Marina N. Nikiforova

Molecular profile and clinical-pathologic features of the follicular variant of papillary thyroid carcinoma. An unusually high prevalence of ras mutations.

The follicular variant (FV) of papillary thyroid carcinoma is characterized by a follicular growth pattern and cytologic features of papillary carcinoma. ret/PTC rearrangements are common in classic papillary thyroid carcinoma (PTC) and PAX8-PPAR gamma and ras mutations in follicular thyroid carcinoma. Their prevalence in FV has not been established. We studied these genetic alterations and clinical-pathologic features in 30 FV cases and compared those with 46 non-FV papillary carcinomas. FV cases revealed 1 ret/PTC rearrangement (3%) and 13 ras mutations (43%). Non-FV cases harbored 13 ret/PTC (28%) (P = .006) and no ras mutations (P = .0002). No PAX8-PPAR gamma was found in either group. FV cases demonstrated a significantly higher prevalence of tumor encapsulation, angiovascular invasion, and poorly differentiated areas and a lower rate of lymph node metastases. These data indicate that the FV of papillary carcinoma has a distinct set of molecular alterations and is characterized by a high frequency of ras point mutations.

PAX8-PPARγ Rearrangement in Thyroid Tumors: RT-PCR and Immunohistochemical Analyses

A PAX8-PPAR&ggr; rearrangement has been recently identified in follicular thyroid carcinomas, but not in follicular adenomas or other thyroid tumors. We report here the analyses of PAX8-PPAR&ggr; in a series of 118 thyroid tumors using a newly developed RT-PCR assay to detect this rearrangement in frozen and paraffin-embedded tissues and using immunostaining with a PPAR&ggr; antibody. PAX8-PPAR&ggr; was detected by RT-PCR in eight of 15 (53%) follicular carcinomas and two of 25 (8%) follicular adenomas but not in 35 papillary carcinomas (including 12 follicular variants), 12 Hurthle cell carcinomas, 12 Hurthle cell adenomas, two anaplastic carcinomas, one poorly differentiated carcinoma, or 16 hyperplastic nodules. The prevalence was higher in follicular carcinomas from patients with a history of radiation exposure (three of three). Strong, diffuse nuclear immunostaining with the PPAR&ggr; antibody correlated with the presence of PAX8-PPAR&ggr; detected by RT-PCR. Most sporadic follicular carcinomas positive for PAX8-PPAR&ggr; were overtly invasive, whereas tumors lacking the rearrangement were predominantly minimally invasive. The two follicular adenomas positive for PAX8-PPAR&ggr; had trabecular growth pattern and thick capsule, but no invasion, and thus may represent “pre-invasive” follicular carcinomas. The absence of PAX8-PPAR&ggr; rearrangements in Hurthle cell tumors and papillary thyroid carcinomas highlights the differences in the molecular pathogenesis of these thyroid tumors.

Solid variant of papillary thyroid carcinoma: incidence, clinical-pathologic characteristics, molecular analysis, and biologic behavior.

Solid variant is a rare and poorly characterized variant of papillary thyroid carcinoma. In this study we analyzed 20 primary cases of the solid variant of papillary carcinoma found in a series of 756 papillary carcinomas operated at the Mayo Clinic between 1962 and 1989. The criteria for classification included predominantly (>70%) solid growth pattern of primary tumor, retention of cytologic features typical of papillary carcinoma, and absence of tumor necrosis. For each case of the solid variant, a control case of classical papillary carcinoma matched by age, sex, tumor size, and length of follow-up was selected. The follow-up ranged from 6 to 32 years. Two patients with the solid variant of papillary carcinoma (10%) died from disease 7 and 10 years after initial surgery, while another two patients (10%) are alive with lung metastases. In contrast, the control group had no cases with distant metastases or death from disease. Molecular analyses showed a similar prevalence of RET/PTC rearrangements in both groups. In conclusion, the solid variant of papillary carcinoma is associated with a slightly higher frequency of distant metastases and less favorable prognosis than classical papillary carcinoma. However, it should be distinguished from poorly differentiated thyroid carcinoma, which has a reported lower survival rate compared with the solid variant of papillary carcinoma.

Prevalence of RET/PTC Rearrangements in Hashimoto's Thyroiditis and Papillary Thyroid Carcinomas

The relationship between Hashimotos thyroiditis (HT) and follicular cell-derived thy roid cancer remains unclear. Recently, 2 studies reported a 95% prevalence of RET/PTC rearrangements in histologically benign tissue affected by HT, suggesting that multiple occult tumors exist in HT patients with high frequency. We tested the prevalence of RET/PTC rearrangements in 26 HT, in 6 papillary carcinomas arising in the background of HT, and in 27 papillary carcinomas not associated with HT. We detected no RET/PTC rearrangements in HT or papillary carcinomas arising in the background of HT, in contrast to a 33% prevalence among papillary carcinomas not associated with HT. However, the expression of wild-type RET was found in more than half of papillary carcinomas. These results suggest that, if the association be tween HT and thyroid cancer exists, its molecular basis is different from RETIPTC rearrangement. Int J Surg Pathol 10(1):15-22, 2002

