Appearances May Be Deceiving: Unmasking the Thyroid Follicular Genotype
University of Pittsburgh
Mutations in carcinomas can be grouped into two major categories: oncogene mutations and
tumor suppressor gene mutations. The types of mutations that affect these genes in tumors are broad, and
include point mutations, deletion mutations, promoter methylation, and other epigenetic changes.
Currently, our understanding of pathogenesis of thyroid follicular tumors is fairly comprehensive;
despite this depth of understanding, however, there are likely to be many additional genetic factors that
remain to be discovered.
The importance of molecular testing in all types of pathology is likely to increase
exponentially in the next 10 years. While much of our prior understanding of tumors and pathogenesis was
based on the protein expression, measured with immunohistochemistry, the future of adjuvant diagnostic
testing in pathology is likely to be at the nucleic acid level, either in DNA or mRNA based testing.
The purpose clinical or translational molecular testing in anatomic pathology can usually
be classified into one or more of the following advantages: Improving our understanding of the disease;
Improving our diagnostic abilities; Improving prognostication; Improving therapeutic decision making.
Importantly, new tests that are developed and proposed should be evaluated for their ability to fulfill
these advantages, particularly if they are going to be used clinically. Any clinical test should have a
definite value to add to the standard algorithms of H&E and immunohistochemical staining that are
current practice in endocrine pathology.
Two major problems exist in thyroid pathology regarding follicular lesions that have been
addressed at multiple levels, from H&E based studies, to DNA and RNA based studies, to expression
profiling using microarray technology.
Because of the problems with inter-observer variation in diagnosing these entities , there
may be even more impetus to design and validate new diagnostic molecular tests that can help pathologists
to make these distinctions. It is with these two major questions in mind that the molecular mutations
that are known to occur in thyroid neoplasia are discussed below.
- Understanding follicular variant of papillary carcinoma and differentiating it from benign and malignant follicular derived tumors.
- Differentiating follicular-derived carcinoma from benign follicular lesions.
The ultimate goal is to incorporating molecular testing into our daily practice to better our
diagnoses and improve management of patients. Early studies have been extremely promising for using the
molecular profiles on both the FNA and the surgical specimens, and even possibly peripheral
|FVPTCa||Follicular variant of papillary thyroid carcinoma|
|PTCa||Papillary thyroid carcinoma|
|AI/MI||Angio-invasive and minimally invasive|
Oncogenes in thyroid carcinomas
Oncogenes are genes that exist normally as wild-type proto-oncogenes, but develop
mutations leading to gene activation; when mutated they are termed as oncogenes. Oncogenes are usually
important in cell growth, differentiation, or motility. Oncogenes are dominant genes, because only one
copy needs to develop a mutation in order for the mutagenic effects to be apparent. In familial
syndromes, oncogene mutations are inherited. A well-studied example of an oncogene that is important in
thyroid pathology is the RET gene in which mutations are inherited in MEN 2a, MEN 2b, and familial
medullary carcinoma syndromes.
Oncogenes can harbor somatic mutations (non-germline) that develop either during
carcinogenesis or as the presumed cause of carcinogenesis. These oncogenes can be activated by a number
of different types of mutations, including point mutations and translocations. Translocations usually
activate oncogenes by coupling part of a gene that drives over-expression to the oncogene.
The RAS proto-oncogenes are commonly mutated in many different types of human
cancers . There are three main RAS genes, H-RAS (chromosome 11p15.5), N-RAS (chromosome
1p13.2) and K-RAS (chromosome 12p12.1). Point mutations are the most common mutations and these usually
occur in the GTP-binding domain (codons 12 and 13), and in the GTPase domain (codon 61), though other
types of mutations, including amplifications and acquired polymorphisms have also been detected
N-RAS mutations may be more frequent than K-RAS and H-RAS point mutations .
Figure 1 - Point Mutations that are Implicated in Thyroid Carcinomas
The occurrence of RAS oncogene mutations in thyroid tumorigenesis is mainly thought to
occur in follicular derived tumors. The mutations are most likely early events, as they are present both
in adenomas and carcinomas . Importantly, however, RAS mutations are found in low frequency
in non-neoplastic conditions such as colloid nodules and goiter, and in papillary carcinomas
Some suggestion that mutations are more frequent in poorly differentiated tumors has also been
BRAF is an oncogene that is located on chromosome 7q34. Point mutations in BRAF are known
to occur in multiple tumor types.
