A novel BRCA2 splice variant identified in a young woman

Background: BRCA1/2 VUSs represent an important clinical issue in risk assessment for the breast/ovarian cancer families (HBOC) families. Among them, some occurring within the intron-exon boundary may lead to aberrant splicing process by altering or creating de novo splicing regulatory elements or unmasking cryptic splice site. Defining the impact of these potential splice variants at functional level is important to establish their pathogenic role. Methods: Genomic DNA was extracted from peripheral blood sample of a young woman affected with breast cancer belonging to a HBOC family and the entire coding regions of the BRCA1 and BRCA2 genes were amplified using the Ion AmpliSeq BRCA1 and BRCA2 Panel. The BRCA2 c.682-2delA variant has been characterized by RT-PCR analysis performed on mRNA extracted from blood and lymphoblastoid cell line. Results: We demonstrated that a novel BRCA2 c.682-2delA variant at the highly conserved splice consensus site in intron 8 disrupts the canonical splice acceptor site generating a truncated protein as predicted by several bioinformatics tools. Segregations analysis in the family and LOH performed on proband breast cancer tissue further confirmed its classification as pathogenic variant. Conclusion: Combining different methodologies, we characterized this new BRCA2 variant and provided findings of clinical utility for its classification as pathogenic variant

Evaluation of polygenic determinants of non-alcoholic fatty liver disease (NAFLD) by a candidate genes resequencing strategy

NAFLD is a polygenic condition but the individual and cumulative contribution of identified genes remains to be established. To get additional insight into the genetic architecture of NAFLD, GWAS-identified GCKR, PPP1R3B, NCAN, LYPLAL1 and TM6SF2 genes were resequenced by next generation sequencing in a cohort of 218 NAFLD subjects and 227 controls, where PNPLA3 rs738409 and MBOAT7 rs641738 genotypes were also obtained. A total of 168 sequence variants were detected and 47 were annotated as functional. When all functional variants within each gene were considered, only those in TM6SF2 accumulate in NAFLD subjects compared to controls (P = 0.04). Among individual variants, rs1260326 in GCKR and rs641738 in MBOAT7 (recessive), rs58542926 in TM6SF2 and rs738409 in PNPLA3 (dominant) emerged as associated to NAFLD, with PNPLA3 rs738409 being the strongest predictor (OR 3.12, 95% CI, 1.8-5.5, P 0.28 was associated with a 3-fold increased risk of NAFLD. Interestingly, rs61756425 in PPP1R3B and rs641738 in MBOAT7 genes were predictors of NAFLD severity. Overall, TM6SF2, GCKR, PNPLA3 and MBOAT7 were confirmed to be associated with NAFLD and a score based on these genes was highly predictive of this condition. In addition, PPP1R3B and MBOAT7 might influence NAFLD severity

po 320 gene panel mutation screening for a better molecular stratification of colorectal cancer patients

Introduction Colorectal carcinoma (CRC) is one of the most commonly diagnosed cancers worldwide. The metastatic disease contributes to the high mortality rate reported for such tumours. Significant benefit on overall survival was brought about the introduction of monoclonal antibodies anti-EGFR and anti-VEGF used in combination with chemotherapy in metastatic CRC (mCRC). While anti-VEGF treatment does not require biomarker-based selection criteria, the potential efficacy of anti-EGFR antibodies is neglected to patients with activating mutations in KRAS and NRAS (RAS) genes, whose molecular analysis became a clinical routine. The advent of Next Generation Sequencing (NGS) instruments, able to reach quick testing of multiple clinically-relevant hotspots, yet maintaining precision and low costs, allows the simultaneous determination of the mutation status of an expanding number of genes. Despite only few of these molecular biomarkers have gained clinical utility in the routine oncological practice, the acquisition of more complex cancer mutational patterns may provide more efficient tumour characterisation for prognostic and predictive purposes and highlight actionable targets. Material and methods We sequenced 639 mCRC samples by IT-PGM platform using a panel of hotspots and targeted regions of 22 genes (including RAS) commonly involved in CRCs. MSI analyses on 89 patients have been performed with a single fluorescent system comprising BAT25 and BAT26 mononucleotide repeats. Results and discussions We identified recurrent mutations (≥1%) in 12/22 genes, being KRAS, TP53 and PIK3CA the most frequently mutated ones. Statistical analysis, indicated that the mutation associations follow a non-random distribution. Categorization of the cases on the base of KRAS and p53 mutation status led us the definition of 8 Mutation Association Patterns (MAPs) characterised by specific mutation associations. Analysis of the clinicopathological data available for 89 out of 639 cases indicates interesting trends for the associations of MAPs with specific parameters, some of which reached statistical significance. Conclusion Application of NGS gene panel as a routine for the characterisation of RAS/BRAF status required for predictive purposes in CRC patients, may provide additional prognostic/predictive information, with no significant extra-costs

