1. Understanding follicular variant of papillary carcinoma and differentiating it from benign and malignant follicular derived tumors.
   
   
2. Differentiating follicular-derived carcinoma from benign follicular lesions.

RAS Proto-oncogene
The RAS proto-oncogenes are commonly mutated in many different types of human cancers [6]. 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 [7, 8] . N-RAS mutations may be more frequent than K-RAS and H-RAS point mutations [9].

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 [10]. Importantly, however, RAS mutations are found in low frequency in non-neoplastic conditions such as colloid nodules and goiter, and in papillary carcinomas [7, 11] . Some suggestion that mutations are more frequent in poorly differentiated tumors has also been made [8].

BRAF
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) [12, 13, 14] .

The BRAF mutations have been reported in between 35% and 69% of papillary thyroid carcinomas [3, 12] . In several large series, BRAF mutations were not found other types of thyroid tumor, except anaplastic carcinomas derived from well-differentiated papillary carcinomas [3, 15] . The BRAF mutation does not appear to have any prognostic significance [16]. 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 [17].

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 [18].

PAX8-PPARγ
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 fusion product.

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 [19, 20, 21, 22, 23] . 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 [24], presumably follicular adenomas, and the protein PPARγ may be overexpressed in both as well [20, 25, 26] . 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 [27]. 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 [28].

Translocation analysis
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 [26]. Protein over-expression, however, can be driven by numerous factors other than translocations [25]. 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 [19]. 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 [29]. The most common partner genes are ELE1 (PTC3) and H4 (PTC1), both of which are also located on chromosome 10 [30, 31, 32, 33] . 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 [34, 35, 36] . Translocations are found in approximately 30-40% of papillary carcinomas; only minor geographic differences have been detected [37, 38] .



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 [39]. 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 [40]. The RET-PTC translocations have not been identified in non-papillary well differentiated tumors [41], though they are present in hyalinizing trabecular tumors [42, 43] . 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 not [44].

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 [22]. 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 described below.

Tumor suppressor gene panel for diagnosis and prognosis
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 [45, 46, 47] . 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 [4, 48, 49] . Fractional allelic loss in well-differentiated papillary carcinomas is most often low, and only increases in the presence of anaplastic transformation [50, 51] .



In thyroid 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% [4]. Furthermore, tumors that recur, metastasize, or are the cause of death in patients with follicular derived carcinomas will usually have a high FAL [52]. 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 [45, 47] .

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 [53]. Anaplastic transformation is a dreaded occurrence in thyroid cancer patients, since most of these patients will die within 6 months of their diagnosis [54, 55] . 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 [51, 56, 57] . Similarly, anaplastic dedifferentiation of a follicular derived tumor is accompanied by a very high FAL [49, 51, 57] . 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 and follow-up.

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 [58]. 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 firm answers.

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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