Section 2 -

Molecular Pathology of Thyroid Tumors Yuri E. Nikiforov Department of Pathology and Laboratory Medicine University of Cincinnati College of Medicine Cincinnati, Ohio

During the last two decades we have witnessed a dramatic progress in our understanding of the molecular biology of thyroid tumors. It has become clear that molecular pathogenesis of thyroid papillary carcinomas involve mutations that activate mitogen-activated protein kinase (MAPK) pathway, some of which represent promising targets for treatment with specific molecular inhibitors. The identification of the gene responsible for the familial forms of medullary carcinomas has led to a dramatic change in the management of patients with this disease, and is one of the first examples when preventive surgery is performed solely on the basis of molecular genetic testing. The progress in molecular biology is expected to affect virtually all aspects of thyroid pathology and provide significant help in the diagnosis of thyroid tumors, determining tumor prognosis, and prediction of tumor response to specific treatment modalities. Papillary Carcinomas. These tumors frequently have mutations of genes coding for proteins which signal along the MAPK pathway. Activating mutations of the BRAF, RET, or RAS genes are found in approximately 70% of all cases and rarely overlap in the same tumor [1]. Point mutations of the BRAF gene coding for serine/threonine kinase are found in ~40% of papillary carcinomas [1, 2, 3]. Virtually all mutations involve nucleotide 1799 and result in a valine-to-glutamate substitution at residue 600 (V600E). Among thyroid tumors, the V600E BRAF mutation is restricted to papillary carcinoma and poorly differentiated and anaplastic carcinomas arising from papillary carcinoma. Therefore, the identification of this mutation in cells from fine needle aspiration of the thyroid gland or in surgical material is virtually diagnostic for papillary carcinoma [4, 5]. BRAF mutations are associated with older patient age, classic papillary histopathology or the tall cell variant, a higher rate of extrathyroidal extension, and more advance tumor stage at presentation [6]. RET/PTC rearrangement is another common genetic alteration in papillary carcinomas [7]. It results in fusion of the 3' portion of the RET receptor tyrosine kinase gene to the 5' portion of various genes. Two most common rearrangement types, RET/PTC1 and RET/PTC3, are paracentric inversions since both RET and its respective fusion partner, H4 or ELE1 (NCOA4), reside on the long arm of chromosome 10. RET/PTC2 and several additional variants of the RET/PTC oncogene are rare. The prevalence of RET/PTC in papillary carcinomas from the general population is ~20%, it has significant geographic variation and is higher in tumors from patients with history of radiation exposure and in pediatric populations [8]. In tumors arising after radiation exposure, RET/PTC1 was found to be associated with classic papillary histology, whereas RET/PTC3 type was more common in the solid variant [9]. Overall, tumors with RET/PTC rearrangements usually presents at younger age, has classic papillary histology, frequent psammoma bodies, and a high rate of lymph node metastases [6]. Although several studies reported the detection of RET/PTC in thyroid adenomas and other benign thyroid lesions, it is generally accepted that strong RET/PTC expression is restricted to papillary carcinomas. Point mutations involving several specific sites (codons 12, 13, and 61) of the N-RAS, H-RAS, or K-RAS genes are found in 10-15% of papillary carcinomas [10, 11]. Tumors with RAS mutations are almost all the follicular variants of papillary carcinoma, this mutation also correlates with significantly less prominent nuclear features, more frequent encapsulation, and low rate of lymph node metastases [6]. Mutations of the RAS gene are not restricted to papillary carcinoma and also found in other benign and malignant thyroid tumors, as well as in neoplasms from other tissues. Follicular Carcinomas: Most frequent genetic alterations in these tumors include point mutations of the RAS genes and PAX8-PPARg rearrangement. RAS mutations, most commonly affecting N-RAS codon 61 or H-RAS codon 61, are found in 40-50% of follicular carcinomas, and may correlate with tumor dedifferentiation and less favorable prognosis [12, 13]. PAX8-PPARg fusion results from the translocation t(2;3)(q13;p25) [14]. It occurs in ~25% of follicular carcinomas and in ~7% of follicular adenomas [15, 16]. It has been suggested that follicular adenomas positive for this rearrangement may be pre-invasive follicular carcinomas or tumors where invasion was overlooked [16]. Tumors with PAX8-PPARg tend to present at a younger age, be smaller in size, and more frequently have vascular invasion than those with mutant RAS [15, 16]. The incidence of RAS and PAX8-PPARg mutations is significantly lower in oncocytic follicular carcinomas. RAS mutations can not be used as a diagnostic marker of follicular carcinoma since they also occur with significant prevalence in follicular adenomas and the follicular variant of papillary