Alessia Ciarrocchi

TERT promoter mutations are associated with distant metastases in Papillary Thyroid Carcinoma

OBJECTIVEnTranscriptional activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene were reported at high frequency in aggressive poorly differentiated and anaplastic thyroid cancers. By contrast, the relevance of these mutations in the metastatic behavior of well-differentiated thyroid cancer is still to be defined. The aim of this work was to investigate the frequency of TERT promoter mutations in a remarkable cohort of well-differentiated papillary thyroid carcinoma that developed distant metastases (DM-PTCs) and to establish whether these mutations may be predictive of metastatic behavior.nnnDESIGNnWe analyzed the frequency of TERT promoter mutations in a group of 43 highly aggressive DM-PTCs. As controls, we analyzed these mutations in a group of 78 PTCs without distant metastases (control-PTCs). The possible correlation between TERT promoter mutations and BRAF V600E mutation was also investigated.nnnMETHODSnTERT promoter mutational status was evaluated by direct sequencing of the hotspot harboring the C228T and the C250T mutations.nnnRESULTSnIn the overall cohort of 121 PTCs analyzed, 17% of cases (21/121) carried a mutation in the TERT promoter. Noticeably, 33% of DM-PTCs were mutated in the TERT promoter while only 9% of the control-PTCs showed a mutation in this locus. We also observed a positive association between BRAF V600E and TERT C228T mutations in the cohort of DM-PTCs.nnnCONCLUSIONSnThese results indicate that TERT promoter mutations are associated with the development of distant metastases in PTCs and may help in predicting aggressive behavior in this type of tumor.

Update on Anaplastic Thyroid Carcinoma: Morphological, Molecular, and Genetic Features of the Most Aggressive Thyroid Cancer

Anaplastic thyroid carcinoma (ATC) is the most aggressive form of thyroid cancer. It shows a wide spectrum of morphological presentations and the diagnosis could be challenging due to its high degree of dedifferentiation. Molecular and genetic features of ATC are widely heterogeneous as well and many efforts have been made to find a common profile in order to clarify its cancerogenetic process. A comprehensive review of the current literature is here performed, focusing on histopathological and genetic features.

Molecular Testing of Thyroid Fine-Needle Aspirations Improves Presurgical Diagnosis and Supports the Histologic Identification of Minimally Invasive Follicular Thyroid Carcinomas

BACKGROUNDnPrevious studies detecting mutations in thyroid nodule fine-needle aspiration (FNA) material differed with respect to the cytologic grading applied to the FNAs, the type of FNA material used, and the prevalence of mutations observed in the samples. Therefore, the aim of the present study was to investigate these differences as possible reasons for the discrepant sensitivities and specificities reported for the ruling-in approach between the previous studies.nnnMETHODSnRNA and DNA was extracted from 347 routine air-dried FNA smears with available histology. PAX8/PPARG and RET/PTC rearrangements were detected by real-time quantitative polymerase chain reaction, while BRAF and RAS mutations were detected by pyrosequencing.nnnRESULTSnBRAF mutations were associated with a carcinoma in 100% of samples; RAS mutations were associated with a carcinoma in 57% of samples. Forty-nine percent of the carcinomas were identified by molecular testing in the group of follicular lesions, which increased the sensitivity from 60% to 80% compared to cytologic FNA evaluation alone. While follicular lesion FNAs had a 28% risk of malignancy, the risk increased to 71% for mutation-positive follicular lesions, and decreased to 18% for mutation-negative follicular lesions.nnnCONCLUSIONnMolecular testing of air-dried FNA samples improves presurgical diagnosis. Discrepant sensitivities and specificities reported previously are most likely related to the use of different grading schemes resulting in different compositions of the cytologic categories, interobserver variability to diagnose follicular variant of papillary thyroid carcinomas and a different prevalence of RAS mutations in follicular carcinomas. The knowledge of the molecular testing might support the histologic identification of minimally invasive follicular carcinomas.

