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Background HER2 overexpression has been investigated as a potential biomarker and therapeutic target in biliary tract cancer (BTC), but a prognostic role of such alteration has not been demonstrated yet. Materials and Methods We retrospectively evaluated HER2 protein expression by immunohistochemistry (IHC) in 100 patients with radically resected BTC. HER2 gene amplification was assessed by fluorescence in situ hybridization (FISH) in 2+ and 3+ cases at IHC. High HER2 protein expression was defined as either IHC 3+ or 2+ associated with FISH positivity. The primary objective of the study was to evaluate the prognostic role of HER2 overexpression in terms of disease-free survival (DFS) and overall survival (OS). Secondary endpoints were the prevalence of HER2 overexpression and the possible correlation with other clinicopathological features. Results HER2 overexpression was identified in 11 patients and was not related to other clinicopathological factors. DFS was significantly shorter in HER2-positive compared with HER2-negative patients (10.6 vs. 20.9 months, log-rank p = .017). HER2 confirmed its prognostic value for DFS at multivariate analysis (hazard ratio 2.512; 95% confidence interval, 1.232–5.125; p = .011) together with nodal stage (p < .001), resection margin (p = .027), and tumor site (p = .030). There was no difference in OS between HER2-positive and -negative patients (p = .068). Conclusion HER2 overexpression represents an independent prognostic factor for disease recurrence in patients with BTC treated with potentially curative surgery.

Pleural effusions are among the first clinical manifestations of malignant pleural mesothelioma (MPM) and often constitute the only available material for diagnosis. Although an MPM diagnosis can be reliable on cytology, the reported sensitivity is low (30% to 75%). Particularly, it can be hard to discriminate epithelioid MPM, the most common histotype, from reactive mesothelial hyperplasia (MH). Currently, BRCA1-associated protein 1 (BAP1) and CDKN2A (p16), evaluated by immunohistochemistry and fluorescent in situ hybridization, respectively, are the most valuable markers to discriminate MPM and MH. Both markers have a high specificity, but their sensitivity is not always satisfying, even when used together. We have recently developed a 117-gene expression panel, based on Nanostring technology, able to differentiate epithelioid MPM from MH pleural tissues better than BAP1 and p16. Herein, we evaluated the efficacy of the same panel on an independent retrospective cohort of 23 MPM and 11 MH pleural effusions (cell blocks and smears). The overall sensitivity and specificity of the panel were equal to 0.9565 and 1, respectively. Moreover, the panel performance was compared with BAP1 and p16 on 25 cell blocks. Sensitivity levels of gene panel, BAP1 alone, p16 alone, and BAP1 plus p16 were 1, 0.5882, 0.4706, and 0.7647, respectively. Specificity was always 1. Although further validation is needed, this gene panel could really facilitate patients' management, allowing a definitive MPM diagnosis directly on pleural effusions.

Background MiRNAs are vital in functioning as either oncogenes or tumor suppressors in the cell cycle. Target transcripts for immune checkpoint molecules such as PD-1/PD-L1 and (programmed cell death-1/its ligand and cytotoxic T-lymphocyte antigen 4) have proven to be beneficial against several solid tumors, including lung adenocarcinoma. Methods Simultaneous quantification of the expression level of miR-33a and PD-1, PD-L1 and CTLA4 mRNAs with NanoString technology was performed in 88 lung adenocarcinoma specimens. A cohort of 323 lung adenocarcinoma patients from the cancer genome atlas (TCGA) database was further analyzed, in order to test our hypothesis. Potential interference of PD-1, PD-L1 and CTLA4 gene expression by miR-33a was predicted using the microRNA target prediction program RNA22. Results High miR-33a expression was significantly associated with younger (p = 0.005), female (p = 0.04), patients with low grade (p

