Technological advances have led to the introduction of next-generation sequencing (NGS) platforms in cancer investigation. NGS allows massive parallel sequencing that affords maximal tumor genomic assessment. NGS approaches are different, and concern DNA and RNA analysis. DNA sequencing includes whole-genome, whole-exome, and targeted sequencing, which focuses on a selection of genes of interest for a specific disease. RNA sequencing facilitates the detection of alternative gene-spliced transcripts, posttranscriptional modifications, gene fusion, mutations/single-nucleotide polymorphisms, small and long noncoding RNAs, and changes in gene expression. Most applications are in the cancer research field, but lately NGS technology has been revolutionizing cancer molecular diagnostics, due to the many advantages it offers compared to traditional methods. There is greater knowledge on solid cancer diagnostics, and recent interest has been shown also in the field of hematologic cancer. In this review, we report the latest data on NGS diagnostic/predictive clinical applications in solid and hematologic cancers. Moreover, since the amount of NGS data produced is very large and their interpretation is very complex, we briefly discuss two bioinformatic aspects, variant-calling accuracy and copy-number variation detection, which are gaining a lot of importance in cancer-diagnostic assessment.

Background-Global DNA hypomethylation has been found in the premalignant stages of some neoplasms and has been implicated as an important factor for tumour progression. Aims-The aim of this study was to evaluate whether DNA hypomethylation occurs during the process of gastric carcinogenesis. Methods-Gastric specimens were obtained from 49

BackgroundNGS technology represents a powerful alternative to the standard Sanger sequencing in the context of clinical setting. The proprietary software that are generally used for variant calling often depend on preset parameters that may not fit in a satisfactory manner for different genes.GATK, which is widely used in the academic world, is rich in parameters for variant calling. However the self-adjusting parameter calibration of GATK requires data from a large number of exomes. When these are not available, which is the standard condition of a diagnostic laboratory, the parameters must be set by the operator (hard filtering). The aim of the present paper was to set up a procedure to assess the best parameters to be used in the hard filtering of GATK. This was pursued by using classification trees on true and false variants from simulated sequences of a real dataset data.ResultsWe simulated two datasets, with different coverages, including all the sequence alterations identified in a real dataset according to their observed frequencies. Simulated sequences were aligned with standard protocols and then regression trees were built up to identify the most reliable parameters and cutoff values to discriminate true and false variant calls. Moreover, we analyzed flanking sequences of region presenting a high rate of false positive calls observing that such sequences present a low complexity make up.ConclusionsOur results showed that GATK hard filtering parameter values can be tailored through a simulation study based-on the DNA region of interest to ameliorate the accuracy of the variant calling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-017-1537-8) contains supplementary material, which is available to authorized users.

We demonstrated that MAs in DNA from EBC of patients with NSCLC are significantly more frequent than in control subjects. More interesting, the MA profile of DNA from EBC corresponds to that from lung cancer tissue of each patient with NSCLC.

As in women, three classes of breast cancer genetic susceptibility (high, moderate, and low penetrance) are recognized in men. However, genes involved and their impact do not exactly overlap in female and male BC. Epigenetic alterations are currently scarcely investigated in MBC, however, the different methylation and miRNA expression profiles identified to date in female and male BCs suggest a potential role for epigenetic alterations as diagnostic biomarkers. Overall, much still needs to be learned about MBC and, because of its rarity, the main effort is to develop large consortia for moving forward in understanding MBC and improving the management of MBC patients on a perspective of gender medicine.

The molecular scenario of breast cancer has become more complex in the last few years. Distinguishing between BRCA-associated, sporadic, HER2-enriched and triple-negative tumors is not sufficient to allow effective clinical management. Basal-like breast cancer, a subtype of triple-negative breast cancer, differs from others grouped under this heading. Commonalities between BRCA-related tumors and basal-like breast cancers (BRCAness phenotype) are highly relevant to ongoing clinical trials, in particular those investigating targeted therapies (e.g. PARP inhibitors) in sporadic breast tumors. The 'gold standard' to identify basal-like phenotype is DNA microarray, but integrated results could provide a panel of biomarkers helpful in identifying 'BRCAness' tumors (e.g. copy number aberrations, abnormal protein localization and altered transcriptional levels) and other molecular targets, such as APE1,the inhibition of which is emerging as an attractive breast cancer treatment in certain therapeutic settings.

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Epigenetic regulation, has been very scarcely explored in familial breast cancer (BC). In the present study RASSF1A and RAR beta promoter methylation and miR17, miR21, miR 124, and let-7a expression were investigated to highlight possible differences of epigenetic regulation between male and female familial BC, also in comparison with sporadic BC. These epigenetic alterations were studied in 56 familial BC patients (27 males and 29 females) and in 16 female sporadic cases. RASSF1A resulted more frequently methylated in men than women (76% vs. 28%, respectively, P = 0.0001), while miR17 and let-7a expression frequency was higher in women than in men (miR17: 66% in women vs. 41% in men, P < 0.05; let-7a: 45% in women vs. 15% in men, P = 0.015). RASSF1A methylation affected 27.6% of familial BC while 83% of familial cases showed high expression of the gene (P = 0.025); on the contrary, only 17% of familial BC presented RAR beta methylation and 55% of familial cases overexpressed this gene (P = 0.005). Moreover, miR17, miR21, and let-7a resulted significantly overexpressed in familial compared to sporadic BC. RASSF1A overexpression (86% vs. 65%, P = 0.13) and RAR beta overexpression (57% vs. 32%, P = 0.11) were higher in BRCA1/2 carriers even if not statistical significance was reached. BRCA mutation carriers also demonstrated significant overexpression of: miR17 (93% vs. 35%, P = 0.0001), let-7a (64% vs. 16%, P = 0.002), and of miR21 (100% vs. 65%, P = 0.008). In conclusion, the present data suggest the involvement of RASSF1A in familial male BC, while miR17 and let-7a seem to be implied in familial female BC.