Proteases have long been associated with tumor progression, given their ability to degrade extracellular matrix components and facilitate invasion and metastasis. However, recent findings indicate that different proteases can also act as tumor-suppressor enzymes. We have recently reported that lung carcinoma cells expressing the ADAMTS-12 metalloprotease show a remarkable impairment of growth in immunodeficient mice as compared with parental cells. Here, we show that ADAMTS12 promoter is hypermethylated in cancer cell lines and tumor tissues. Interestingly, ADAMTS12 expression in the stromal cells surrounding epithelial malignant cells is higher than in the paired normal tissues. Moreover, the expression of this metalloprotease in colon fibroblasts co-cultured with colon cancer cell lines is higher than in those cultured alone. Furthermore, the expression of ADAMTS-12 by these fibroblasts is linked with an anti-proliferative effect on tumor cells. Based on these findings, we hypothesize that ADAMTS-12 is a novel anti-tumor protease that can reduce the proliferative properties of tumor cells. This function is lost by epigenetic silencing in tumor cells, but concurrently induced in stromal cells, probably as part of a response of the normal tissue aimed at controlling the progression of cancer.

The analytical algorithm of Lynch syndrome (LS) is increasingly complex. BRAF V600E mutation and MLH1 promoter hypermethylation have been proposed as a screening tool for the identification of LS. The aim of this study was to assess the clinical usefulness and cost-effectiveness of both somatic alterations to improve the yield of the diagnostic algorithm of LS. A total of 122 colorectal tumors from individuals with family history of colorectal cancer that showed microsatellite instability and/or loss of mismatch repair (MMR) protein expression were studied. MMR germline mutations were detected in 57 cases (40 MLH1, 15 MSH2 and 2 MSH6). BRAF V600E mutation was assessed by single-nucleotide primer extension. MLH1 promoter hypermethylation was assessed by methylation-specific multiplex ligation-dependent probe amplification in a subset of 71 cases with loss of MLH1 protein. A decision model was developed to estimate the incremental costs of alternative case-finding methods for detecting MLH1 mutation carriers. One-way sensitivity analysis was performed to assess robustness of estimations. Sensitivity of the absence of BRAF mutations for depiction of LS patients was 96% (23/24) and specificity was 28% (13/47). Specificity of MLH1 promoter hypermethylation for depiction of sporadic tumors was 66% (31/47) and sensitivity of 96% (23/24). The cost per additional mutation detected when using hypermethylation analysis was lower when compared with BRAF study and germinal MLH1 mutation study. Somatic hypermethylation of MLH1 is an accurate and cost-effective pre-screening method in the selection of patients that are candidates for MLH1 germline analysis when LS is suspected and MLH1 protein expression is absent.

BACKGROUND: Concomitant quantification of multiple mutant KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) alleles may provide information in addition to that provided by standard mutationdetection procedures. We assessed the feasibility of a nanofluidic digital PCR array platform to detect and quantify KRAS mutations simultaneously in clinically relevant samples.

Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eighty-three genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes.

Purpose The majority of genomic alterations causing intratumoral heterogeneity (ITH) in colorectal cancer (CRC) are thought to arise during early stages of carcinogenesis as a burst but only after truncal mutations in APC have expanded a single founder clone. We have investigated if the initial source of ITH is consequent to multiple independent lineages derived from different crypts harboring distinct truncal APC and driver KRAS mutations, thus challenging the prevailing monoclonal monocryptal model. Experimental design High-depth next-generation sequencing and SNP arrays were performed in whole lesion extracts of 37 FAP colorectal adenomas. Also, ultra-sensitive genotyping of hotspot mutations of APC and KRAS was performed using nanofluidic PCRs in matched bulk biopsies (n=59) and crypts (n=591) from 18 adenomas and 7 carcinomas and adjacent normal tissues. Results Multiple co-occurring truncal APC and driver KRAS alterations were uncovered in whole lesion extracts from adenomas and subsequently confirmed to belong to multiple clones. Ultra-sensitive genotyping of bulk biopsies and crypts revealed novel undetected APC mutations that were prominent among carcinomas, whereas abundant wild-type APC crypts were detected in adenomas. KRAS mutational heterogeneity within crypts was evident in both adenomas and carcinomas with a higher degree of concordance between biopsy and crypt genotyping in carcinomas. Non-random heterogeneity among crypts was also observed. Conclusions The striking degree of non-random intercrypt heterogeneity in truncal and driver gene mutations observed in adenomas and carcinomas is consistent with a polycryptal model derived from multiple independent initiation linages as the source of early ITH in colorectal carcinogenesis.

Background: There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. Methods:We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. Results:Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice.Conclusions: Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

CHEK2 variants are associated with intermediate breast cancer risk, among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for their classification. First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next-generation sequencing in 1848 prospective patients with HC suspicion. We refined ACMG-AMP criteria and applied different combined rules to classify CHEK2 variants and define risk alleles. We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)-pathogenic; two can also be considered "established risk-alleles" and one as "likely risk-allele." The prevalence of (likely)-pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary nonpolyposis colorectal cancer patients). Here, we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this study would be useful for variant classification of other genes with low effect variants.

TPS9122 Background: Lung cancer (LC) is a rare disease among young adults. Polymorphisms in genes involved in carcinogenic metabolism of tobacco and/or xenobiotic metabolism have been identified in those patients. The growing use of next generation sequencing (NGS) unravelled germline mutations in genes associated with hereditary cancer in patients with LC. However, germline DNA mutations have not been systematically studied in young adults with LC. Early onset LC patients are likely to harbor actionable somatic mutations. Broad hybrid NGS can identify actionable genomic alterations in LC patients deemed driver negative by routine molecular analysis. Trial Design: This is a multicentre, prospective, single-cohort study to determine whether young patients with LC harbor germline mutations and to evaluate the impact of NGS using Foundation One platform on the therapeutic options and overall survival for this specific patient population. Major inclusion criteria: 1) patients already or newly diagnosed of non-small cell lung cancer at any stage at age < 51; 2) to have sufficient tumor sample to perform standard molecular diagnosis ( EGFR/ALK/ROS1/BRAF); 3) to provide written informed consent for the study. Methods: After signing the informed consent form, family history of cancer and smoking history will be collected and a peripheral blood sample will be obtained. Germline targeted sequencing will be carried out in our centre using a customized hereditary cancer panel (I2HCP) of 136 genes designed to cover the coding exons and intron-exon boundaries of genes associated with moderate or high risk of hereditary cancer. Patients with clinical criteria for hereditary cancer or harboring pathogenic or probably pathogenic germline alteration will be referred to the genetic counselling unit. Patients will follow the usual clinical pathway for biomarker analysis and in case of remaining tumour material it will be used for conducting comprehensive genomic profiling through Foundation One platform. When tumour material available will not be sufficient to perform NGS, a tumour re-biopsy will be considered. Enrolment begun on June 2018 and, to date, a total of 41 patients have been included in the study.