Lung cancer is the leading cause of cancer-associated mortality worldwide, and the mainstay of treatment remains to be personalised therapy. Tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR-TKIs) have been reported to exert a significant impact in the treatment of non-small cell lung cancer (NSCLC), particularly in patients harbouring mutations in the EGFR gene. The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) gene translocation has been described in a subset of patients with NSCLC and possesses potent oncogenic activity. This translocation represents one of the most novel molecular targets in the treatment of NSCLC. Patients who harbour the EML4-ALK rearrangement possess lung tumours that lack EGFR or K-ras mutations. The present study reports the case of a patient possessing the EML4-ALK rearrangement that was initially treated with erlotinib and achieved a lasting clinical response. To the best of our knowledge, the current study is the first report of a clinical response to EGFR-TKI in a patient with lung adenocarcinoma harbouring the EML4-ALK fusion gene, but no EGFR mutations. However, as the disease progressed, the ALK gene status of the tumour was investigated, and based upon a positive result, the patient was treated with crizotinib and achieved a complete response. In conclusion, the present study suggests that the EML4-ALK rearrangement is not always associated with resistance to EGFR-TKIs. Further studies are required to clarify the biological features of these tumours and to investigate the mechanisms underlying the primary resistance to EGFR-TKIs when the EML4-ALK rearrangement is present.

The follicular variant (FV) of papillary thyroid cancer (PTC) is one of the most common variants of PTC. Clinically, non-infiltrative FVPTC is considered a low-risk variant of PTC, and the non-invasive encapsulated forms of FVPTC represent a group of thyroid tumors with a particularly good prognosis. Consequently, these neoplasms have been very recently reclassified as non-invasive follicular neoplasms with papillary-like nuclear features (NIFTP). From a molecular standpoint, NIFTP appears to be similar to follicular neoplasms. However, only limited data are currently available regarding their gene expression profile.The aim of this study was to identify specific molecular signatures of 26 NIFTPs compared to those of 19 follicular adenomas (FAs) and 18 infiltrative FVPTCs (IFVPTCs). A nanoString custom assay was used to perform mRNA expression analysis. All cases were also genotyped for BRAF, N-, H-, and K-RAS mutations. Samples were grouped on the basis of gene expression profiles by Pearson's correlation and non-negative matrix factorization clustering analysis. Finally, the uncorrelated shrunken centroid machine-learning algorithm was used to classify the samples.The results revealed distinct expression profiles of FAs and IFVPTCs. NIFTP samples can exhibit different expression profiles, more similar to FAs (FA-like) or to IFVPTCs (IFVPTC-like), and these different expression profiles largely depend on the presence of different mutations (RAS or BRAF).In conclusion, although further validation of the model is required by using a larger group of prospective cases, these data reinforce the hypothesis that IFVPTC-like NIFTPs might represent precursors of IFVPTC.

Echinoderm microtubule associated proteinlike 4-anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation has been described in a subset of patients with non-small cell lung cancer (NSCLC) and has been shown to have oncogenic activity. Fluorescence in situ hybridization (FISH) is used to detect ALK-positive NSCLC, but it is expensive, time-consuming, and difficult for routine application.To evaluate the potential role of immunohistochemistry (IHC) as a screening tool to identify candidate cases for FISH analysis and for ALK inhibitor therapy in NSCLC.We performed FISH and IHC for ALK and mutational analysis for epidermal growth factor receptor (EGFR) and KRAS in 523 NSCLC specimens. We conducted IHC analysis with the monoclonal antibody D5F3 (Ventana Medical Systems, Tucson, Arizona) and a highly sensitive detection system. We also performed a MassARRAY-based analysis (Sequenom, San Diego, California) in a small subset of 11 samples to detect EML4-ALK rearrangement.Of the 523 NSCLC specimens, 20 (3.8%) were positive for ALK rearrangement by FISH analysis. EGFR and KRAS mutations were identified in 70 (13.4%) and 124 (23.7%) of the 523 tumor samples, respectively. ALK rearrangement and EGFR and KRAS mutations were mutually exclusive. Of 523 tumor samples analyzed, 18 (3.4%) were ALK(+) by IHC, 18 samples (3.4%) had concordant IHC and FISH results, and 2 ALK(+) cases (0.3%) by FISH failed to show ALK protein expression. In the 2 discrepant cases, we did not detect any mass peaks for the EML4-ALK variants by MassARRAY.Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis.

