
for advanced disease.
Methods: A review of published literature was performed.
Results: A subset of MTC cases is hereditary and due to
germline mutations in the RET tyrosine kinase receptor gene.
Somatic mutations in either RET or RAS are also present in
most sporadic tumors.
Conclusions: Molecular genetic testing is routinely
performed to identify hereditary cases. In addition,
understanding the molecular basis of both hereditary
and sporadic MTC has led to the development of targeted
therapy with tyrosine kinase inhibitors. Although additional
data are needed, tumor mutation status may affect response
to targeted therapy. Therefore, it is possible that genetic
testing of tumor tissue to predict treatment response, as
is currently done for other cancer types, may come into
practice in the future.
768 Am J Clin Pathol 2015;143:768-777
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Upon completion of this activity you will be able to:
• recognize the histologic features of medullary thyroid carcinoma
(MTC), including its variants that may mimic other thyroid lesions.
• describe the genetic basis of the multiple endocrine neoplasia types
2A and type 2B and familial MTC syndromes and the correlation
between genotype and phenotype.
• list the indications and rationale for RET mutation analysis to detect
patients with a hereditary syndrome.
• discuss the major genetic changes in sporadic MTC and their
potential impact on tyrosine kinase inhibitor therapy.
The ASCP is accredited by the Accreditation Council for Continuing
Medical Education to provide continuing medical education for physicians.
The ASCP designates this journal-based CME activity for a maximum of
1 AMA PRA Category 1 Credit ™ per article. Physicians should claim only
the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance
of Certification Part II Self-Assessment Module.
The authors of this article and the planning committee members and staff
have no relevant financial relationships with commercial interests to disclose.
Questions appear on p 905. Exam is located at www.ascp.org/ajcpcme.
Medullary thyroid carcinoma (MTC) is a relatively
uncommon tumor type, accounting for 5% or less of thyroid
malignancies.1 However, it warrants attention because it
causes a disproportionate number of thyroid cancer deaths
due to its more aggressive clinical behavior compared with
well-differentiated papillary and follicular thyroid carcinomas.2 Furthermore, the clinical management of MTC differs from nonmedullary thyroid carcinoma. Thus, accurate
diagnosis is important so that patients can be appropriately
treated.
Treatment is, in part, dictated by the unique molecular
pathology of these tumors. A significant subset is hereditary, due to germline mutations in the RET proto-oncogene,
and patients with MTC are routinely referred for genetic
testing.3-6 Patients who are found to have hereditary disease can then be monitored for other associated endocrine
© American Society for Clinical Pathology
AJCP / Review Article
A
B
❚Image 1❚ Histologic features of typical medullary thyroid carcinoma. A, The tumor cells form nests and are plasmacytoid
with abundant pale pink cytoplasm, round to oval nuclei, and granular chromatin (H&E, ×40). B, Pale pink, amorphous amyloid
deposits are frequently seen (H&E, ×40).
abnormalities, and at-risk family members can be screened
and treated prior to the development of MTC (with prophylactic thyroidectomy). Somatic (nongermline) RET mutations also occur in a significant proportion of sporadic
MTCs.7 More recently, RAS mutations have been identified
in RET wild-type sporadic tumors.8 Genetic testing of tumor
tissue for somatic mutations is not routinely performed since
none of these alterations currently affects patient management. However, RET activation in these tumors has made it
possible to use targeted therapy to treat advanced and metastatic MTC.9,10 It is possible that genetic testing of tumor
tissue to predict response to a particular targeted agent, as is
currently done for other cancer types such as colon and lung,
may come into practice in the future.
Our aim is to briefly review the pathologic features of
MTC and then explore the molecular basis of both hereditary and sporadic disease. In addition, we address the role of
diagnostic molecular testing to identify and manage hereditary cases, as well as the development of targeted therapies for treatment of both hereditary and sporadic tumors.
Finally, the prospect of molecular testing of tumor tissue to
predict response to therapy is explored.
