RET/PTC Gene Rearrangements in Thyroid Carcinogenesis: Assessment and Clinico-Pathological Correlations.

Rearranged during transfection (RET) is a proto oncogene implicated in thyroid carcinogenesis of papillary type (PTC). The RET proto-oncogene in PTC is constitutively activated by fusion of its tyrosine kinase domain with the 5 ´region of another gene thereby generating chimeric products collectively named RET/PTCs. RET/PTC1 and RET/PTC3 are best characterized among all RET/PTC rearrangements. Kashmir valley has witnessed an alarming increase in thyroid cancer incidence in young women. Therefore, we investigated the occurrence of RET/PTC 1 & 3 rearrangements by semi quantitative and qPCR in thyroid cancer patients (n = 48) of Kashmiri population and interrelated results with various clinicopathological characteristics. We observed that all the RET/PTC rearrangements were confined to PTC cases (10/40). Presence of RET/PTC rearrangement significantly correlated with gender, elevated TSH levels and lymph node metastasis. Overall, our study advocates that RET/PTC3 rearrangement is a frequent event in the carcinogenesis of thyroid gland in Kashmiri population although a study with a larger sample size is needed to get a clear scenario.

Detection of RET rearrangements in papillary thyroid carcinoma using RT-PCR and FISH techniques - A molecular and clinical analysis.

INTRODUCTION: Oncogenic BRAF and RAS mutations as well as multiple known (and yet unknown) RET fusion oncogenes comprise the majority of causative molecular alterations in papillary thyroid carcinoma (PTC). Apparently "mutation-negative" PTCs encompass a heterogenous group impeding analysis of prognostic significance of underlying genetics. MATERIAL AND METHODS: BRAF wild type PTC tissue of 56 patients was analyzed using two established methods: hybrid-specific RT-PCR for the predominant rearrangement RET/PTC1 and fluorescent in situ hybridization (FISH). Clinical features of the cases with and without RET rearrangement were compared (patient age, gender, tumor size, focality, lymph node affection, and iodine avidity). RESULTS: RT-PCR revealed RET/PTC1 rearrangements in five of 56 tumors (9%). FISH confirmed these, and identified four additional RET rearrangements (9/56; 16%). Loss of the iodine avidity only occurred in cases of RET/PTC hybrids (7/9 tumors), but not in RET/PTC-negative PTCs (0/41 tumors with available uptake information; p = 0.029). The risk to develop lymph node metastases was eight times higher in presence of RET rearrangements (p = 0.010). CONCLUSIONS: FISH analysis, in contrast to hybrid-specific RT-PCR, revealed infrequent and unknown RET fusion genes. The presence of RET rearrangements was associated with a significantly elevated risk to develop iodine refractory disease and lymph node metastases. Of note, significant clinical discrimination was only achievable when taking the FISH results into account; differences would have been missed when using the RT-PCR method only. Increasing evidence of the clinical impact of RET/PTC-positivity may influence the decision on the extent of surgical resection, especially on lymph node dissection, in PTCs.

A novel RET/PTC variant detected in a pediatric patient with papillary thyroid cancer without ionization history.

Papillary thyroid carcinoma (PTC) is the most frequent type of thyroid cancer. Its development is often caused by the formation of RET/PTC fused genes. RET/PTC1 is the most prevalent form, where exon 1 of CCDC6 gene is fused with the intracellular portion of RET protooncogene starting with exon 12. We have discovered a novel RET/PTC1 variant which we have named RET/PTC1ex9 in metastatic PTC of 8-year-old boy. RET/PTC1ex9 detection was performed by real-time polymerase chain reaction with melting curve analysis and subsequent Sanger and next-generation sequencing. A fusion of exon 1 of CCDC6 with exon 9 of extracellular domain of RET followed by exon 12 of RET was revealed. This is the first RET/PTC variant among PTC cases that contain the extracellular part of RET. This observation could be probably explained by incorrect splicing of RET due to the somatic 32-bp deletion in exon-intron 11 boundary of RET.

Analysis of the factors associated with clinicopathological characteristics of papillary thyroid carcinoma / 中华内分泌代谢杂志

Objective BRAFV600E mutation, RET/PTC rearrangement, and the concomitant of Hashimoto's thyroiditis(HT) could influence clinicopathological features of papillary thyroid carcinoma (PTC).This study is to investigate the distribution of three factors in PTC and to analyze their associations with clinicopathological characteristics.Methods Fine-needle aspiration samples were collected in a total of 122 conventional PTC patients, who were confirmed by surgery.The clinicopathological features were collected to analyze its association with different factors.BRAFV600E mutation and RET/PTC rearrangement were detected by pyrosequencing and Taqman-qPCR, respectively.Results BRAFV600E mutation was significantly correlated with an older age and a less coexistence with HT(P＜0.05).In contrast, RET/PTC rearrangement was more prevalent in young patients and was associated with the concomitant of HT(P＜0.05).In the age of ≥20 year and＜45 year groups, BRAFV600E mutation was significantly associated with extrathyroidal invasiveness.RET/PTC rearrangement was significantly associated with bilateral lymph node metastasis and the number of metastatic lymph node.Conclusions The distribution of three factors were different in PTC patients.In addition to the age at diagnosis, all of three factors should also be considered together to analyze the association of clinicopathological features of PTC.

