Section 3 -

Molecular Pathology of Thyroid Tumors Dr. Manuel Sobrinho-Simões Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) Porto , Portugal

This review is restricted to well differentiated carcinomas derived from follicular-cells, thus excluding the C-cell derived tumors (medullary carcinomas). The classification of thyroid tumors advanced in the recently published WHO book [1] is used as a frame. In this new classification, oncocytic (Hürthle cell) tumors are included as variants of their non-oncocytic counterparts (e.g. oncocytic variant of follicular adenoma, papillary carcinoma – PTC – and follicular carcinoma). Follicular carcinoma Despite the huge amount of information collected at the immunohistochemical level [1, 2, 3], the diagnosis of minimally invasive follicular carcinoma still depends on the identification of histological signs of invasiveness. There is no immunohistochemical stain or any molecular feature that may be used as a reliable marker of invasion [1]. There are, nevertheless, some promising attempts to find a combination of molecular features that may be used, even in fine needle aspiration samples, to diagnose malignancy in follicular tumors [4]. It remains to be seen whether or not such an objective will be achievable in concrete, difficult cases. Aneuploidy, ras mutations and PAX 8 PPARγ can not also be used for diagnostic purposes (for a review on this see ref. 5). As with the early data on aneuploidy [6, 7], the occurrence of PAX8 PPARγtranslocation was, at first, thought to be restricted to follicular carcinomas, thus constituting a sign of malignancy [8]. This concept was disproved in several studies that showed the presence of the translocation in a number of follicular adenomas [9]. Curiously, Nikiforova et al [10] advanced that follicular carcinomas with PAX 8 -PPARγ rearrangement tended to be very vascular invasive ("overtly" invasive) and to overexpress galectin 3. Despite still waiting for confirmation in other, independent, series (see below the clinical data on the presence of PAX 8-PPARγ in follicular variant of PTC) these results are important because they reinforce the prognostic meaning of the evaluation of the number and type of vessels invaded in encapsulated follicular carcinomas as it has been stressed by Juan Rosai and other pathologists [1, 2]. Finally, regarding the cyto-architectural features, it should be stressed that follicular adenomas with a fetal/embryonal growth pattern present, like follicular carcinomas, in about 60% of the cases, an aneuploid DNA content which frequently falls within the triploid range [11]. Since it has been shown that both follicular carcinomas and PTC can apparently arise from pre-existing follicular adenomas, it remains to be seen whether or not such malignant transformation may be associated with the fetal-like growth pattern and/or the aneuploid DNA content. From a practical standpoint, it is important to search carefully in follicular lesions with a fetal growth pattern for the existence of PTC-nuclei and for the presence of signs of vascular invasion. Papillary carcinoma The diagnosis of PTC still depends almost exclusively on the identification of the typical nuclei [1, 2]. The differential diagnosis is particularly difficult in the field of encapsulated follicular-patterned lesions that may correspond to follicular adenoma, follicular carcinoma or follicular variant of PTC [1, 2, 12]. At variance with follicular carcinoma, almost every PTC is diploid or quasi-diploid [7]. PTC display, furthermore, microsatellite stability [13]. Taking these facts together with the tendency of PTC to occur as multicentric neoplasms, clonally independent from each other [14], it is tempting to advance that PTC tumorigenesis reflects the end-product of (very) few oncogenic steps [5]. Two major types of molecular alterations have been described in PTC: genetic structural abnormalities, epitomized by RET/PTC and TRK rearrangements and BRAF mutations [15, 16, 17, 18], and epigenetic changes, namely over-expression of c-met and EGR-R, and down-regulation of E-cadherin (for a thorough review see ref. 5). Structural abnormalities are diagnostically more interesting than epigenetic changes. Among the former, RET/PTC rearrangements, which have been shown to serve as an initiating event, are diagnostically better than BRAF mutations which appear to occur as a late event in PTC neoplastic development [19, 20]. Unfortunately, none of the aforementioned molecular features is useful in PTC diagnosis. This holds true even for RET/PTC rearrangements that besides being present only in 16 to 40% of PTC cases [18], are also detected, without leading per se to the diagnosis of PTC, in dispersed cells in foci of Hashimoto's thyroiditis or nodular goiters displaying PTC-like nuclei [3, 21, 22]. It remains to be clarified whether or not some of those epithelial foci with PTC-like nuclei will evolve towards clinically evident PTC. Fusco et al [22] showed that there is indeed RET activation in the nuclei of the focal areas of follicular tumors showing PTC-like nuclei, and concluded that such foci may precede the development of invasive papillary cancer. The putative diagnostic and prognostic significance of BRAF mutations in PTC remains to be clarified [20, 23]. The results on record show that, after melanoma, PTC is the human cancer displaying the highest prevalence of BRAF mutations which occur almost always in the same hot spot (V600E) [16, 17]. Curiously, the BRAF V600E is quasi-restricted to PTC with a conventional (papillary or mixed follicular-papillary) growth pattern, and is particularly prevalent in PTC cases with a prominent papillary structure, as is the case for Warthin-like PTC [17]. The prevalence of the BRAF mutation depends also on the age of patients with PTC, being rarely detected in young patients, both in the post-Chernobyl setting and in sporadic cases occurring in western Europe [19, 20]. Follicular variant of papillary carcinoma In contrast to conventional PTC, a distinct BRAF mutation (K601E) has been detected in 7 to 9% of cases of FVPTC [17, 20]. E-cadherin immunoexpression in FVPTC resembles that of follicular carcinoma (and normal thyroid) rather than that of conventional PTC [24]. The same holds true regarding some cytogenetic alterations [25, 26]. The frequency of ras mutations is much higher in FVPTC than in conventional PTC [26, 27] thus approaching the FVPTC to follicular carcinoma. Finally, we have recently demonstrated that the PAX 8 -PPAR γ rearrangement is detected in about one third of cases of FVPTC [26, 28]. These data support the conclusion that FVPTC shows genetic differences from conventional PTC and suggest that at least a subset of FVPTC shares some of the molecular features of follicular carcinomas [26]. It remains to be seen if the subset of FVPTC displaying genetic alterations of the follicular carcinoma-type encompasses cases particularly prone to give rise to lung and/or bone metastases together with nodal metastases. The peculiar blood born metastatic pattern of some cases of FVPTC has been highlighted many years ago by Carcangiu et al [29]. Recently, the group of Virginia LiVolsi has suggested the existence of a so-called hybrid carcinoma – "Are there tumors that show features of both follicular and papillary cancer? Yes, the so-called hybrid carcinomas (solitary encapsulated tumors displaying vascular invasion and some nuclear features suggesting papillary carcinoma)" [30]. The idea of a "hybrid" carcinoma in the thyroid, sharing the molecular pathways and the clinicopathologic features of papillary and follicular carcinomas is very appealing from a conceptual standpoint [31]; however, we think that, for the moment, there is not enough evidence to go further than acknowledging that a subset of FVPTC resembles follicular carcinoma clinically, histopathologically and molecularly [26, 31]. Oncocytic (Hürthle cell) tumors This topic has been addressed very recently by our group [32, 33, 34]. Briefly, tumors with oncocytic (Hürthe cell) features may be benign (oncocytic variant of follicular adenoma) or malignant (oncocytic variant of papillary carcinoma and oncocytic variant of follicular carcinoma). Medullary and poorly differentiated carcinomas may also display oncocytic features [34]. The criteria used in the diagnosis of the oncocytic variants of PTC and follicular carcinoma are those used in the diagnosis of conventional tumors, i.e., the nuclear characteristics in PTC and the signs of capsular and/or vascular invasion in follicular carcinoma [32, 34] The presence of abundant (and abnormal) mitochondria in the cytoplasm of the neoplastic cells may be seen throughout the entire tumor - Primary oxyphilia, indicating that the carcinogenic hit has occurred in cells with abnormalities of the mtDNA, or mitochondrial abnormalities resulting from mDNA encoding mitochondrial enzymes –, or just in some parts of the tumor – Secondary oxyphilia, indicating that the mitochondrial abnormalities have occurred after tumor development [32, 34, 35]. The oncocytic variant of PTC has RET/PTC rearrangements and BRAF mutations with similar frequency as non-oncocytic papillary carcinomas [17, 36]. 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