Most notably, mutations are common in melanoma, cholangiocarcinoma and
in papillary thyroid carcinoma. The most common point mutation is a T àA transversion at nucleotide 1796
in exon 15 (V599E)
The BRAF mutations have been reported in between 35% and 69% of papillary thyroid
. In several large series, BRAF mutations were not found other types of
thyroid tumor, except anaplastic carcinomas derived from well-differentiated papillary carcinomas
The BRAF mutation does not appear to have any prognostic significance . But,
given its high prevalence and the high specificity for papillary thyroid carcinoma, it may be useful as a
diagnostic marker, either in FNA specimens or in surgical specimens .
BRAF mutation analysis has a potential role in follicular lesions, but this role is
limited due to the fact that it has not been found in a high percentage of follicular variants of
papillary carcinoma .
Translocations that occur in germline tissues during embryogenesis can be completely
silent, and are called balanced translocations when the normal number of each chromosome part is
maintained. In carcinogenesis, somatic translocations can be the mutational event responsible for tumor
development. Carcinogenic translocations can occur between genes on different chromosomes or on the same
chromosome (intrachromosomal rearrangements). Usually, one of the partners of the fusion gene is a
proto-oncogene, and the other product is a gene that drives over-expression of the resulting oncogene
Figure 3 - Translocations that are Implicated in Thyroid Carcinomas
The PAX8-PPARγ chromosomal translocation has been found to be present in approximately 40% of
follicular thyroid carcinomas
. PAX8 is a thyroid transcription factor that is critical
for thyroid regulation of growth, differentiation and function. The nuclear receptor PPARγ
(peroxisome proliferators-activated receptor γ ) is a ligand dependent transcription factor that is
highly expressed in adipose tissue. The translocation may also be present in benign
lesions , presumably follicular adenomas, and the protein PPARγ may be overexpressed in
both as well
. However, because adenomas are thought to be the precursors of
follicular carcinomas and there are often very subtle differences in the histology of adenoma vs.
carcinoma, it is unclear what the significance of the translocation in histologically benign tumors.
The difference between follicular thyroid tumors that have the PAX8-PPARγ translocation and those
that do not has been explored at several levels. Some have suggested that it may be associated with
lower grade tumors . Other groups have examined the expression profile differences by
microarray analysis between tumors with and without the translocation and have also found that there are
significant, though incompletely understood differences that are highly uniform .
Cytogenetic analysis is a reliable test to detect specific translocations (especially
those between two different chromosomes) and to identify chromosomal numeric changes. Cytogenetic
analysis requires that the cells be capable of dividing, and therefore necrotic areas should be avoided.
PCR-based analysis for translocations may be more practical than cytogenetics in thyroid tumors.
Many translocation breakpoints occur in large introns and may not be tightly clustered.
When the area that needs to be examined for translocations is this large (i.e., sometimes thousands of
basepairs), routine PCR from genomic DNA may be nearly impossible. The best strategy for testing for
these mutations is to utilize the messenger RNA, in which the introns are spliced out, resulting in
consistent juxtaposition of exons from the two different partner genes in the fusion mRNA. For these
assays, reverse transcription is followed by PCR. Because mRNA is extremely fragile, frozen tissue is
optimal for this assay. However, in our experience, RNA can successfully be extracted with fairly good
results from paraffin-embedded tissue in about 60-80% of routine surgical cases.
In some circumstances, the expression of a fusion protein, or over-expression of the
normal protein driven by the translocation, can be detected by immunohistochemistry. PPARγ
over-expression has been associated with translocations and lack of expression is associated with the
absence of the mutation . Protein over-expression, however, can be driven by numerous
factors other than translocations . Therefore, the most accurate assessment of
translocations remains at the nucleic acid level.
Another test for translocations that can used in paraffin embedded tissues is fluorescent in situ
hybridization (FISH), which can be used to detect the PAX8-PPARγ translocation . Dual
color probes, or chromosome specific paints, will localize each chromosome involved in the translocation,
and after hybridization, abnormal fusions can be identified. In the presence of translocations, the two
separate colors are positioned next to each other in the majority of cells examined. There are several
caveats to analyzing paraffin embedded tissues for translocations using FISH. First, because the cells
can be fragmented or cut in several planes or overlapping, the signals for the two gene partners can be
entirely absent or artificially overlapped with each other. Therefore, a certain number of cells must be
examined before a definitive molecular call is made and this cutoff should be established in each
laboratory that does the assay.
RET-PTC1 and RET-PTC3
The RET-PTC translocations are the other major translocations that occur in thyroid
carcinomas. These are seen in papillary carcinomas. The translocation is between the tyrosine kinase
domain of the RET proto-oncogene on chromosome 10 and multiple different partner genes . The
most common partner genes are ELE1 (PTC3) and H4 (PTC1), both of which are also located on chromosome
. These mutations are therefore more accurately termed intrachromosomal
rearrangements, but historically they have been referred to as translocations. These two partners (ELE1
and H4) account for over 90% of the translocations that have been identified in papillary carcinomas.