PIK3CA somatic mutation in sinonasal teratocarcinosarcoma.

Sinonasal Teratocarcinosarcoma (SNTCS) is a rare and histologically heterogeneous tumor of uncertain origin and unknown molecular pathogenesis. Its location and aggressiveness, with frequent recurrences, high rate for metastasis and short mean survival, make SNTCS a tumor highly difficult to treat. Thus, the identification of underlying genetic changes could potentially provide successful adjuvant or alternative precision medicine treatment options for patients with this tumor. We report here a 55-year-old male with a naso-ethmoidal SNTCS that invaded right maxillary sinus, orbital cavity and cranial anterior fossa and that was treated with surgery followed by radiotherapy and chemotherapy in which we evaluated the mutational profile by multigene panel sequencing. Tumor and adjacent normal mucosa were screened for hotspots and targeted regions of 22 cancer related genes by multigene panel sequencing. The analysis revealed a somatic pathogenic mutations in the PIK3CA gene (p.His1047Leu) and a germline alteration in the DDR2 gene (p.Pro476Leu) whose oncogenic function is considered unknown. This study suggests the involvement of PIK3CA gene mutation in SNTCS tumorigenesis highlighting a potential target for individualized molecular therapy for patients with this tumor

An integrative in-silico analysis discloses a novel molecular subset of colorectal cancer possibly eligible for immune checkpoint immunotherapy

Background: Historically, the molecular classification of colorectal cancer (CRC) was based on the global genomic status, which identified microsatellite instability in mismatch repair (MMR) deficient CRC, and chromosomal instability in MMR proficient CRC. With the introduction of immune checkpoint inhibitors, the microsatellite and chromosomal instability classification regained momentum as the microsatellite instability condition predicted sensitivity to immune checkpoint inhibitors, possibly due to both high tumor mutation burden (TMB) and high levels of infiltrating lymphocytes. Conversely, proficient MMR CRC are mostly resistant to immunotherapy. To better understand the relationship between the microsatellite and chromosomal instability classification, and eventually discover additional CRC subgroups relevant for therapeutic decisions, we developed a computational pipeline that include molecular integrative analysis of genomic, epigenomic and transcriptomic data. Results: The first step of the pipeline was based on unsupervised hierarchical clustering analysis of copy number variations (CNVs) versus hypermutation status that identified a first CRC cluster with few CNVs enriched in Hypermutated and microsatellite instability samples, a second CRC cluster with a high number of CNVs mostly including non-HM and microsatellite stable samples, and a third cluster (7.8% of the entire dataset) with low CNVs and low TMB, which shared clinical-pathological features with Hypermutated CRCs and thus defined Hypermutated-like CRCs. The mutational features, DNA methylation profile and base substitution fingerprints of these tumors revealed that Hypermutated-like patients are molecularly distinct from Hypermutated and non-Hypermutated tumors and are likely to develop and progress through different genetic events. Transcriptomic analysis highlighted further differences amongst the three groups and revealed an inflamed tumor microenvironment and modulation Immune Checkpoint Genes in Hypermutated-like CRCs. Conclusion: Therefore, our work highlights Hypermutated-like tumors as a distinct and previously unidentified CRC subgroup possibly responsive to immune checkpoint inhibitors. If further validated, these findings can lead to expanding the fraction of patients eligible to immunotherapy

