Clonal origin of non-medullary thyroid tumours assessed by non-random X-chromosome inactivation

Abstract

Objective: X-chromosome inactivation analysis was performed in order to assess the clonal origin of non-medullary thyroid tumours and to distinguish between multicentricity and multifocality in multiple papillary thyroid carcinoma (PTC). Methods: One hundred and thirteen tumour samples from 31 patients with isolated PTC, 16 patients with multinodular PTC, 14 patients with follicular thyroid adenoma (FTA) and 15 patients with follicular thyroid carcinoma (FTC) were collected. The corresponding normal thyroid tissues were analysed, and in 14 cases, tumour-surrounding tissue was also studied. Genomic DNA was digested with HpaII and HhaI previous to PCR amplification of the polymorphic CAG repeat, on exon 1 of the human androgen receptor gene (HUMARA). PCR products were analysed by denaturing gel electrophoresis, silver staining and densitometric analysis. PCR products were also used to determine the number of CAG repeats of patients with isolated PTC, FTA, FTC and of 41 healthy volunteers. Results: Heterozygosity for the HUMARA polymorphism was found in 64/76 (84%) cases. Lyonization of the thyroid was observed in 15/76 (20%) cases, which were excluded from clonal analysis. Except for two cases of isolated PTC, all tumour samples studied presented monoclonal X-inactivation patterns, while normal thyroid tissue was polyclonal. Monoclonal patterns were also found in 4/14 tumour-surrounding tissues. No difference was found in the length of CAG alleles between patients and controls. Of eight informative cases of multinodular PTC, three showed evidence of multicentricity and five revealed patterns consistent with multifocality. Conclusions: Both isolated and multinodular PTC as well as FTA and FTC are of monoclonal origin. Our results also suggest that approximately one-third of multiple PTC have an independent origin for the different nodules (multicentricity). Monoclonality was also found in tissues surrounding some PTC nodules. No association was found between the length of CAG alleles and thyroid malignancies.

Clonal analysis of bilateral, recurrent, and metastatic papillary thyroid carcinomas

“…To account for possible differences in fluorescence signal acquisition and gel loading of each allele, we modified to uniform the volume of internal size marker (Genescan-500Liz; ABI Perkin Elmer, Warrington, United Kingdom) and calculated a corrected allele ratio as follows: (peak 1 height of undigested sample ÷ peak 2 height of undigested sample ÷ Liz 200 bp height of undigested group)/(peak 1 height of digested sample ÷ peak 2 height of digested sample ÷ Liz 200 bp height of digested group). We defined tumor samples as nonrandom XCI if this corrected ratio was less than 0.5 or greater than 2 [27]. Tumors were considered to be of single clonal origin if the same inactivation pattern was detected in each separate tumor from individual patient.…”

Heterogeneity of Thyroid Cancer

“…Both hypotheses, as presented by Kuhn et al [39], were confirmed in several studies. Despite the differences in molecular techniques among these studies, the common conclusion was that PTC multiplicity could be a result of either multicentricity or intrathyroidal spread [40-42]. One particularly interesting finding was that the ipsilateral PTC foci showed the same X chromosome inactivation pattern, and discordance was observed between contralateral nodules [39].…”

Molecular pathology of well-differentiated thyroid carcinomas

“…Furthermore, PTCs display microsatellite stability [56]. Taking these facts together with the tendency of PTC to occur as multicentric neoplasms, which are clonally independent from each other [37,54], it is tempting to advance that PTC tumorigenesis reflects the end product of (very) few oncogenic steps.…”