Both translocations are more frequently seen in radiation-induced tumors, and the RET-PTC3 translocation
may be more common in solid variant of papillary carcinomas
. Translocations are found in
approximately 30-40% of papillary carcinomas; only minor geographic differences have been
The most important use for the RET-PTC translocation analysis in the setting of follicular
lesions should be to aid in the differential diagnosis between follicular variant of papillary carcinoma
and follicular adenoma. The nuclear features of papillary carcinoma have been linked to the RET-PTC
translocation experimentally . An extremely controversial area, follicular lesions with
focal atypical nuclear features, has been assessed by microdissecting these isolated atypical areas out
from the surrounding follicular lesion. Interestingly, the RET-PTC translocation could be found
specifically in these areas of incompletely developed nuclear features of papillary
carcnioma . The RET-PTC translocations have not been identified in non-papillary well
differentiated tumors , though they are present in hyalinizing trabecular tumors
Studies of the translocation in Hashimoto's thyroiditis are controversial, with some
suggesting that mutations can be found in this benign condition, and others suggesting they do
Unfortunately, the use of this translocation analysis in the follicular lesion is limited
because the RET-PTC translocations do not appear to be very common in follicular variant of papillary
carcinoma . The relative absence of the RET-PTC translocation in FVPTCA challenges our
understanding of this lesion.
Tumor Suppressor Genes in Thyroid Carcinomas
Tumor suppressor genes are recessive genes, because inactivation of both copies is
necessary for the deleterious effects to be realized. Inactivation can occur via multiple different
mechanisms, including deletion mutations, point mutations, promoter methylation, and other epigenetic
changes that decrease the function of the tumor suppressor gene. While many of these mutations are
measurable, the assessment can be tedious and labor intensive. Therefore, the most common way to detect
presumptive tumor suppressor gene inactivation is to assess the tumor for loss of heterozygosity, also
termed allelic imbalance.
Most tumor suppressor genes are difficult to measure directly, because they do not contain
polymorphisms that enable us to differentiate the maternally inherited copy from the paternally inherited
copy. Without being able to differentiate the two copies, we would be unable to detect if one copy were
lost through a deletion mutation. For loss of heterozygosity assessment, therefore, surrogate
polymorphic markers are used, such as short tandem repeat units that co-localize with the tumor
suppressor genes. The closer the short tandem repeat is to the tumor suppressor gene, the better a
marker it will be for the gene.
Loss of heterozygosity can be utilized in several other ways to assess neoplasia beyond
just understanding the genes that are involved in carcinogenesis of a particular tumor type. When
utilized in a panel format with a significant number of markers, one can assess for generalized genetic
tumor instability and molecular mutagenesis. It can also be used to identify the clonal patterns of loss
in a tumor. In these clonality assessments, different areas within the same tumor can be compared to one
another, or tumors and their metastatic deposits can be compared. This method of analysis is an
alternative to studying X-inactivation (in women) for clonality assessments.
Many different tumor suppressor genes have been reported to be involved in thyroid
neoplasia. In addition, many genetic loci that are not necessarily related to tumor suppressor genes
have also been described. In fact, recent studies using comparative genomic hybridization have examined
nearly the entire genome to identify specific areas that are unstable, showing either gain or loss in
tumors, as compared to the normal counterparts. Some applications of studying tumor suppressor genes are
Tumor suppressor gene panel for diagnosis and
In many tumors, carcinogenesis is either caused by, or accompanied by instability in tumor suppressor
genes. In thyroid carcinomas, loss of heterozygosity deletion mutations are noted in all types of
tumors, including papillary carcinomas, follicular carcinomas and Hürthle cell carcinomas, medullary
carcinomas, and anaplastic carcinomas
. One calculation that can be used to assess the
overall genetic instability is the fractional allelic loss in a panel of tumor suppressor genes. This is
the number of tumor suppressor gene loci with loss divided by the number of loci that were informative.
In general, follicular derived tumors that are more aggressive histologically and clinically have more
measurable levels of loss of heterozygosity and higher fractional allelic losses
Fractional allelic loss in well-differentiated papillary carcinomas is most often low, and only increases
in the presence of anaplastic transformation
follicular carcinomas, the fractional allelic loss (FAL) correlates well with the histologic and clinical
outcome for the tumors. Follicular adenomas have a mean FAL of approximately 10%, while minimally
invasive and angioinvasive tumors have a mean of approximately 30%, and widely invasive tumors have a
mean FAL of >50% . Furthermore, tumors that recur, metastasize, or are the cause of death
in patients with follicular derived carcinomas will usually have a high FAL . Therefore, a
thyroid tumor suppressor gene panel can be used to aid in distinguishing the histologic category of the
tumor and can also serve as a marker for the potential aggressiveness of the tumor.