High-Sensitivity BRAF Mutation Analysis: BRAF V600E Is Acquired Early During Tumor Development but Is Heterogeneously Distributed in a Subset of Papillary Thyroid Carcinomas

CONTEXTnThe homogeneous distribution of BRAF V600E in papillary thyroid carcinoma (PTC) has been called into question by recent reports. These studies claim that BRAF V600E is heterogeneous and is limited to tumor cell subsets in the majority of PTCs.nnnOBJECTIVEnThe objective of the study was to understand the allele distribution of BRAF V600E by evaluating the percentage of mutated neoplastic cells in a group of PTCs using two different highly sensitive analytical approaches: allele-specific locked nucleic acid PCR and 454 next-generation sequencing targeted to BRAF exon 15.nnnSTUDY DESIGNnBRAF V600E was investigated using allele-specific locked nucleic acid PCR on 155 consecutive samples of PTC. Mutated cases were reanalyzed by 454 next-generation sequencing and immunohistochemistry. Because the evaluation of genetic heterogeneity in tumor samples can be profoundly biased by contamination with normal cells, all mutation frequency data were normalized to the real amount of neoplastic cells within each tumor.nnnRESULTSnEighty-five of 155 PTCs (54.8%) were BRAF V600E mutated. The distribution of mutated neoplastic cells within the tumor was as follows: greater than 80% in 37 of 85 (43.5%), 30-80% in 39 of 85 (45.9%), and less than 30% in 9 of 85 (10.6%). In most of the PTCs with less than 80% BRAF V600E-positive neoplastic cells, the mutation was present in large neoplastic cell subpopulations. Tumors with less than 30% mutated neoplastic cells were smaller than tumors with a percentage of mutated cells greater than 80% or between 30% and 80% (P < .05).nnnCONCLUSIONSnBRAF V600E is heterogeneously distributed in some PTCs. The large BRAF V600E neoplastic cell subpopulations found in mutated cases is consistent with the view that the BRAF V600E is acquired early during PTC development.

Papillary thyroid microcarcinoma with fatal outcome: evidence of tumor progression in lymph node metastases Report of 3 cases, with morphological and molecular analysis

Papillary thyroid microcarcinoma generally carries an excellent prognosis, and fatal cases are becoming increasingly rare. Their pathologic and molecular features, however, remain largely unknown. We describe 3 cases of papillary thyroid microcarcinoma that, despite surgical and radioiodine treatment, recurred, metastasized, and eventually caused the death of the patients. In addition to morphology, immunohistochemical (cyclin D1 and p53) and molecular analyses (BRAF [v-raf Murine sarcoma viral oncogene homolog B1], KRAS [V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog], HRAS [v-Ha-ras Harvey rat sarcoma viral oncogene homolog], NRAS [neuroblastoma RAS viral oncogene homolog], and PIK3CA [phosphoinositide-3-kinase, catalytic, alpha polypeptide]) were performed. Interestingly, all 3 cases presented with massive lymph node metastases that showed morphological evidence of tumor progression (tall cell features, poorly differentiated areas, and high-grade cytologic features). Cyclin D1 was consistently immunoreactive in both primary and metastatic site, whereas p53 was negative. BRAF V600E was absent in both sites, and KRAS, HRAS, NRAS, and PIK3CA were consistently wild type. These data suggest that, in cases of metastatic papillary thyroid microcarcinoma, an accurate morphologic analysis of the metastatic deposits could contribute to a more accurate prediction of tumor behavior.

Cadherin-6 promotes EMT and cancer metastasis by restraining autophagy

The transdifferentiation of epithelial cells toward a mesenchymal condition (EMT) is a complex process that allows tumor cells to migrate to ectopic sites. Cadherins are not just structural proteins, but they act as sensors of the surrounding microenvironment and as signaling centers for cellular pathways. However, the molecular mechanisms underlying these signaling functions remain poorly characterized. Cadherin-6 (CDH6) is a type 2 cadherin, which drives EMT during embryonic development and it is aberrantly re-activated in cancer. We recently showed that CDH6 is a TGFβ target and an EMT marker in thyroid cancer, suggesting a role for this protein in the progression of this type of tumor. Papillary thyroid carcinomas (PTCs) are usually indolent lesions. However, metastatic spreading occurs in about 5% of the cases. The identification of molecular markers that could early predict the metastatic potential of these lesions would be strategic to design more tailored approaches and reduce patients overtreatment. In this work, we assessed the role of CDH6 in the metastatic progression of thyroid cancer. We showed that loss of CDH6 expression profoundly changes cellular architecture, alters the inter-cellular interaction modalities and attenuates EMT features in thyroid cancer cells. Using a yeast two-hybrid screening approach, based on a thyroid cancer patients library, we showed that CDH6 directly interacts with GABARAP, BNIP3 and BNIP3L, and that through these interactions CDH6 restrains autophagy and promotes re-organization of mitochondrial network through a DRP1-mediated mechanism. Analysis of the LIR domains suggests that the interaction with the autophagic machinery may be a common feature of many cadherin family members. Finally, the analysis of CDH6 expression in a unique cohort of human PTCs showed that CDH6 expression marks specifically EMT cells. and it is strongly associated with metastatic behavior and worse outcome of PTCs.

