MEN2A or Sipple’s syndrome is an autosomal dominant disorder characterized by MTC and pheochromocytomas and hyperparathyroidism in about 50 % and 30 % of affected individuals, respectively. Other less common manifestations of MEN2A include cutaneous lichen amyloidosis and Hirschsprung’s disease [159]; 97 % of MEN2A is caused by activating missense germline RET mutations in exon 10, codons 609, 611, 618, and 620, and in exon 11, codons 630 and 634, all of which enhance receptor dimerization, with increased receptor autophosphorylation and hence increased kinase activity [139–148, 160]. Molecular diagnostic testing for MEN2A is usually directed at these specific mutations with methods described above.

MEN2B or Wermer’s syndrome is characterized by MTC, pheochromocytoma, mucosal neuromas (of the tongue, lips, conjuctivae), ganglioneuromas of the intestines, characterized by special facial appearance, and marfanoid habitus. Interestingly, the MTC of MEN2B is more aggressive than those seen in MEN2A or FMTC; 98 % of MEN2B carry RET mutations in either exon 16, codon 918, or exon 15, codon 833 [140–148, 160]. Directed testing for these mutations is the most sensitive, rapid, and accurate method for identifying individuals with MEN2B [154].

MTC is the only manifestation of FMTC and typically follows a very indolent course. A kindred should have 10 or more RET mutation carriers with MTC and sufficient history to rule out pheochromocytoma or hyperparathyroidism [159]. FMTC is characterized by the same mutations as are found in MEN2A, with a few individuals having germline mutations in codons 768, 790, 791, 804, and 891 in RET exons 13, 14, and 15 [139–148, 160]. Molecular diagnostic testing is usually directed at the more frequently occurring of these mutations.

Although not presently employed in TC molecular diagnostic testing, differences have been detected in the levels of specific microRNAs in PTC, ATC, MTC, oncocytic tumors, and FTC, with each histopathological TC type showing a unique microRNA expression pattern [161–164]. Additionally, the microRNA expression pattern changed with concomitant BRAF, RET/PTC, and PAX8-PPAR-gamma, but not with RAS mutations, showing possible microRNA expression pattern alterations with mutational status [164]. Although not presently employed in the molecular diagnostics of TC, different microRNA patterns are well established in TC and are likely to be incorporated into TC molecular testing in the future. Interestingly, microRNA studies have proven valuable in increasing our understanding of some thyroid lesions. For example, the hyalinizing trabecular adenoma is a distinctive thyroid neoplasm that shows some morphological features of PTC, including similar nuclear characteristics, psammoma bodies, and RET/PTC rearrangements [165–167]. Sheu et al. [168] analyzed microRNA expression and BRAF V600E mutation and RET/PTC-1 and -3 rearrangements in 18 hyalinizing trabecular adenomas. They found that microRNAs -146b, -181b, -21, -221, and -222, which are all significantly up-regulated in PTC, were suppressed in hyalinizing trabecular adenomas. Additionally, no BRAF V600E mutation and RET/PTC-1 and -3 rearrangements were detected. Based on this, the authors concluded that the hyalinizing trabecular adenoma and PTC are separate entities. The reasons for the discrepant findings of this study vs. previous studies concerning RET/PTC-1 and -3 rearrangements were not explained [167, 168].

HRPT2 is a recently identified PC tumor suppressor found at 1q25 and has 17 exons that encode a 61 kDa nuclear protein parafibromin [174]. Germline mutations of the gene have been identified in kindreds with hyperparathyroidism-jaw tumor syndrome, which is characterized by childhood parathyroid hyperplasia, fibro-osseous jaw tumors, renal lesions, with a ~ 15 % lifetime chance of developing PC [175, 176]. Howell et al. [177] analyzed 60 parathyroid tumors , including five from individuals with hyperparathyroidism-jaw tumor syndrome, three with familial isolated hyperparathyroidism, three with MEN1, one with MEN2A, 25 sporadic PAs, 17 hyperplastic parathyroid glands, two lithium-associated tumors, and four sporadic PCs. All 17 exons and intron-exon boundaries were PCR amplified and analyzed by denaturing high-performance liquid chromotography. HRPT2 mutations were found in all four sporadic PCs, all five tumors from individuals with hyperparathyroidism-jaw tumor syndrome, and in one of the two tumors from individuals with familial isolated hyperparathyroidism. The authors hypothesized that HRPT2 mutation is an early event in PC carcinogenesis and may be a potential marker for both familial and sporadic PCs. Interestingly, parafibromin immunostaining is reduced in PCs compared to PA, with positive parafibromin nuclear staining largely excluding carcinoma and reduced staining likely indicating carcinoma or rare PAs with low staining [178, 179].

Cyclin D1 or PRAD1 is located at 11q13 and encodes a cyclin-dependent kinase that plays a central role in regulating and promoting G₁S phase cell cycle progression [180]. Chromosomal rearrangements of the cyclin D1 regulatory region have been detected in 8 % of PAs and 20–40 % of PCS, and 31 % of hyperplastic parathyroid glands overexpress cyclin D1 [181, 182]. The overexpression appears to result from the effects of growth factor stimulation and calcium-sensing receptor activation [183].

Ki-67 and PCNA immunohistochemical staining may have some value in separating PC from benign hyperplasia and adenomas, although the literature is contradictory and due to the rarity of PC most studies include relatively few cases [184, 185]. Last, the proapototic gene product FHIT appears to be preferentially mutated in PC compared to hyperplastic or adenomatous parathyroid lesions [185].

PC and PAs show gains and losses in specific chromosomal locations, often where tumor suppressor genes (at 1p, 3q, 4q, 13q, and 21q) and oncogenes are located (at 1q, 5q, 9q, 16p, 19p, and Xq). Interestingly, these areas tend to be different between PCs and PAs, likely indicating a different molecular pathogenesis of these neoplasms [186]. Presently, none of the above molecular alterations identified in PC, PA, or hyperplasia are used in molecular diagnostics, and many of these alterations appear to be quite similar between PC and PAs. Further research is required before molecular diagnostic tests for parathyroid neoplastic lesions can be implemented.