carcinoma. However, detection of PAX8-PPARg rearrangement may be of diagnostic value since it occurs almost exclusively in follicular carcinomas. The rearrangement results in overexpression of PPARg protein that can be detected by immunohistochemistry [14]. However, only strong diffuse nuclear staining correlates with the presence of rearrangement [17]. The usage of this immunostaining may be challenging since not all commercially available PPARg antibodies give a reliable result. The presence of strong diffuse PPARg staining in the tumor nodule, especially if confirmed by RT-PCR or FISH, should justify the submission of additional sections of the capsule and obtaining deeper levels of all suspicious areas in search for capsular or vascular invasion. Follicular carcinomas are characterized by a considerable rate of loss of heterozygosity (LOH) and frequent losses of multiple chromosomal regions. The average rate of LOH per chromosome arm is ~20% in follicular carcinomas, as compared to 6% in follicular adenomas, and only 3% in papillary carcinomas. The most commonly deleted regions in follicular carcinomas are on chromosomes 2p, 3p, 9q, 10q, 11p, 15q, and 17p. conventional follicular tumors. The presence of numerous mitochondria on oncocytic tumors may be a consequence of somatic mutations and sequence variants in mitochondrial DNA [18]. Poorly Differentiated Carcinomas: These tumors show a variable frequency of BRAF, RAS, and RET/PTC mutations, which are characteristic of well differentiated papillary and follicular carcinomas, as well as a substantial rate of p53 and β-catenin mutations, which are common in anaplastic carcinomas [19]. No genetic mutations unique for poorly differentiated carcinoma have been identified to date. This suggests that poorly differentiated carcinoma, as a group, represents a distinct step in the evolution from well differentiated to anaplastic thyroid carcinoma, rather than an entirely separate type of thyroid malignancy. Undifferentiated (Anaplastic) Carcinomas: These tumors typically have a highly unstable, complex karyotype with numerous gains and losses of whole chromosomes and smaller chromosomal regions. Point mutations of RAS are found in approximately 60% of undifferentiated carcinomas and mutations in exons 5-8 of p53 in 70-80% of cases. Mutations in exon 3 of the β-catenin (CTNNB1) gene have been reported in 66% of undifferentiated carcinomas [20]. Follicular Adenomas: Molecular alterations include point mutations of the RAS genes, most frequently involving N-RAS codon 61, which are found in about 30% of adenomas. They are not specific for adenomas and also occur in follicular and papillary carcinomas. PAX8-PPARg rearrangement was reported in ~7% of adenomas, and the identification of PAX8-PPARg in a follicular tumor should prompt an exhaustive search for vascular or capsular invasion. Hyperfunctioning adenomas frequently show point mutations in the TSH receptor gene and occasionally in the GSα (GSP) gene. Cytogenetic alterations are found in less than half of adenomas and most frequently manifest as trisomy 7 or translocations involving the long arm of chromosome 19. Oncocytic adenomas frequently show multiple numerical chromosomal abnormalities and somatic mutations and sequence variants in mitochondrial DNA [18]. Hyalinizing Trabecular Tumors: This tumor, previously known as hyalinizing trabecular adenoma, had been suspected for a long time to be related to papillary carcinoma, since it shares with it several diagnostic nuclear features. Several recent reports have identified RET/PTC rearrangement in a significant portion of these tumors, suggesting their link to papillary carcinoma [21, 22]. However, the great majority of these tumors behave as benign neoplasms, and their linkage to papillary carcinoma has not been fully proven. Medullary Carcinomas: RET proto-oncogene as a key molecule in the development of medullary carcinoma, including both familial and sporadic forms of the disease. In these tumors, RET is activated by point mutation, in contrast to its activation by chromosomal rearrangement in papillary thyroid carcinomas. Germline mutations in specific functional regions of RET are found in almost all patients with familial forms of medullary carcinoma. In MEN 2A an familial medullary carcinoma, mutations are typically located in the extracellular domain, within the cysteine rich region [23]. Almost 90% of MEN 2A mutations affect a single codon 634, whereas in familial medullary carcinoma they are more evenly distributed along the cysteine rich region [24]. In MEN 2B, most of the mutations involve codon 918 in the intracellular tyrosine kinase domain. In sporadic medullary carcinomas, somatic mutations of RET are found in 23-70% of cases. The vast majority of those affect codon 918, although they have also been identified in few other regions. References Kimura ET, et al. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 2003 63: 1454-7. Cohen Y, et al. BRAF mutation in papillary thyroid carcinoma. J Natl Cancer Inst 2003 95: 625-7. Nikiforova MN, et al. 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