Yes-associated protein-1 (YAP-1) is a player of the Hippo pathway and is involved in regulating cell proliferation. YAP-1 is overexpressed in papillary and anaplastic thyroid cancers. However, a correlation between YAP-1 expression and outcome in thyroid carcinoma has not been conclusively demonstrated.This study was designed to clarify whether YAP-1 may be considered a marker of worse prognosis and outcome in thyroid cancer.A large series of cases of thyroid cancer with a long follow-up were investigated for YAP-1 expression.The study was carried out in the Pathology section of a referral Italian center for Endocrine Surgery and Endocrinology.The study included a consecutive series of 105 patients who underwent thyroidectomy from 1985 to 1992. The mean follow-up was 15 years. For all patients, clinicopathologic features were considered. All patients completed the study.The study also included a consecutive series of 52 patients who underwent thyroidectomy from 2012 to 2013 in order to analyze more deeply the correlation of YAP-1expression with BRAF mutation.The 105 thyroid tumors were immunohistochemically investigated for YAP-1 expression.We expected a correlation between YAP-1 expression and worse prognosis.Among 105 tumors, 77 scored positive for YAP-1 expression, of which 68 papillary thyroid carcinomas and 9 anaplastic thyroid carcinomas were YAP-1 positive. The correlation of YAP-1 expression with clinicopathologic characteristics was significant for the absence of a tumoral capsule, gender, and extrathyroid invasion.Interestingly, significant correlations were found between YAP-1 and both persistence of disease and death from carcinoma.The data show an association of YAP-1 expression with worse clinicopathologic features of thyroid tumors that seem to have a specific impact on outcome.

Papillary thyroid carcinoma (PTC) is the most common type of endocrine malignancy and accounts for ~80% of thyroid carcinomas in adults and 90% in children. Risk stratification is important for identifying patients at higher risk and, for this reason, recent advances in molecular genetics of thyroid cancer can be applied to provide novel biomarkers useful in understanding tumor behavior. B-Raf proto-oncogene, serine/threonine kinase (BRAF) and rat sarcoma (RAS) mutations have been widely studied and appear to have an important role in thyroid tumorigenesis. Somatic telomerase reverse transcriptase (TERT) promoter mutations have been recently identified in several types of malignant tumors, including thyroid neoplasia; however, the actual role of TERT mutations in thyroid tumorigenesis is still under debate. In the present study, the mutational status of BRAF, RAS and TERT was analyzed in order to elucidate the roles of these genes in thyroid tumorigenesis. The TERT mutational analysis was also correlated with an immunohistochemical study of TERT protein expression. According to the literature, our data provide evidence of the BRAF and RAS roles in thyroid tumorigenesis, supporting an association between BRAF (V600E) mutations and the more aggressive clinical and pathological features of thyroid tumors. By contrast, TERT mutations were not significantly associated with any clinical parameters; therefore, its role in initial tumorigenesis should be further investigated.

Non‑small cell lung cancer (NSCLC) accounts for ~70% of all lung cancer‑associated mortalities worldwide. The serine/threonine protein kinase tumor progression locus 2 [TPL2/MAP3 kinase 8 (MAP3K8)] may impact oncogenic events; however the role of TPL2 in lung carcinogenesis remains unclear. The present study was focused on the potential prognostic role of TPL2 in 101 patients with early‑stage NSCLC. Since TPL2 is a potential target of miR‑21, the association between TPL2 and miR‑21 expression was also examined. TPL2 and miR‑21 mRNA expression was quantified using reverse transcription quantitative polymerase chain reaction (RT‑qPCR). TPL2 protein levels were evaluated by immunohistochemistry (IHC). The present study identified that the mRNA expression of TPL2 was low in 52/101 (51%) cases and high in 49/101 (49%) cases. IHC analysis of TPL2 protein expression often demonstrated identical mRNA results. No statistically significant associations were observed between the mRNA expression of TPL2 and the predominant clinicopathological characteristics of the patients with NSCLC, as well as identifying no association between TPL2 and miR‑21. TPL2 mRNA expression was significantly higher in patients with NSCLC with good prognosis (disease‑free interval P=0.009; overall survival P=0.024), when compared with those of poor prognosis. Focusing on the difference in mRNA expression of TPL2 among the adenocarcinomas in affected patients, TPL2 was more highly expressed in lepidic adenocarcinomas compared with in the other subtypes (P=0.012). The present study is the first examination, to the best of our knowledge, of TPL2 in early‑stage NSCLC in relation to miR‑21, and in different adenocarcinoma subtypes. Future studies must clarify the mechanism by which TPL2 is involved in lung carcinogenesis due to its important translational implications.

The recently described telomerase reverse transcriptase (TERT) promoter mutations are recurrent in cutaneous melanoma. Several authors have described an association between these molecular alterations, some histological parameters, and patient survival. BRAF mutations are very frequent in melanoma, but their actual role in the evolution of the disease is still unclear. Here, we investigated the relationship of TERT promoter mutations and BRAF mutations with the most relevant clinicopathological parameters, individually and coexisting, in order to evaluate their role as independent prognostic markers and to determine the effect of their coexistence. A TERT promoter alteration was found in 20 of 53 cases (38 %), significantly associated with histological type, increasing tumor thickness and mitotic rate, more advanced pathologic tumor (pT) stage, and absence of regression. A BRAF mutation was found in 21 of 53 cases (40 %), significantly associated with tumor thickness and presence of metastases in the sentinel lymph node. Coexistence of a TERT promoter and BRAF mutation was detected in 11 of 53 cases (21 %). This was associated with increasing thickness, high mitotic rate, lymph node metastasis, presence of ulceration, and absence of regression. Coexistence of a mutation in the TERT promoter and in the BRAF gene correlated with more prognostically relevant factors than either mutation alone. Our data lead us to hypothesize that TERT promoter and BRAF mutations cooperate in cutaneous melanoma. Further studies in larger cohorts of patients are needed to investigate how this synergistic effect is involved in the evolution of the disease.

e20656 Background: In lung cancer the evaluation of MET, a tyrosine kinase receptor involved in tumor growth and invasiveness, for copy number amplification and point mutations is relevant for prognosis and therapy. In lung adenocarcinoma (ADC) MET amplification is observed in 1-2% and MET mutations in 3-4% of cases. Mutations mainly occur in exon 14 (ex14), which encodes for the juxtamembrane negative regulatory domain, including splice site alterations and missense mutations within the exon.Commonly, MET ex14 alterations, in particular splice site ones, are mutually exclusive with other driver mutations, but co-occurrence with MET and MDM2 amplification and KRAS mutations was described. We evaluated METex14 mutation frequency and coexistence with additional driver alterations in a prospective cohort of Italian ADC patients. Methods: 315 ADC patients were tested (January-December 2016) for MET ex14 alterations by Sanger Sequencing on formalin-fixed paraffin-embedded tissues and cytological smears (tumor cells > 40%). MET positive cases were screened also for other oncogenes, among which KRAS, BRAF and PIK3CA, using a MALDI-TOF platform, and for ROS1 translocations and MET amplification by FISH. Results: 16 patients (5%) were MET positive: 7 splice site mutations (43%) and 9 (57%) other alterations within ex14. Among splice site mutations 1 co-occurred with ROS1 translocation and 1 with MET amplification. Among other mutations we found 5 T1010I missense mutations: 3 co-occurring with G12D/G13C/Q61H KRAS, 1 with G469A BRAF and 1 with a MET intron 13 point mutation; 2 R988C mutations: 1 with G13C KRAS and 1 with G13C KRAS plus ROS1 translocation; 1 P1026S mutation and 1 frameshift deletion (p.A991del;R992fs*999), both with G12C KRAS. Conclusions: Splice site mutations cause MET activation by exon skipping and increase sensitivity to tyrosine-kinase inhibitors, whereas how other ex14 mutations affect MET function is not fully understood. In our cohort we found a high rate of METex14 mutations together with alterations in other oncogenes, mostly KRAS. The interaction of MET with cancer signaling pathways deserve further investigation in order to better define the role of ex14 mutations in the context of target therapy.

