Clonal Analysis Of Papillary Thyroid Carcinoma

Thyroid cancer is the most common endocrine neoplasm which accounts for roughly 1% of all new malignant disease. With the incidence increasing by 6.2% per year, thyroid cancer is currently the seventh most common malignancy diagnosed in women in U.S. population.Previous studies have shown that thyroid cancer displays several highly prevalent genetic alterations, like RAS mutations in follicular thyroid carcinoma; rearrangements of the RET in papillary thyroid carcinoma and thyroid adenoma; and P53 mutations in anaplastic thyroid carcinoma. Among these alterations, mutations in BRAF gene are the most common. The point mutation in exon 15 of BRAF gene has been found to result in a valine to glutamine conversion at codon 600 (BRAFV600E mutation) with an average prevalence of 45% in papillary thyroid cancer. It has been reported that the BRAFV600E mutation is associated with one or more conventional high-risk clinicopathological characteristics of PTC, like lymph node metastasis, extrathyroidal invasion, and advanced disease stages. Until now, there is few study with a relative larger sample size to investigate the prevalence and possible prognostic value of BRAFV600E mutation in Chinese population.Papillary thyroid carcinoma (PTC) is the most common histotype of thyroid cancer. PTC is often multifocal, with a reported frequency varying widely from 18 to 87 percent. Multifocality in PTC is associated with an increased risk of lymph node metastases and regional recurrence. However, as a specific subtype of multifocal PTCs, bilateral papillary thyroid carcinoma has not been widely investigated. In addition, in patients with local or distant recurrence after lobectomy, a tumor is found in more than 60% of the time in the resected contralateral lobe. The clonality of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe remains unknown.Part one. BRAF mutation in Chinese population of thyroid cancerPurpose:To determine the incidence and define the possible role of BRAFV600E mutation in Chinese population of thyroid cancer.Methods:We established a computer-based database comprising 1006 thyroid cancer patients and clinically followed them up with a mean time of six years (range from 2.5 years to 13.5 years). Two hundreds and twenty patients were enrolled from this database containing 208 papillary thyroid cancers,8 follicular thyroid cancers,1 medullary thyroid cancers and 3 anaplastic thyroid cancers. Also, a control group of 46 cases of benign thyroid tumors were studied. After compared the distinct methods of extracting genomic DNA from formalin-fixed paraffin-embedded tissues (FFPET), we screened the BRAF gene by DNA sequencing.Results:One hundred and fifteen PTCs were positive for the BRAFV600E mutation (55.3%,115/208). We confirmed previous reports that BRAFv600E mutation did not occur in benign disorders. This mutation was associated with older age (P= 0.004) and poorer prognosis (close to statistic difference, P= 0.068).Conclusions:BRAFV600E mutation was also common in Chinese population of thyroid cancer and might be an indicator of poorer prognosis.Part two. Clonal analysis of bilateral papillary thyroid carcinomasPurpose:To address the issue of clonal origin of tumors appearing simultaneously in both lobes or recurring in the contralateral lobe.Methods:we examined twenty-five pairs of bilateral papillary thyroid carcinomas (synchronous or metachronous) and 15 matched metastatic lymph nodes. BRAF gene mutation analysis combined with X-chromosome inactivation was used to evaluate these tumors' clonal origins. Genomic DNA was prepared from paraffin-embedded tissues after microdissection.Results:In total,62 tumors yielded DNA of adequate quality. Eighteen out of 21 informative cases (18/21,85.7%) showed concordant BRAF status in tumors from both thyroid lobes, be either BRAF mutation-positive (12 patients) or BRAF mutation-negative (6 patients). Metastatic lymph nodes in 12 patients (12/15,80%) had a complete concordance of BRAF state with their primaries. Of the 18 studied female patients, eleven were suitable for X-chromosome inactivation assay. Nine cases (9/11, 81.1%) showed the same pattern of inactivation in bilateral tumors. A close correlation was found between BRAF mutation and X-chromosome inactivation analysis.Conclusions:our data provide evidence that bilateral, recurrent and metastatic papillary thyroid carcinomas often arise from a single clone, and that intrathyroidal metastasis may play an important role in the development of bilateral tumors, as well as in the recurrence of this malignancy.Part three. Molecular characterization of novel BRAFV599Ins mutationPurpose:In the study of Part two, we report a rare BRAF gene mutation in a bilateral PTC, namely a 1795GTT insertion, resulting in BRAFV599Ins, and here we describe its biochemical characterization.Methods:We studied the MAPK cascade in HEK293 cells transiently transfected with the various BRAF constructs. Then MTT assay and flow cytometry were done to test the cell viability and the possible change of cell cycle. Results:Phosphorylation of ERK was virtually low in vector-transfected cells, while the MAPK cascade was significantly more active in cells expressing BRAFV600E or BRAFV5991ns than in cells expressing BRAFWT. Cells transfected with BRAFV600E or BRAFV599Ins obviously promoted cell growth and had a higher rate of S phase. Conclusions:This study demonstrated that BRAFV599Ins mutation, as BRAFV600E, is a "gain of function" mutation...