The use of the genotyping panel is limited in papillary carcinomas since these tumors, including the
follicular variant of papillary carcinomas, appear to have a low fractional allelic loss
Molecular panel for detecting anaplastic de-differentiation
Anaplastic transformation can occur in all types of thyroid carcinoma, though it is
probably more often recognized in association with papillary carcinomas . Anaplastic
transformation is a dreaded occurrence in thyroid cancer patients, since most of these patients will die
within 6 months of their diagnosis
. Despite the fact that we cannot predict which
cancers are at risk for anaplastic transformation, we do have a fairly good understanding of the
molecular events that accompany this dedifferentiation. In particular, it is clear that a significant
mutational burden accompanies anaplastic transformation
. Similarly, anaplastic
dedifferentiation of a follicular derived tumor is accompanied by a very high FAL
The hope for future studies will be discovering the specific mutational events that initiate
transformation, or defining at risk well-differentiated tumors in the early stages, to optimize treatment
Reporting issues for clinical molecular testing in thyroid disease
False negatives and false positives
Molecular testing for translocations and point mutations is highly sensitive and very specific as
well. But, the mutation itself is not a highly sensitive predictor of carcinoma (though it is probably
quite specific), since a significant proportion of malignant tumors will not harbor a definite somatic
point mutation or translocation. Importantly, therefore, the absence of mutations should not be seen as
a reliable indicator of the absence of malignancy. Furthermore, the absence of detection of the fusion
mRNA, particularly in paraffin samples, may mean that a component of the reaction did not
work . Robust controls are needed for every step of the assay in order to have confidence in
the final result.
Again, our ability to maximize the benefit of molecular testing in anatomic pathology rests entirely
on our willingness to integrate all of the data and interpret the novel testing in light of the cytologic
and histologic findings.
Diagnostic implications of testing result: Unmasking the follicular genotype
For years and years, the gold standard has been the H&E, with even very little
supplementation from immunohistochemistry or other adjuvant tests, such as electron microscopy. And,
even today and into the foreseeable future, the H&E (or Pap stain) will remain the gold standard. In
today's era, however, we know a lot about the mutational activity in thyroid follicular lesions and these
can be useful to us as diagnosticians. There are many questions left unanswered, however, and many of
them center on the lesion that we call follicular variant of papillary carcinoma. Are these follicular
lesions, with papillary nuclei? Are they papillary lesions with follicular growth? The molecular data
on these tumors has aroused the curiosity of the entire pathology community and we are still waiting for
What is the follicular genotype? Follicular tumors do have a distinctive genotype. Many
tumors will harbor at least one of the follicular mutations. For example, we do know that follicular
tumors often have RAS mutations, or PAX8-PPARg translocations; we also know that they have tumor
suppressor gene mutations that increase in frequency as tumors become more aggressive. Finally, we know
that they do not usually harbor BRAF mutations or RET-PTC translocations. All of these features come
together as the follicular genotype.
How can we use the follicular genotype? I envision the use as a supplemental test in fine needle
aspirations and surgical pathology material. An important question is how we can utilize these
molecular tests in the pre-operative setting on FNA specimens. Will we be confident enough that false
positives are minimized to recommend a total thyroidectomy based upon the molecular results? In the
pre-operative setting, cases with a specific follicular mutation or a high burden of tumor suppressor
gene mutations may be triaged to surgery, since these are associated at least with neoplasia, if not
frank carcinoma. In the post-operative setting, the follicular genotype can help in making diagnoses in
subtle cases, cases with incompletely developed histologic features, and in cases in which the tumor is
fragmented or incompletely sampled (after attempting to rectify this histologically).
Summary: "Lasagna Diagnostics"
The diagnostic use of molecular markers for thyroid carcinomas has not been fully
realized. Though there will certainly be false positives, many have argued that these are pre-malignant
changes that should still be taken seriously in the setting of a thyroid nodule. In clinical practice,
it is likely that the mutation assessment in thyroid surgical specimens will be useful not only
diagnostically, but also will have value for treatment planning in the future.
In all of pathology, molecular testing is becoming more available as a tool to aid in
diagnostics, therapeutics, and prognostics for patient care. In the best practice, however, the adjuvant
assays will be utilized in conjunction with the solid foundation of the cytologic, frozen section,
well-characterized histologic, and immunohistochemical findings. Because of the common problems of false
negatives, false positives, or early genetic changes associated with pre-neoplastic histology, the
morphology should always guide interpretation of the molecular tests. I view this layered approach to
diagnostics as "lasagna diagnostics". Without the solid foundation and each intervening layer, the
molecular test will have no value.
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