Introduction Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm associated with asbestos exposure. Currently, the molecular mechanisms that induce MPM development are still unknown. The purpose of this study was to identify new molecular biomarkers for mesothelial carcinogenesis. Methods We analyzed a panel of 84 genes involved in extracellular matrix remodeling and cell adhesion by polymerase chain reaction (PCR) array in 15 samples of epithelioid mesothelioma and 10 samples of reactive mesothelial hyperplasia (MH; 3 of 25 samples were inadequate for mRNA analysis). To validate the differentially expressed genes identified by PCR array, we analyzed 27 more samples by immunohistochemistry, in addition to the 25 samples already studied. Results Twenty-five genes were differentially expressed in MPM and MH by PCR array. Of these we studied matrix metalloproteinase 7 (MMP7), MMP14, CD44, and integrin, alpha3 expression by immunohistochemistry in 26 epithelioid MPM and 26 MH samples from the entire series of 52 cases. We observed higher MMP14 and integrin, alpha3 expression in MPM samples compared with MH samples ( p = 0.000002 and p = 0.000002, respectively). Conversely, CD44 expression was low in most (57.7%) mesothelioma samples but only in 11.5% of the MH samples ( p = 0.0013). As regards MMP7, we did not observe differential expression between MH and MPM samples. Conclusions We have extensively studied genes involved in cell adhesion and extracellular matrix remodeling in MPM and MH samples, gaining new insight into the pathophysiology of mesothelioma. Moreover, our data suggest that these factors could be potential biomarkers for MPM.

The EML4-ALK gene translocation was described in a non small cell lung cancer (NSCLC) subset, with a potent oncogenic activity. It represents one of the newest molecular targets in NSCLC. We report on the case of a metachronous second primary lung sarcomatoid carcinoma after resection of lung adenocarcinoma both with ALK translocation, in a non-smoking patient. EML4-ALK rearrangement was detected with immunohistochemistry and confirmed with fluorescent in situ hybridization (FISH). To assess the clonal relationship between the two tumors, both adenocarcinoma and sarcomatoid carcinoma were analyzed by array comparative genomic hybridization (aCGH). We observed different genomic profiles suggesting that the tumors arose independently and were thus multiple primaries. To the best of our knowledge, this is the first report concerning the presence of the EML4-ALK fusion gene in a sarcomatoid carcinoma of the lung. Crizotinib, the ALK tyrosine kinase inhibitor, is highly effective in ALK-rearranged NSCLC; therefore, it may be imperative to identify all NSCLC that harbor ALK translocations in the near future. Starting from our evidence, tumors with sarcomatoid histology may need to be screened for the presence of EML4-ALK rearrangement.

MicroRNAs (miRNAs) play a key role in cancer pathogenesis and are involved in several human cancers, including non-small cell lung cancer (NSCLC). This study evaluated Let-7g and miR-21 expression by quantitative real-time PCR in 80 NSCLC patients and correlated the results with their main clinicopathological and molecular features. MiR-21 expression was significantly higher in NSCLC tissues compared to non-cancer lung tissues (p

Background: P2X7, a purinergic receptor, plays important roles in inflammatory diseases, but recently its expression has been found in several tumors, suggesting a potential role as a cancer cell biomarker. Moreover, the relative amount of P2X7 varies among human individuals due to numerous single nucleotide polymorphisms resulting in either a loss- or gain-of-function; the P2X7 gene is highly polymorphic, and polymorphisms in the promoter or coding region may modify its expression or function. A polymorphism in exon 13 of the P2X7 receptor gene at the +1513 position (Glu496Ala substitution, corresponding to SNP rs3751143) has been shown to eradicate the function of this receptor and has been correlated with histological variants and clinical parameters in thyroid cancer. Until now, no data regarding P2X7 expression and polymorphisms in lung cancer have been published; based on these premises, we decided to evaluate the impact of the P2X7 expression and polymorphisms in ninety-seven cases of non-small cell lung cancer (NSCLC). Results: No significant difference in the genotype frequency of the A1513C polymorphism was found between the two histological variants of NSCLC, adenocarcinoma and squamous cell carcinoma, and no statistically significant associations were observed between P2X7 protein expression and the main clinico-pathological characteristics of the NSCLC patients. Conclusions: Based on our results, P2X7 expression and polymorphisms seem to have no potential impact in patients with non-small cell lung cancer; however, further studies will surely provide deeper insights regarding the role of this receptor at the clinical level in NSCLC.