Clinical laboratory reports in molecular pathology

CONTEXT Molecular pathology is a rapidly growing area of laboratory medicine in which DNA and RNA are analyzed. The recent introduction of array technology has added another layer of complexity involving massive parallel analysis of multiple genes, transcripts, or proteins. OBJECTIVE As molecular technologies are increasingly implemented in clinical settings, it is important to bring uniformity to the way that test results are reported. DATA SOURCES The College of American Pathologists Molecular Pathology Resource Committee members summarize elements that are already common to virtually all molecular pathology reports, as set forth in the College of American Pathologists checklists used in the laboratory accreditation process. Consensus recommendations are proposed to improve report format and content, and areas of controversy are discussed. Resources are cited that promote use of proper gene nomenclature and that describe methods for reporting mutations, translocations, microsatellite instability, and other genetic alterations related to inherited disease, cancer, identity testing, microbiology, and pharmacogenetics. CONCLUSIONS These resources and recommendations provide a framework for composing patient reports to convey molecular test results and their clinical significance to members of the health care team.

Establishment of a non-tumorigenic papillary thyroid cell line (FB-2) carrying the RET/PTC1 rearrangement.

A novel human thyroid papillary carcinoma cell line (FB‐2) has been established and characterized. FB‐2 cells harbor the RET/PTC1 chimeric oncogene in which the RET kinase domain is fused to the H4 gene. FB‐2 cells neither formed colonies in semisolid media nor induced tumors after heterotransplant into severe combined immunodeficient mice. However, HMGI(Y), HMGI‐C and c‐myc genes, which are associated to thyroid cell transformation, were abundantly expressed in FB‐2 cells but not in normal thyroid cells. FB‐2 cells only partially retained the differentiated thyroid phenotype. In fact, the PAX‐8 gene, which codes for a transcriptional factor required for thyroid cell differentiation, was expressed, while thyroglobulin, TSH‐receptor and thyroperoxidase genes were not. Moreover, FB‐2 cells produced high levels of interleukin (IL)‐6 and IL‐8.

Prevalence of minisatellite and microsatellite instability in radiation-induced post-Chernobyl pediatric thyroid carcinomas

Exposure to ionizing radiation induces different forms of genomic instability in cultured cells and experimental animals. A higher rate of germline mutations at human hypervariable minisatellite loci was reported in children born from parents exposed to radiation after Chernobyl, implicating genome destabilization as a possible mechanism responsible for late radiation effects in humans. To test if radiation-induced carcinogenesis in the thyroid gland may be associated with somatic minisatellite instability or microsatellite instability, we utilized a PCR-based approach to study normal and tumor DNA from 17 pediatric post-Chernobyl papillary thyroid carcinomas for mutations at three different minisatellite loci (D1S80, D17S30, ApoB), and 27 microsatellite loci of di-, tri-, or tetranucleotide repeats. Minisatellite instability was found in three (18%) tumors, with one of them exhibiting mutations in all three minisatellite loci, whereas two others showed mutations in one of two informative markers. By contrast, none of 20 sporadic thyroid cancers from patients with no history of radiation exposure was positive for minisatellite instability. Microsatellite analysis of post-Chernobyl tumors revealed a mutation in one (6%) tumor only at the locus of D10S1412, whereas all other 26 microsatellite markers showed identical patterns in each normal/tumor pair. Our results suggest that somatic cell microsatellite instability does not contribute to radiation-induced thyroid carcinogenesis. However, somatic minisatellite mutation events are present in a subset of radiation-induced, but not sporadic, thyroid cancers, suggesting that this type of genomic instability may play a role in radiation-induced tumorigenesis in the thyroid gland.

Detection of SYT-SSX Rearrangements in Synovial Sarcomas by Real-Time One-Step RT-PCR

Synovial sarcomas are aggressive tumors of adolescent and young adults that account for up to 10% of soft tissue sarcomas. Cytogenetically, they are characterized by translocation t(X;18), which is found in more than 95% of tumors. In most cases, it results in fusion of the SYT gene with the SSX1 or SSX2 gene, thus creating SYT-SSX1 or SYT-SSX2 rearrangement. The 2 types of gene fusion have been correlated with histologic variants and prognosis of synovial sarcomas. In this study, we developed a simple and rapid method for the simultaneous detection of SYT-SSX1 and SYT-SSX2 rearrangements by using a LightCycler real-time one-step reverse transcriptase polymerase chain reaction (RT-PCR) technology (Roche). Oligonucleotide probes were designed so that the donor probe would span a fusion point and the acceptor probe would be complementary to the SSX1 sequence but have 2 nucleotide mismatches with SSX2 sequence. Such a design allows simultaneous amplification of 2 types of rearrangement in the same reaction but distinguishes them based on differences in melting temperature detected by melting curve analysis after PCR. With this method, 27 tumors (9 synovial sarcomas and 18 nonsynovial sarcomas) were studied and showed SYT-SSX1 rearrangement in 6 cases and SYT-SSX2 in 3 cases. These results had complete correlation with the finding of conventional RT-PCR and direct sequencing. In conclusion, we have developed a fast, accurate, and simple method for the detection of 2 major types of SYT-SSX rearrangement by using LightCycler RT-PCR and melting curve analysis.