Clinical implications of rearrangements during transfection of papillary thyroid cancer (RET/PTC) 1,3 detected with FQ-PCR in fine-needle aspiration specimen of thyroid nodules / 中华内分泌代谢杂志

Objective To evaluate the clinical significance of rearrangements during transfection of papillary thyroid cancer (RET/PTC) 1,3 in fine needle aspiration (FNA) specimen from regional thyroid nodules by FQ-PCR.Methods Two hundred and eighty-five FNA samples were collected from patients with thyroid nodules during January 2012 to January 2013.RET/PTC1,3 rearrangements were detected with FQ-PCR.Results The frequencies of the RET/PTC1 and RET/PTC3 rearrangements in 285 FNA samples were 17.2％ (49/285) and 1.4％ (4/285),respectively.During 21.7 months of follow-up,19 (40.4％,19/47) RET/PTC1 positive patients were confirmed to have papillary thyroid carcinoma(PTC) after operation.In the patients with RET/PTC1 rearrangement PTC was found in Thy5 and Thy4 groups.In Thy 2 group,22.6％ cases with RET/PTC1 rearrangements developed PTC as compared with 3.2％ cases without it(x2 =6.667,P＜0.01).In addition,8.5％ (4/47) RET/PTC1 rearrangements emerged in benign lesions.Conclusions It is convenient and reliable to detect RET/PTC1,3 rearrangements by FQ-PCR using FNA samples.RET/PTC1 rearrangement frequently occurs in PTC,however it would be detected in benign lesions occasionally.

The correlation between BRAF mutations, RET/PTC rearrangements and platelet-derived growth factor B expression in papillary thyroid carcinomas / 中华内科杂志

Objective To investigate the prevalence of BRAF T1799A mutation and RET/PTC rearrangement in Qingdao and detect the expression of platelet-derived growth factor B (PDGF-B) in order to investigate the correlation between gene mutation and PDGF-B.Methods Fresh tissue from 48 papillary thyroid carcinomas (PTC) patients was examined for BRAF mutation RET rearrangements (RET/PTC1 and RET/PTC3) by PCR,followed by direct-sequence analysis.The expression of PDGF was analyzed by immunohistochemistry.Results Among the 48 patients,14 (29.2％) were micro PTC; 18 (37.5％) had BRAF T1799A mutations and 23(47.9％) had RET/PTC rearrangement.There were 17 (35.4％) cases of RET/PTC1 and 6 (12.5％) of RET/PTC3,with no multiple rearrangements.Both BRAF T1799A mutation and RET/PTC rearrangement were present in 6 (12.5％) cases of non-micro PTC.The level of PDGF-B expression in BRAF T1799A positive was higher than that in the negative,and the level of PDGF-B expression in RET/PTC3 was higher than that in RET/PTC1 (P ＜ 0.05).The more advanced neoplasm stage was,the stranger PDGF-B expression was.Conclusions The incidence of BRAF T1799A mutation and RET/PTC rearrangement is higher in Qingdao.BRAF T1799A mutation and RET/PTC3 rearrangement in patients suggests a poorer prognosis than the negative one.The BRAF T1799A mutation and RET/PTC3 rearrangement may strengthen the expression of PDGF-B.Both variations suggest a poor prognosis.

DNA instability at chromosomal fragile sites in cancer.

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.

Molecular determination of benign and malignant thyroid tumors.

Recent molecular studies have revolutionized our understanding of the pathogenesis of thyroid tumors and particular advances have been made in three areas. First, toxic thyroid nodules, which originate from constitutive activation of thyroid-stimulating hormone receptor/Gs α signaling and represent the dominant cause of thyrotoxicosis in regions with iodine deficiency. Second, papillary thyroid cancer, the most frequent thyroid malignancy, which is characterized by a common fingerprint of constitutive mitogen-activated protein kinase activation. Importantly, this is caused by distinct genetic alterations in radiation-induced (RET/PTC, NTRK and AKAP9/BRAF rearrangements) and sporadic tumors (BRAF and RAS point mutation) and, recently, there exciting in vitro have emerged explaining the structural basis for this. These findings suggest a scenario in which the fate of a thyroid tumor is determined by the specific genetic defect at the beginning. Third, application of microarray analysis in nodular pathologies in which the oncogenic pathway is less clear, notably follicular neoplasia, has led to the identification of a number of promising genetic markers (TFF-3, Gal-3, PLAB, CCND2 and PCKD2) for the diagnostic distinction of follicular adenoma and carcinoma. In addition to the diagnostic perspective, the identification of molecular fingerprints of thyroid tumors opens novel avenues for an improved therapeutic approach; for example, selective antagonism of cell signaling in treatment-refractory thyroid cancer.