Optimizing the identification of risk-relevant mutations by multigene panel testing in selected hereditary breast/ovarian cancer families

The introduction of multigene panel testing for hereditary breast/ovarian cancer screening has greatly improved efficiency, speed, and costs. However, its clinical utility is still debated, mostly due to the lack of conclusive evidences on the impact of newly discovered genetic variants on cancer risk and lack of evidence-based guidelines for the clinical management of their carriers. In this pilot study, we aimed to test whether a systematic and multiparametric characterization of newly discovered mutations could enhance the clinical utility of multigene panel sequencing. Out of a pool of 367 breast/ovarian cancer families Sanger-sequenced for BRCA1 and BRCA2 gene mutations, we selected a cohort of 20 BRCA1/2-negative families to be subjected to the BROCA-Cancer Risk Panel massive parallel sequencing. As a strategy for the systematic characterization of newly discovered genetic variants, we collected blood and cancer tissue samples and established lymphoblastoid cell lines from all available individuals in these families, to perform segregation analysis, loss-of-heterozygosity and further molecular studies. We identified loss-of-function mutations in 6 out 20 high-risk families, 5 of which occurred on BRCA1, CHEK2 and ATM and are esteemed to be risk-relevant. In contrast, a novel RAD50 truncating mutation is most likely unrelated to breast cancer. Our data suggest that integrating multigene panel testing with a pre-organized, multiparametric characterization of newly discovered genetic variants improves the identification of risk-relevant alleles impacting on the clinical management of their carriers

Presumed TP53 mosaicism: variants detected using a NGS hereditary cancer multigene panel

Aims/Context: NGS multigene panels are routinely used to identify germline pathogenic variants in cancer susceptibility genes. In addition, NGS allows the identification of low-level mosaicism events that may not be detectable by conventional Sanger sequencing. We describe two cases of presumed TP53 mosaic variants detected by NGS on blood-derived DNA, and confirmed by ARMS-PCR and Sanger sequencing. Case 1: female, 87 years old, colon cancer at 83 and metachronous breast cancer at 86, no history of familial cancer. Case 2: female, 75 years old, ovarian cancer at 71, local relapse at 74. Methods: NGS using TruSight® Cancer Sequencing Panel and TruSight® Rapid Capture kit (Illumina) and paired-end sequencing on MiSeq® platform (Illumina). Bioinformatic analysis with MiSeq Reporter, Enrichment, VariantStudio, VEP, Alamut Visual, VarAFT, VarSome and IGV. ARMS-PCR and Sanger sequencing were used to confirm the TP53 variants. Results and Conclusions: Two cases of presumed TP53 mosaic variants were studied. Case 1: the missense alteration TP53: c.764T>G, p.(Ile255Ser) was detected with a variant allele frequency (VAF) of 26% (39/150 reads). This variant is described in ClinVar as a somatic alteration, classified as likely pathogenic. It is not reported in gnomAD and VarSome software classified it as a variant of uncertain significance. Case 2: missense variant TP53: c.524G>A, p.(Arg175His) detected with a VAF of 15% (10/58 reads). This variant is described as pathogenic in HGMD Professional, LOVD and ClinVar, in association with Li-Fraumeni syndrome. These two cases seem to represent TP53 mosaicism, supported by: i) VAF lower than 30%, ii) detection at the sensitivity limit of Sanger sequencing and iii) confirmation by ARMS-PCR. Confirming this hypothesis by studying tumor and other tissue samples and offspring analysis (underway in both cases), is essential for disease diagnosis, assessing recurrence risk and genetic counseling. The hypothesis of acquired aberrant clonal expansion limited to the hematologic compartment, versus a germline variant should be considered in similar cases, and confirmatory methodologies are mandatory.info:eu-repo/semantics/publishedVersio

A Robust Gene Expression Prognostic Signature for Overall Survival in High-Grade Serous Ovarian Cancer.

The objective of this research was to develop a robust gene expression-based prognostic signature and scoring system for predicting overall survival (OS) of patients with high-grade serous ovarian cancer (HGSOC). Transcriptomic data of HGSOC patients were obtained from six independent studies in the NCBI GEO database. Genes significantly deregulated and associated with OS in HGSOCs were selected using GEO2R and Kaplan-Meier analysis with log-rank testing, respectively. Enrichment analysis for biological processes and pathways was performed using Gene Ontology analysis. A resampling/cross-validation method with Cox regression analysis was used to identify a novel gene expression-based signature associated with OS, and a prognostic scoring system was developed and further validated in nine independent HGSOC datasets. We first identified 488 significantly deregulated genes in HGSOC patients, of which 232 were found to be significantly associated with their OS. These genes were significantly enriched for cell cycle division, epithelial cell differentiation, p53 signaling pathway, vasculature development, and other processes. A novel 11-gene prognostic signature was identified and a prognostic scoring system was developed, which robustly predicted OS in HGSOC patients in 100 sampling test sets. The scoring system was further validated successfully in nine additional HGSOC public datasets. In conclusion, our integrative bioinformatics study combining transcriptomic and clinical data established an 11-gene prognostic signature for robust and reproducible prediction of OS in HGSOC patients. This signature could be of clinical value for guiding therapeutic selection and individualized treatment

Multi-gene panel testing for hereditary cancer predisposition in unsolved high-risk breast and ovarian cancer patients.