Pathologic Features of MTC
Histopathology and Cytopathology
MTC is a neuroendocrine carcinoma that arises from the
neural crest–derived parafollicular C cells. C cells are located in the upper one-third of the thyroid lobes bilaterally but
© American Society for Clinical Pathology
cannot normally be seen on routine H&E sections because
they are so infrequent and morphologically similar to the
much more numerous follicular cells. Their function is to
produce calcitonin, which has a poorly defined physiologic
role. Calcitonin is, however, an important serum tumor
marker for patients with MTC because it is also secreted by
these tumors.
The typical MTC is solid with sheets or nests of cells
that are plasmacytoid and have abundant pale pink cytoplasm and round to oval nuclei with granular chromatin
❚Image 1❚. Tumor cells are usually uniform but may show
pleomorphism. Amyloid is present in approximately 80%
of the tumors and is composed of calcitonin secreted by the
tumor cells (Image 1).11 On fine-needle aspiration biopsy,
the cells are usually singly dispersed or loosely cohesive,
and red cytoplasmic granules may be present (Diff-Quik
stain). The amyloid may resemble the dense colloid that is
often seen in cytologic preparations of papillary thyroid carcinomas. However, amyloid, unlike colloid, may be metachromatic on Diff-Quik–stained slides. Hereditary tumors
are more often multifocal than sporadic tumors but otherwise do not have distinctive histologic or cytologic features.
MTCs can have a wide variety of divergent histologic
patterns. Described histologic variants include spindle cell,
papillary or pseudopapillary, follicular or glandular, clear
cell, oncocytic, mucin producing, melanin producing, paraganglioma-like, and small cell, among others. In some cases,
these variants may mimic other types of thyroid carcinomas
or lesions ❚Image 2❚. For example, the follicular variant can
resemble follicular carcinoma or adenoma when it has a
capsule or even nodular hyperplasia when it lacks a capsule.
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Chernock et al / Medullary Thyroid Carcinoma
A
B
C
D
❚Image 2❚ Select variants of medullary thyroid carcinoma (MTC) that can mimic other thyroid lesions. A, Low-power image of a
follicular variant that has variably sized cystic spaces filled with colloid-like material and that resembles nodular hyperplasia (H&E,
×20). B, High-power image of the same follicular variant MTC that has “scalloping” at the periphery of the colloid-like material
within pseudofollicles. The tumor cells, however, have a pale pink cytoplasm and a plasmacytoid appearance typical of MTC
(H&E, ×40). C, A papillary or pseudopapillary variant of MTC that has finger-like projections with fibrovascular cores (H&E, ×40).
D, Intranuclear pseudoinclusions may occasionally be seen in MTC as well (arrow) (H&E, ×60).
Benign entrapped thyroid follicles may also be present
within the tumor, and this should not be mistaken as evidence of follicular differentiation. Similarly, the papillary or
pseudopapillary variant may raise the possibility of papillary
thyroid carcinoma. Although intranuclear cytoplasmic pseudoinclusions and nuclear contour irregularity may be seen
in MTC, other cytologic features are usually present that
are not typical of papillary thyroid carcinoma (plasmacytoid
cells with pale pink cytoplasm and granular chromatin).
Other entities in the differential diagnosis include metastatic
renal cell carcinoma for the clear cell variant, metastatic
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melanoma for the melanin-producing variant, and paraganglioma for the paraganglioma-like variant. Metastasis to the
thyroid gland should also be considered when the small cell
variant is encountered.
Because of the wide variety of histologic patterns that
have been reported, it is important to have a low threshold
for performing immunohistochemistry in any thyroid tumor
that lacks definite colloid or is simply not typical of another
type of thyroid carcinoma. Useful positive immunostains
include calcitonin and monoclonal carcinoembryonic antigen, which are negative in other thyroid tumors. Calcitonin,
© American Society for Clinical Pathology
AJCP / Review Article
hereditary disease. When C-cell hyperplasia is not visible on
routine H&E sections (because the C cells are not atypical
and are therefore indistinguishable from adjacent follicular
cells), it should be considered reactive, rather than neoplastic, C-cell hyperplasia. In such cases, C-cell hyperplasia is
only seen by calcitonin immunohistochemistry. The distinction between reactive C-cell hyperplasia and neoplastic
C-cell hyperplasia may be difficult in some cases. When
there is uncertainty, genetic testing for a constitutional RET
mutation can exclude hereditary disease.