Circulating tumor cells in right-and left-sided colorectal cancer

Molecular alterations are not randomly distributed in colorectal cancer (CRC), but rather clustered on the basis of primary tumor location underlying the importance of colorectal cancer sidedness. We aimed to investigate whether circulating tumor cells (CTC) characterization might help clarify how different the patterns of dissemination might be relative to the behavior of left-(LCC) compared to right-sided (RCC) cancers. We retrospectively analyzed patients with metastatic CRC who had undergone standard baseline CTC evaluation before starting any first-line systemic treatment. Enumeration of CTC in left-and right-sided tumors were compared. The highest prognostic impact was exerted by CTC in left-sided primary cancer patients, even though the lowest median number of cells was detected in this subgroup of patients. CTC exhibit phenotypic heterogeneity, with a predominant mesenchymal phenotype found in CTC from distal compared to proximal primary tumors. Most CTC in RCC patients exhibited an apoptotic pattern. CTC in left-sided colon cancer patients exhibit a predominant mesenchymal phenotype. This might imply a substantial difference in the biology of proximal and distal cancers, associated with different patterns of tumor cells dissemination. The poor prognosis of right-sided CRC is not determined by the hematogenous dissemination of tumor cells, which appears to be predominantly a passive shedding of non-viable cells. Conversely, the subgroup of poor-prognosis left-sided CRC is reliably identified by the presence of mesenchymal CTC

RAS mutation conversion in bevacizumab-treated metastatic colorectal cancer patients: a liquid biopsy based study

Liquid biopsies have shown that, in RAS mutant colorectal cancer, the conversion to RAS wild-type * status during the course of the disease is a frequent event, supporting the concept that the evolutionary landscape of colorectal cancer can lead to an unexpected negative selection of RAS mutant clones. The aim of the present study was to clarify whether the negative selection of RAS mutation in plasma might be drug-dependent. For this purpose, we used liquid biopsy to compare the rate of conversion from RAS mutant to RAS wild-type * in two groups of originally RAS mutant mCRC patients: the first treated with chemotherapy alone, while the second was treated with chemotherapy combined with bevacizumab. Serial liquid biopsies were performed at 3 months (T1), 6 months (T2), 9 months (T3), and 12 months (T4) after starting first line treatments. We found that the only independent variable significantly associated to RAS status conversion was the use of bevacizumab. RAS conversion was not found associated to tumor burden reduction, although bevacizumab-treated patients who converted to RAS wild-type * had a significantly longer PFS compared to patients who remained RAS mutant. The appearance of a “RAS wild-type * window”, mainly in bevacizumab-treated patients, might present them as candidates for second line treatment with anti-EGFR, which was otherwise precluded

A multidisciplinary approach for the differential diagnosis between multiple primary lung adenocarcinomas and intrapulmonary metastases

Purpose: The distinction between multiple primary lung cancers (MPLCs) and intrapulmonary metastases has a significant impact on tumor staging and therapeutic choices. Several criteria have been proposed to solve this diagnostic issue, but a definitive consensus is still missing. We tested the efficacy of a combined clinical, histopathological and molecular (“real world”) approach for the correct classification of multiple lung tumors in a selected cohort of patients. Methods: 24 multiple lung tumors with a diagnosis of adenocarcinoma from 10 patients were retrospectively reviewed. Radiological, pathological and clinical information, including follow-up, were integrated with molecular profiling via a routine multigene panel sequencing. Results: Comprehensive histologic assessment revealed readily distinguishable histologic patterns between multiple tumors suggesting unrelated lesions in 7 cases, in agreement with clinical, radiological and molecular data, thus leading to final diagnosis of MPLCs. In the remaining 3 cases, the differential diagnosis between MPLCs and intrapulmonary metastases was challenging, since the histologic features of the lesions were similar or identical. The final interpretation (2 MPLCs and 1 most likely intrapulmonary metastases) was reached thanks to the integration of all available data, and was confirmed by follow-up. Conclusions: A multidisciplinary approach including a routinely affordable multigene panel sequencing is a useful tool to discriminate MPLCs from intrapulmonary metastases in multiple lung nodules sharing the adenocarcinoma histotype