TERT Promoter Mutations in Papillary Thyroid Microcarcinomas

BACKGROUNDnSmall papillary thyroid carcinomas have contributed to the worldwide increased incidence of differentiated thyroid cancer observed over the past decades. However, the mortality rate has not changed over the same period of time, raising questions about the possibility that thyroid cancer patients, especially those with small tumors, are overdiagnosed and overtreated. Molecular prognostic marker able to discriminate aggressive thyroid cancers from those with an indolent course would be of great relevance to tailor the therapeutic approach and reduce overtreatment. Mutations in the TERT promoter were recently reported to correlate strongly with aggressiveness in advanced forms of thyroid cancer, holding promise for a possible clinical application. The occurrence and potential clinical relevance of TERT mutations in papillary thyroid microcarcinomas (mPTCs) is currently unknown. This study aimed to analyze the occurrence of two TERT promoter mutations (-124C>T and -146C>T) and their potential association with unfavorable clinical features in a large cohort of mPTCs.nnnMETHODSnA total of 431 mPTCs cases were collected from six Italian institutions, and TERT promoter mutational status was assessed by a next-generation sequencing approach.nnnRESULTSnTERT promoter mutations were found in 4.7% of the analyzed mPTCs, showing that even microcarcinomas carry mutations in this gene. Correlation analysis showed that TERT promoter mutations are not associated with aggressive features or clinical outcome in the cohort analyzed.nnnCONCLUSIONSnTERT mutations are present but uncommon in mPTCs. Apparently, in mPTCs, the occurrence of TERT mutations is not correlated with unfavorable clinical features.

Time to re-consider the meaning of BRAF V600E mutation in papillary thyroid carcinoma

The BRAF V600E mutation, resulting from the BRAFT1799A transversion, is the most common genetic mutation in papillary thyroid carcinoma (PTC), with a mean frequency close to 50% among all cases. A large number of studies in the past decade have tried to dissect the relevance and the function of the V600E mutation in controlling oncogenesis and progression of thyroid cancer. However, several works published in the latest years have provided new evidence, in partial conflict with the previous knowledge, suggesting the need of reconsidering the meaning of the BRAF V600E mutation in PTC. In this work, we attempt to discuss some of the most recent molecular, preclinical and clinical evidence to construct a more exhaustive model of function for the BRAF V600E in development, progression and therapeutic approach of thyroid cancer.

Tollip Is a Mediator of Protein Sumoylation

Tollip is an interactor of the interleukin-1 receptor involved in its activation. The endosomal turnover of ubiquitylated IL-1RI is also controlled by Tollip. Furthermore, together with Tom1, Tollip has a general role in endosomal protein traffic. This work shows that Tollip is involved in the sumoylation process. Using the yeast two-hybrid technique, we have isolated new Tollip partners including two sumoylation enzymes, SUMO-1 and the transcriptional repressor Daxx. The interactions were confirmed by GST-pull down experiments and immunoprecipitation of the co-expressed recombinants. More specifically, we show that the TIR domain of the cytoplasmic region of IL-1RI is a sumoylation target of Tollip. The sumoylated and unsumoylated RanGAP-1 protein also interacts with Tollip, suggesting a possible role in RanGAP-1 modification and nuclear-cytoplasmic protein translocation. In fact, Tollip is found in the nuclear bodies of SAOS-2/IL-1RI cells where it colocalizes with SUMO-1 and the Daxx repressor. We conclude that Tollip is involved in the control of both nuclear and cytoplasmic protein traffic, through two different and often contrasting processes: ubiquitylation and sumoylation.

Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.