Hereditary vs Sporadic Disease
❚Image 3❚ C-cell hyperplasia is characterized by nodular
aggregates of C cells that have more abundant pale pink
cytoplasm than the adjacent follicular cells and often granular
chromatin (H&E, ×40). Stromal fibrosis is absent and, if
present, suggests an invasive microcarcinoma.
however, is not specific to MTC and can be positive in
neuroendocrine carcinomas from other organ systems.12 If
material for immunohistochemistry is not available on fineneedle aspiration biopsy but is felt to be needed, one can
request that the clinician check a serum calcitonin level,
since it is almost always elevated in patients with MTC.
C-Cell Hyperplasia
Histologically recognizable C-cell hyperplasia is generally considered a neoplastic process and a precursor lesion
to MTC (neoplastic C-cell hyperplasia). It is characterized
by nodular aggregates of C cells that are enlarged and have
more abundant pale pink to clear cytoplasm than the adjacent
follicular cells and often granular chromatin ❚Image 3❚. The
cells encircle thyroid follicles and are confined by basement
membrane. Stromal fibrosis should not be present since this
is a sign of invasion (medullary thyroid microcarcinoma).
Calcitonin immunohistochemistry can be used to highlight
the foci of C-cell hyperplasia: greater than 50 C cells per
low-power (×100) field should be seen to make this diagnosis.13 Neoplastic C-cell hyperplasia is commonly present in
patients with hereditary tumors but also occasionally occurs
adjacent to sporadic tumors or even incidentally in thyroids
removed for other reasons. C-cell hyperplasia should be
noted when identified on routine H&E sections since it
represents an indication for genetic testing (see below).14
Neoplastic C-cell hyperplasia is not, however, diagnostic of
© American Society for Clinical Pathology
Approximately 25% of MTCs are hereditary.15 The
hereditary cases occur in the setting of three syndromes:
multiple endocrine neoplasia syndrome (MEN) type 2A
(MEN2A), MEN type 2B (MEN2B), and familial medullary
thyroid carcinoma syndrome (FMTC). These syndromes
are autosomal dominant and highly penetrant; virtually all
patients will develop MTCs if their thyroid glands are not
removed.16 MEN2B is the least common (~5% of hereditary
MTC), and patients develop carcinomas at the youngest
age, often before 10 years. Both MEN2A and MEN2B syndromes are associated with other endocrine abnormalities
and clinical features (these are summarized in ❚Table 1❚).
Pheochromocytoma is seen in about half of patients with
MEN2A and MEN2B.16 A subset of patients with MEN2A
develop parathyroid hyperplasia, which is not seen in
MEN2B.16 Patients with MEN2B have a marfanoid habitus,
ocular abnormalities, mucosal neuromas, and gastrointestinal ganglioneuromatosis. Patients with FMTC do not have
other associated features. However, it is thought that FMTC
represents a mild variant of MEN2A with low penetrance of
pheochromocytoma and hyperparathyroidism. If pheochromocytoma or hyperparathyroidism is seen in a family with
FMTC, reclassification as MEN2A is warranted.
Sporadic tumors usually occur in the fifth to sixth
decades of life and lack other associated MEN features or
family history of MTC. There is overlap between the age of
presentation of MTCs in sporadic and hereditary cases. In
fact, approximately 5% to 10% of patients with apparently
sporadic tumors (no family history or stigmata of an MEN
syndrome) have hereditary disease (FMTC or MEN2A)
upon genetic testing.17
The initial clinical management of both hereditary and
sporadic MTC is the same but is different from that of follicular-derived thyroid tumors. All patients with a fine-needle
aspiration biopsy or serum calcitonin level diagnostic or suspicious for MTC will be screened for MEN-associated diseases (pheochromocytoma and hyperparathyroidism) prior
to thyroid surgery unless the patient has already undergone
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Chernock et al / Medullary Thyroid Carcinoma
❚Table 1❚
Clinical Features of Hereditary Medullary Thyroid Carcinoma Syndromes
Syndrome (% of Cases)
Characteristic
MEN2A (70%-80%)
MEN2B (5%)
FMTC (15%-30%)
Medullary thyroid carcinoma (onset)
Pheochromocytoma, %
Parathyroid hyperplasia, %
Marfanoid habitus
Other features
Yes (often <20-30 y)
Yes (~50)
Yes (20-30)
No
Hirschsprung disease (rare),
cutaneous lichen amyloidosis
Yes (<10 y)
Yes (~50)
No
Yes
Mucosal neuromas, gastrointestinal
ganglioneuromatosis, thickened corneal nerves
Yes (adults)
No
No
No
None
FMTC, familial medullary thyroid carcinoma; MEN2A, multiple endocrine neoplasia type 2A; MEN2B, multiple endocrine neoplasia type 2B.
genetic testing and has a negative family history.14 If a
pheochromocytoma is identified, it should be treated first to
avoid a hypertensive crisis. If preoperative serum calcitonin
is high (>400 pg/mL) or there is nodal disease on imaging,
the patient will be evaluated for distant metastases.14 Surgery typically consists of total thyroidectomy and prophylactic central (level VI) lymph node dissection for clinically
detected organ-confined disease.14
Molecular Basis of Hereditary Disease
and Genotype-Phenotype Correlation
In the early 1990s, activating mutations in the Rearranged during Transfection, or RET, proto-oncogene were
identified as the genetic basis for MEN2A, MEN2B, and
FMTC syndromes.3-6 Since then, germline RET mutations
have been identified in 98% of patients with MEN2A, 95%
of patients with MEN2B, and 88% of patients with FMTC.18
RET is located on chromosomal band 10q11.2 and encodes
a single-pass transmembrane receptor tyrosine kinase that
plays an important role in the development of the parathyroids, urogenital system, and neural crest—including brain,
para- and sympathetic ganglia, adrenal medulla, enteric ganglia, and thyroid C cells.19 Not surprisingly, many of these
organ systems are affected in the MEN syndromes with the
notable exceptions of the brain and urogenital system.
More than 80 different RET mutations in exons 5, 8, 10,
11, 12 to 16, and 19 have been associated with hereditary
MTC.20 Most are single-nucleotide missense mutations, and
all lead to constitutive activation of the RET tyrosine kinase
and downstream signaling pathways. A database of RET mutations, including variants of unknown significance, is maintained at the University of Utah/ARUP Laboratories (http://
www.arup.utah.edu/database/MEN2/MEN2_welcome.php).20
The RET gene also harbors several common polymorphisms, benign genetic variants that should not be overinterpreted as causes of familial cancer (eg, G691S, which
is present in as many as one in five healthy individuals).21
In general, if a RET variant is detected by sequencing,
particularly at a noncanonical position, it is important to
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Cadherin-like
domain
Cysteine-rich
domain
Plasma
membrane
Tyrosine
kinase 1
Tyrosine
kinase 2
Codon
Exon
MEN2A/FMTC
(609, 611, 618,
620, 630, 634)
10 + 11
MEN2A/FMTC
(768, 790, 791,
804)
13 + 14
MEN2B
(M918T)
16
❚Figure 1❚ The RET tyrosine kinase receptor with common
codon mutations that occur in the 3 hereditary syndromes
and their corresponding exons in the RET gene. FMTC,
familial medullary thyroid carcinoma; MEN2A, multiple
endocrine neoplasia type 2A; MEN2B, multiple endocrine
neoplasia type 2B.
consult a database of common polymorphisms, such as the
Single Nucleotide Polymorphism Database (http://www.
ncbi.nlm.nih.gov/SNP/) or the Exome Variant Server from
the National Institutes of Health’s National Heart, Lung,
and Blood Institute (http://evs.gs.washington.edu/EVS/), to
search for evidence that the variant may be a polymorphism
without significance.
The specific location of the mutations within the RET
gene is likely responsible for the different constellation of
phenotypes in each hereditary syndrome ❚Figure 1❚. The
RET protein has an extracellular domain composed of a
cadherin-like region that is important for ligand binding and
a highly conserved cysteine-rich domain. The intracellular
region contains two tyrosine kinase domains (TK1 and TK2).
Upon ligand binding, the extracellular cysteine-rich domain
facilitates receptor dimerization, which leads to autophosphorylation and activation of the intracellular TK1 and TK2.
This activates multiple signaling pathways, including RAS/
RAF/MAP kinase, PI3K/AKT, and STAT3, that lead to cell
survival, proliferation, migration, and differentiation.
© American Society for Clinical Pathology
AJCP / Review Article
Several RET mutations give rise to MEN2A syndrome,
the majority of which are located in cysteines within the
extracellular cysteine-rich domain (corresponding to exons
10 and 11).22 These mutations abolish intramolecular disulfide bridge formation and allow for constitutive dimerization
of two RET proteins in the absence of ligand.23 Other mutations in exons 13, 14, and, rarely, 5, 8, 12, 15, and 19 have
also been reported in MEN2A.20,22 Thirty-nine different
mutations have been associated with FMTC in exons 5, 8,
10, 11, and 13 to 16, with most being located in the cysteinerich domain or TK1 domain.18 There is overlap between
the mutations that are associated with MEN2A and FMTC.
Some mutations can be associated with either MEN2A or
FMTC in different families, which is why FMTC is considered a variant of MEN2A. The mutations that can give rise
to either syndrome are associated with low penetrance of
pheochromocytoma and hyperparathyroidism.
In contrast to the diversity of mutations seen in MEN2A/
FMTC, 95% of patients with MEN2B have a single mutation
in exon 16 (M918T) that causes a conformational change in
the intracellular TK2 binding pocket and allows for constitutive kinase activation in the absence of dimerization, as
well as altered substrate binding.23,24 Much less commonly
(2%-3% of cases), MEN2B is caused by a dinucleotide substitution in codon 883 (exon 15) in the TK1 domain.25 Very
rarely, simultaneous mutations in two different codons (eg,
V804M/E805K mutated in cis) can cause an MEN2B-like
phenotype (“atypical” MEN2B).26
It is thought that the different mechanisms of RET
activation induced by these mutations trigger different patterns of phosphorylation and cell signaling. For example, the
M918T codon mutation that accounts for the vast majority
of MEN2B is associated with increased PI3K/AKT pathway
activation compared with MEN2A-associated mutations.27,28
This may explain differences in disease phenotype among
the syndromes. Since some mutations are associated with
either MEN2A or FMTC in a given family, there are likely
additional modifying factors that affect phenotype as well.
Furthermore, within each of the hereditary syndromes,
the specific RET mutation is associated with variation in
disease phenotype. In MEN2A, the specific codon mutated
roughly correlates with the age of presentation with MTC
and the probability of developing pheochromocytoma or
hyperparathyroidism. Codon 634 in exon 11, corresponding to a cysteine in the extracellular cysteine-rich domain,
is the most commonly altered codon in MEN2A. Mutations at codon 634 are associated with the earliest onset
of MTC, often before age 20 years, and the highest risk of
pheochromocytoma and hyperparathyroidism in this syndrome.24,29,30 In addition, cutaneous lichen amyloidosis is
almost exclusively seen in patients with codon 634 mutations. Hirschsprung disease, a genetically heterogeneous
© American Society for Clinical Pathology
disease, can co-occur with MTC in patients with mutations
in exon 10.31,32
The M918T mutation that accounts for 95% of MEN2B
cases is associated with the earliest onset of MTC of any
RET mutation, in some cases within the first year of life.30 In
addition, the tumors are more aggressive and typically present at a more advanced stage.33 MEN2B patients with the
less common A883F mutations appear to develop MTCs at
an older age and have less aggressive tumors compared with
those with M918T.25 The rare “atypical” MEN2B syndrome
caused by simultaneous mutations in two different codons
has also been associated with a more mild phenotype.26,34
In FMTC, those mutations not involving the cysteinerich domain (usually in exon 13, 14, or 15) are associated
with the latest onset of MTC.35 This group of mutations
shows significant clinical overlap with the age of onset
of sporadic MTC. Therefore, routine genetic screening of
patients with apparently sporadic disease may reveal unexpected germline mutations in a subset of cases, usually in
patients with de novo and/or noncysteine mutations.17
Diagnostic Molecular Testing
for Hereditary Disease
The American Thyroid Association (ATA) recommends
that all patients with a personal history of MTC, primary
C-cell hyperplasia, or clinical features of MEN2 or FMTC
should be offered genetic testing for germline RET mutations.14 Furthermore, anyone with a family history of MEN2
or FMTC in a first-degree relative should also undergo
genetic testing.14 The rationale for screening all patients with
MTC or C-cell hyperplasia is based on the evidence that
approximately 5% to 10% of patients with MTC who lack
clinical features to suggest hereditary disease will harbor a
germline RET mutation, often de novo and/or noncysteine
mutations (as described above).17 Screening of at-risk family members should also be emphasized since those family
members found to harbor a RET mutation can be treated
with prophylactic thyroidectomy before carcinoma develops
and be monitored for pheochromocytoma or hyperparathyroidism, if indicated for the specific mutation identified.