PurposeMany women with an elevated risk of hereditary breast and ovarian cancer have previously tested negative for pathogenic mutations in BRCA1 and BRCA2. Among them, a subset has hereditary susceptibility to cancer and requires further testing. We sought to identify specific groups who remain at high risk and evaluate whether they should be offered multi-gene panel testing.MethodsWe tested 300 women on a multi-gene panel who were previously enrolled in a long-term study at UCSF. As part of their long-term care, all previously tested negative for mutations in BRCA1 and BRCA2 either by limited or comprehensive sequencing. Additionally, they met one of the following criteria: (i) personal history of bilateral breast cancer, (ii) personal history of breast cancer and a first or second degree relative with ovarian cancer, and (iii) personal history of ovarian, fallopian tube, or peritoneal carcinoma.ResultsAcross the three groups, 26 women (9%) had a total of 28 pathogenic mutations associated with hereditary cancer susceptibility, and 23 women (8%) had mutations in genes other than BRCA1 and BRCA2. Ashkenazi Jewish and Hispanic women had elevated pathogenic mutation rates. In addition, two women harbored pathogenic mutations in more than one hereditary predisposition gene.ConclusionsAmong women at high risk of breast and ovarian cancer who have previously tested negative for pathogenic BRCA1 and BRCA2 mutations, we identified three groups of women who should be considered for subsequent multi-gene panel testing. The identification of women with multiple pathogenic mutations has important implications for family testing

A simplified genomic profiling approach predicts outcome in metastatic colorectal cancer

The response of metastatic colorectal cancer (mCRC) to the first-line conventional combination therapy is highly variable, reflecting the elevated heterogeneity of the disease. The genetic alterations underlying this heterogeneity have been thoroughly characterized through omic approaches requiring elevated efforts and costs. In order to translate the knowledge of CRC molecular heterogeneity into a practical clinical approach, we utilized a simplified Next Generation Sequencing (NGS) based platform to screen a cohort of 77 patients treated with first-line conventional therapy. Samples were sequenced using a panel of hotspots and targeted regions of 22 genes commonly involved in CRC. This revealed 51 patients carrying actionable gene mutations, 22 of which carried druggable alterations. These mutations were frequently associated with additional genetic alterations. To take into account this molecular complexity and assisted by an unbiased bioinformatic analysis, we defined three subgroups of patients carrying distinct molecular patterns. We demonstrated these three molecular subgroups are associated with a different response to first-line conventional combination therapies. The best outcome was achieved in patients exclusively carrying mutations on TP53 and/or RAS genes. By contrast, in patients carrying mutations in any of the other genes, alone or associated with mutations of TP53/RAS, the expected response is much worse compared to patients with exclusive TP53/RAS mutations. Additionally, our data indicate that the standard approach has limited efficacy in patients without any mutations in the genes included in the panel. In conclusion, we identified a reliable and easy-to-use approach for a simplified molecular-based stratification of mCRC patients that predicts the efficacy of the first-line conventional combination therapy

Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids

© 2012 Matyášek et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Advanced diagnostic genetic testing in inherited retinal disease: experience from a single tertiary referral centre in the UK National Health Service

In 2013, as part of our genetic investigation of patients with inherited retinal disease, we utilized multigene panel testing of 105 genes known to cause retinal disease in our patient cohorts. This test was performed in a UK National Health Service (NHS) accredited laboratory. The results of all multigene panel tests requested between 1.4.13 and 31.8.14 were retrospectively reviewed. All patients had been previously seen at Moorfields Eye Hospital, London, UK and diagnosed with an inherited retinal dystrophy after clinical examination and detailed retinal imaging. The results were categorized into three groups: (i) Testing helped establish a certain molecular diagnosis in 45 out of 115 (39%). Variants in USH2A (n = 6) and RP1 (n = 4) were most common. (ii) Definitive conclusions could not be drawn from molecular testing alone in 13 out of 115 (11%) as either insufficient pathogenic variants were discovered or those identified were not consistent with the phenotype. (iii) Testing did not identify any pathogenic variants responsible for the phenotype in 57 out of 115 (50%). Multigene panel testing performed in an NHS setting has enabled a molecular diagnosis to be confidently made in 40% of cases. Novel variants accounted for 38% of all identified variants. Detailed retinal phenotyping helped the interpretation of specific variants. Additional care needs to be taken when assessing polymorphisms in genes that have been infrequently associated with disease, as historical techniques were not as rigorous as contemporary ones. Future iterations of sequencing are likely to offer higher sensitivity, testing a broader range of genes, more rapidly and at a reduced cost

Targeted disruption of py235ebp-1: Invasion of erythrocytes by Plasmodium yoelii using an alternative Py235 erythrocyte binding protein

Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression