Background: Small 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. Methods: A total of 431 mPTCs cases were collected from six Italian institutions, and TERT promoter mutational status was assessed by a next-generation sequencing approach. Results: TERT 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. Conclusions: TERT mutations are present but uncommon in mPTCs. Apparently, in mPTCs, the occurrence of TERT mutations is not correlated with unfavorable clinical features.

Several biomarkers have been proposed as useful parameters to better specify the prognosis or to delineate new target therapy strategies for glioblastoma patients. MicroRNAs could represent putative target molecules, considering their role in tumorigenesis, cancer progression and their specific tissue expression. Although several studies have tried to identify microRNA signature for glioblastoma, a microRNA profile is still far from being well‐defined.In this work the expression of 19 microRNAs (miR‐7, miR‐9, miR‐9∗, miR‐10a, miR‐10b, miR‐17, miR‐20a, miR‐21, miR‐26a, miR‐27a, miR‐31, miR‐34a, miR‐101, miR‐137, miR‐182, miR‐221, miR‐222, miR‐330, miR‐519d) was evaluated in sixty formalin‐fixed and paraffin‐embedded glioblastoma samples using a locked nucleic acid real‐time PCR. Moreover, a comparison of miRNA expressions was performed between primary brain neoplasias of different grades (grades IV–I).The analysis of 14 validated miRNA expression in the 60 glioblastomas, using three different non‐neoplastic references as controls, revealed a putative miRNA signature: mir‐10b and miR‐21 were up‐regulated, while miR‐7, miR‐31, miR‐101, miR‐137, miR‐222 and miR‐330 were down‐regulated in glioblastomas. Comparing miRNA expression between glioblastoma group and gliomas of grades I–III, 3 miRNAs (miR‐10b, mir‐34a and miR‐101) showed different regulation statuses between high‐grade and low‐grade tumors. miR‐10b was up‐regulated in high grade and significantly down‐regulated in low‐grade gliomas, suggesting that could be a candidate for a GBM target therapy.This study provides further data for the identification of a miRNA profile for glioblastoma and suggests that different‐grade neoplasia could be characterized by different expression of specific miRNAs.

AIM: To summarize the current knowledge about the potential relationship between hepatitis C virus (HCV) infection and the risk of several extra-liver cancers. METHODS: We performed a systematic review of the literature, according to the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) Statement. We extracted the pertinent articles, published in MEDLINE and the Cochrane Library, using the following search terms: neoplasm/cancer/malignancy/tumor/carcinoma/adeno-carcinoma and non-Hodgkin lymphomas, kidney/renal-, cholangio-, pancreatic-, thyroid-, breast-,oral-, skin-, prostate-, lung-, colon-, stomach-, haematologic. Case series, case-series with control-group, case-control, cohort-studies as well as meta-analyses, written in English were collected. Some of the main characteristics of retrieved trials, which were designed to investigate the prevalence of HCV infection in each type of the above-mentioned human malignancies were summarised. A main table was defined and included a short description in the text for each of these tumours, whether at least five studies about a specific neoplasm, meeting inclusion criteria, were available in literature. According to these criteria, we created the following sections and the corresponding tables and we indicated the number of included or excluded articles, as well as of meta-analyses and reviews: (1) HCV and haematopoietic malignancies; (2) HCV and cholangiocarcinoma; (3) HCV and pancreatic cancer; (4) HCV and breast cancer; (5) HCV and kidney cancer; (6) HCV and skin or oral cancer; and (7) HCV and thyroid cancer. RESULTS: According to available data, a clear correlation between regions of HCV prevalence and risk of extra-liver cancers has emerged only for a very small group of types and histological subtypes of malignancies. In particular, HCV infection has been associated with: (1) a higher incidence of some B-cell Non-Hodgkin-Lymphoma types, in countries, where an elevated prevalence of this pathogen is detectable, accounting to a percentage of about 10%; (2) an increased risk of intra-hepatic cholangiocarcinoma; and (3) a correlation between HCV prevalence and pancreatic cancer (PAC) incidence. CONCLUSION: To date no definitive conclusions may be obtained from the analysis of relationship between HCV and extra-hepatic cancers. Further studies, recruiting an adequate number of patients are required to confirm or deny this association.

Selection of lung cancer patients for therapy with tyrosine kinase inhibitors directed at EGFR requires the identification of specific EGFR mutations. In most patients with advanced, inoperable lung carcinoma limited tumor samples often represent the only material available for both histologic typing and molecular analysis. We defined a next generation sequencing protocol targeted to EGFR exons 18-21 suitable for the routine diagnosis of such clinical samples. The protocol was validated in an unselected series of 80 small biopsies (n=14) and cytology (n=66) specimens representative of the material ordinarily submitted for diagnostic evaluation to three referral medical centers in Italy. Specimens were systematically evaluated for tumor cell number and proportion relative to non-neoplastic cells. They were analyzed in batches of 100-150 amplicons per run, reaching an analytical sensitivity of 1% and obtaining an adequate number of reads, to cover all exons on all samples analyzed. Next generation sequencing was compared with Sanger sequencing. The latter identified 15 EGFR mutations in 14/80 cases (17.5%) but did not detected mutations when the proportion of neoplastic cells was below 40%. Next generation sequencing identified 31 EGFR mutations in 24/80 cases (30.0%). Mutations were detected with a proportion of neoplastic cells as low as 5%. All mutations identified by the Sanger method were confirmed. In 6 cases next generation sequencing identified exon 19 deletions or the L858R mutation not seen after Sanger sequencing, allowing the patient to be treated with tyrosine kinase inhibitors. In one additional case the R831H mutation associated with treatment resistance was identified in an EGFR wild type tumor after Sanger sequencing. Next generation sequencing is robust, cost-effective and greatly improves the detection of EGFR mutations. Its use should be promoted for the clinical diagnosis of mutations in specimens with unfavorable tumor cell content.

Role of microRNAs in the main molecular pathways of hepatocellular carcinoma

The use of endoscopic ultrasonography has allowed for improved detection and pathologic analysis of fine needle aspirate material for pancreatic lesion diagnosis. The molecular analysis of KRAS has further improved the clinical sensitivity of preoperative analysis. For this reason, the use of highly analytical sensitive and specific molecular tests in the analysis of material from fine needle aspirate specimens has become of great importance. In the present study, 60 specimens from endoscopic ultrasonography fine needle aspirate were analyzed for KRAS exon 2 and exon 3 mutations, using three different techniques: Sanger sequencing, allele specific locked nucleic acid PCR and Next Generation sequencing (454 GS-Junior, Roche). Moreover, KRAS was also tested in wild-type samples, starting from DNA obtained from cytological smears after pathological evaluation. Sanger sequencing showed a clinical sensitivity for the detection of the KRAS mutation of 42.1%, allele specific locked nucleic acid of 52.8% and Next Generation of 73.7%. In two wild-type cases the re-sequencing starting from selected material allowed to detect a KRAS mutation, increasing the clinical sensitivity of next generation sequencing to 78.95%. The present study demonstrated that the performance of molecular analysis could be improved by using highly analytical sensitive techniques. The Next Generation Sequencing allowed to increase the clinical sensitivity of the test without decreasing the specificity of the analysis. Moreover we observed that it could be useful to repeat the analysis starting from selectable material, such as cytological smears to avoid false negative results.