Although de novo mutations are common in MEN2B, only
5% to 10% of MEN2A/FMTC cases represent new mutations.36,37 Therefore, siblings and even parents may be at
risk in addition to children of the index case. The ATA has
guidelines for the age of recommended prophylactic thyroidectomy in mutation carriers based on the specific RET
mutation ❚Table 2❚.
Approximately 60 laboratories in the United States
perform constitutional genetic testing for RET mutations on
peripheral blood (www.ncbi.nlm.nih.gov/gtr). However, the
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❚Table 2❚
ATA Recommendations for Age at Prophylactic Thyroidectomy Based on Genotype Risk Category
ATA Risk Level
(MTC Aggressiveness)
Codon Mutation
Syndrome
MTC Age
of Onset
Age of Prophylactic
Thyroidectomy
A (least high)
B (higher)
C (high)
D (highest)
768, 790, 791, 804, 649, 891
609, 611, 618, 620, 630, 631
634
918, 883
MEN2A/FMTC
MEN2A/FMTC
MEN2A/FMTC
MEN2B
Adults
5y
<5 y
<1 y
When calcitonin rises/>5 y
5y
<5 y
As soon as possible (<1 y)
ATA, American Thyroid Association; FMTC, familial medullary thyroid carcinoma; MEN2A, multiple endocrine neoplasia type 2A; MEN2B, multiple endocrine neoplasia
type 2B; MTC, medullary thyroid carcinoma.
laboratories vary in methodology, with a few laboratories
sequencing the entire coding region, and the majority select
exons. Laboratories that perform sequencing of select exons
also vary in which specific exons are sequenced. Most laboratories routinely sequence exons 10, 11, 13, and 14 (cysteine-rich and TK1 domains) to exclude MEN2A/FMTC.
Some also sequence exons 15 and 16 and rarely exons 5 and
8. It is helpful to know which exons an individual laboratory
sequences. For example, it may be important for patients
with apparently sporadic MTC undergoing genetic screening to have sequencing that includes the noncysteine region
(especially codons 13, 14, and 15), since these regions are
most likely to harbor unexpected germline mutations in this
patient population.17 For those with apparently sporadic
MTC or suspected MEN2A/FMTC, the ATA recommends
that initial testing should include sequencing of MEN2specific exons but does not specify the exons that should
be included.14 Sequencing of the entire coding region of
RET is recommend only for those who meet clinical criteria
for MEN2 but in whom initial select exon sequencing was
negative.14
For patients with suspected MEN2B or those at risk
for a known mutation (due to a kindred with a known RET
mutation), genetic testing is more straightforward. Patients
with suspected MEN2B (usually identified by their characteristic clinical features) can be specifically tested for the
M918T mutation, present in 95% of cases, with additional
testing for rare MEN2B-associated mutations only if the
M918T mutation is not found. Furthermore, the ATA recommends sequencing the entire coding region of RET for
patients who meet clinical criteria for MEN2B but in whom
testing for known mutations is negative.14 Patients with a
kindred harboring a known RET mutation can be specifically
tested for that mutation.
Molecular Basis of Sporadic Disease
While patients with sporadic MTC do not harbor germline RET mutations, up to half have somatic RET mutations in their tumors.7 The M918T mutation (seen in most
patients with MEN2B) is also the most common mutation
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RET+
RET–
RET–
RET+
RET+
RET–
RET+
❚Figure 2❚ RET mutational heterogeneity has been reported
in sporadic tumors harboring somatic mutations. This means
that a subset of tumor cells may harbor a RET mutation
that is not present throughout the entire tumor and may be
present in some but not all metastases.
in sporadic tumors, accounting for 75% to 95% of RET
mutations.7 Many other RET mutations have been reported
in sporadic tumors, including in exons 10, 11, 15, and 16.7
As in MEN2B, M918T mutations are associated with more
aggressive/advanced stage disease in sporadic tumors.38
Somewhat surprisingly, RET mutations in sporadic tumors
may not necessarily drive tumorigenesis but rather appear
important for progression of disease. This is supported by
the fact that RET mutations are present in a lower proportion of sporadic microcarcinomas than of larger tumors and,
when present, often show mutational heterogeneity, even
in advanced disease.39,40 This means that RET mutations
may be found in subpopulations of tumor cells rather than
the entire tumor, indicating that the mutation arose later in
the course of disease. Indeed, in half of patients with RET
mutations and multiple metastases, the mutation was found
in some but not all metastatic deposits ❚Figure 2❚.39
Much more recently, activating RAS mutations have also
been identified in a subset of sporadic MTCs. These mutations
may be associated with clinically less aggressive tumors.8,41
Mutations occur within known hotspots in exons 2, 3, and 4 and
predominantly involve HRAS and KRAS.42 NRAS mutations
© American Society for Clinical Pathology
AJCP / Review Article
are rare.41 RAS mutations appear to be almost always mutually exclusive with RET mutations and are present in approximately 10% to 45% of RET wild-type sporadic tumors.8,41-43
This leaves a small group of RET and RAS mutation-negative
tumors for which the main genetic changes are undefined. It is
important to note that the RAS signaling pathway is activated
by the RET tyrosine kinase receptor. Therefore, activation of
this pathway, either by RET or RAS mutation, appears important in both hereditary and sporadic tumors.
Molecular Targeted Therapy
Molecular targeted therapy for MTC has been long
sought due to significant shortcomings of traditional treatment strategies with a lack of effective systemic therapy.
Conventional chemotherapy is of limited benefit and associated with significant toxicity. Radioactive iodine is not
useful because MTC is not derived from thyroid follicular
cells and therefore does not take up iodine. Currently, complete surgical resection (thyroidectomy and lymph node
dissection) is the only chance for cure. However, about
half of patients have lymph node metastases, and only 10%
of these cases are cured by surgery.44 A subset of patients
have an indolent clinical course despite incurable disease.
Long-term, 10-year survival is more than 90% for those with
localized disease, decreasing to 78% and 40% in patients
with regional and distant metastases, respectively.45 Nevertheless, there is a need for additional treatment modalities in
patients who have residual or recurrent disease after surgery.
In the past several years, two targeted tyrosine kinase
inhibitors (TKIs), vandetanib and cabozantinib, have been
approved by the US Food and Drug Administration for the
treatment of symptomatic, advanced, or progressive MTC.
This approval was based on the results of two large phase
III clinical trials that showed increased progression-free survival of each drug compared with placebo.9,10 Both of these
TKIs are nonselective and target multiple kinases in addition
to RET. Vandetanib also targets VEGFR2, VEGFR3, and
EGFR, while cabozantinib additionally targets c-MET and
VEGFR2. It is thought that the efficacy of these drugs results
in part from their ability to target multiple kinases. Unfortunately, both TKIs induce partial responses with resistance
eventually developing, and an increase in overall survival
has not yet been shown.9,10 Furthermore, since the design
of the two phase III clinical trials was not identical and the
two drugs were not compared head to head, it is difficult to
determine which drug is more effective.9,10 However, the
side effect profile for each is different and may be considered when selecting a treatment option. The most significant
side effect associated with vandetanib is QT interval prolongation with rare torsades de pointes and sudden death; with
© American Society for Clinical Pathology
cabozantinib, the side effects are rare fistula formation, gastrointestinal perforation, and hemorrhage.9,10 Nevertheless,
these TKIs represent a significant advance in the treatment
of MTC, and it seems likely that in the future, they will be a
part of multidrug therapy.
One question that has been raised by the advent of TKIs
for the treatment of MTC is whether RET and RAS mutation
status affects tumor response to the drug. As of yet, this question has not been fully answered, but there is some evidence
that mutation status may affect treatment response. In the
phase III clinical trial of vandetanib, RET mutation status was
unavailable or incomplete in nearly half (45.3%) of patients
with sporadic tumors, so much is unknown about drug
response by mutation status.10 Nevertheless, M918T-mutated
tumors (seen in MEN2B and the majority of RET mutationpositive sporadic tumors) appeared to have increased sensitivity to the drug (overall response rate of 54.5% vs 30.9%).10 In
vitro studies have also shown that V804M and V804L RET
mutations conferred resistance to vandetinib.46 All RET mutation subgroups, including RET mutation-negative tumors,
showed benefit in the cabozantinib clinical trial, although the
benefit was marginal in the RET-negative group and best in
the M918T codon mutation group.9,47 Clearly, further data are
needed to address response by particular RET mutation.
TKI response by RAS mutation status was not assessed
initially in either clinical trial, likely because the discovery
of RAS mutations in MTC was relatively recent. Subsequent
analysis of a subgroup of RAS-mutated tumors showed comparable, although slightly shorter, progression-free survival
with cabozantinib treatment than for RET-mutated tumors.47
However, because RAS mutations have been associated with
resistance to TKI therapy in other types of tumors, such as
colon and lung cancer, a more thorough investigation in
MTC is warranted.48,49
Although there is some evidence to suggest that TKI
response varies by mutation status, additional information
is needed. With more data, it is possible that RET and/or
RAS mutation analysis of tumor tissue from formalin-fixed,
paraffin-embedded blocks or fine-needle aspiration biopsy
specimens for somatic mutations may become part of clinical practice in the future. If this is the case, several issues
should be considered. One is the reported mutational heterogeneity that can be seen with RET mutations in sporadic
tumors.39 Caution should be exercised when RET mutation
analysis is performed on a primary tumor in a patient being
treated for metastatic disease, since the mutation status may
be different in the metastasis. Another consideration is the
method of testing, since next-generation sequencing may
have higher sensitivity than Sanger sequencing50 and allows
several genes to be assayed on the same platform. The relative cost and effectiveness of these two methods should be
considered before testing guidelines are established.
Am J Clin Pathol 2015;143:768-777775
DOI: 10.1309/AJCPHWACTTUYJ7DD
Chernock et al / Medullary Thyroid Carcinoma
Conclusion
In summary, molecular diagnostic testing has been a
mainstay of the clinical management of MTC for many years.
Patients are routinely referred for RET mutation analysis of
constitutional DNA to identify hereditary cases so that they
can be monitored for other associated endocrine abnormalities,
and at-risk family members can be screened and treated prior
to the development of MTC (with prophylactic thyroidectomy). Somatic (nongermline) mutations in either RET or RAS
also occur in most sporadic MTCs. However, genetic testing of
tumor tissue for somatic mutations is not routinely performed.
It is possible that, with the advent of TKIs now in use to treat
advanced and metastatic MTC, genetic testing of tumor tissue
to predict response to a particular drug may come into practice
in the future. However, further information about the impact of
RET and RAS mutations on TKI treatment response is needed.
Address reprint requests to Dr Chernock: Dept of Pathology and
Immunology, Washington University School of Medicine, 660 S
Euclid Ave, Campus Box 8118, St Louis, MO; rchernock@path.
wustl.edu.
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