Benchmarking of Whole Exome Sequencing and Ad Hoc Designed Panels for Genetic Testing of Hereditary Cancer

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. Eightythree 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

Molecular profiling and feasibility using a comprehensive hybrid capture panel on a consecutive series of non-small-cell lung cancer patients from a single centre

Background: Targeted next-generation sequencing (NGS) is recommended to screen actionable genomic alterations (GAs) in patients with non-small-cell lung cancer (NSCLC). We determined the feasibility to detect actionable GAs using TruSight™ Oncology 500 (TSO500) in 200 consecutive patients with NSCLC. Materials and methods: DNA and RNA were sequenced on an Illumina® NextSeq 550 instrument and processed using the TSO500 Docker pipeline. Clinical actionability was defined within the molecular tumour board following European Society for Medical Oncology (ESMO) guidelines for oncogene-addicted NSCLC. Overall survival (OS) was estimated as per the presence of druggable GAs and treatment with targeted therapy. Results: Most patients were males (69.5%) and former or current smokers (86.5%). Median age was 64 years. The most common histological type and tumour stage were lung adenocarcinoma (81%) and stage IV (64%), respectively. Sequencing was feasible in most patients (93.5%) and actionable GAs were found in 26.5% of patients. A high concordance was observed between single-gene testing and TSO500 NGS panel. Patients harbouring druggable GAs and receiving targeted therapy achieved longer OS compared to patients without druggable GAs. Conversely, patients with druggable GAs not receiving targeted therapy had a trend toward shorter OS compared with driver-negative patients. Conclusions: Hybrid capture sequencing using TSO500 panel is feasible to analyse clinical samples from patients with NSCLC and is an efficient tool for screening actionable GAs

Benchmarking of Whole Exome Sequencing and Ad Hoc Designed Panels for Genetic Testing of Hereditary Cancer

Acknowledgements: We thank all patients who contributed to this study. The work was supported by grants from the Instituto de Salud Carlos III (ISCIII, MINECO) (operating grants: PI13/00285 and RD12/0036/0008 awarded to C.L. and PIE13/00022 and RD12/0036/0031 awarded to G.C.) and confunded by FEDER funds/European Regional Development Fund (ERDF) - a way to Build Europe-"// FONDOS FEDER "una manera de hacer Europa", the Generalitat de Catalunya (Government of Catalonia) (operating grant 2014SGR338, awarded to G.C.) and the Asociación Española Contra el Cáncer (operating grants, 2010 Grupos Estables, awarded to G.C.). J.B. received a Spanish Society of Medical Oncology grant. This activity is sponsored by the ISCIII Ministerio de Economía y Competitividad (PT13/0001/0044).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. Eightythree 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

Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma

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 evaluated. 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

Biomarcadors en càncer colorectal hereditari: aplicacions clíniques i estudi de la dinàmica tumoral

[cat] • L’anàlisi de la hipermetilació del promotor d’MLH1 utilitzant el mètode de l’MS-MLPA ha demostrat ser un mètode de selecció precís, i més rendible que l’estudi de la mutació BRAF en la selecció dels pacients que són candidats a ser estudiats a nivell germinal en el gen MLH1, quan hi ha sospita de ser síndrome de Lynch i quan no hi ha expressió de la proteïna MLH1 en el tumor. • Alguns casos de síndrome de Lynch poden no ser detectats amb aquesta estratègia, el que posa de relleu la importància del criteri clínic en l’estudi de casos par tal de minimitzar els falsos negatius. • La PCR digital proporciona una mesura robusta i quantitativa de la proporció d’al·lels de KRAS mutats en mostres de tumors sent capaç de detectar al·lels mutats minoritaris. L’ús d’aquesta metodologia dóna una nova visió de l’heterogeneïtat intratumoral en hotspots seleccionats durant la tumorogènesi. • L’estudi de la dinàmica de les metapoblacions mostra com el càncer és un procés evolutiu on la càrrega mutacional sembla estar augmentada en les primeres etapes de la tumorogènesi, tot i que, en general, aquesta variabilitat no es tradueix en selecció d’aquestes mutacions. • L’anàlisi de l’heterogeneïtat basada en el doble enfoc de l’estudi de criptes amb tècniques d'alta sensibilitat analítica contribueix a comprendre els mecanismes mutacionals, destacant l'augment de la càrrega mutacional present en etapes primerenques de la tumorogènesi. • L'heterogeneïtat observada reflecteix els antecedents genètics de predisposició al càncer. • L’estudi de les metapoblacions tumorals basat en l'aparició de mutacions i la seva comparació, ens pot ajudar a entendre millor el procés d'expansió en les primeres etapes de la tumorogènesi.[eng] • 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. • Analysis of MLH1 hypermethylation using MS-MLPA has very few false negative results, making this technique a reasonable option in the diagnostic algorithm of LS. In any case, clinicians must be aware that some LS cases may not be identified. • Digital PCR provides a robust, quantitative measure of the proportion of KRAS mutant alleles in routinely obtained samples. • Digital PCR has allowed the identification of multiple alleles in a substantial proportion of the analyzed samples. The use of techniques of increased sensitivity provides new insights into the intratumoral heterogenity. • Study of mutational dynamics has shown that there is a striking degree of heterogeneity of cell populations in early stages of colorectal tumorigenesis that is nonrandom and depends on the genetic background. • The vast majority of mutated subpopulations identified are not selected during tumor progression. • The study of metapopulations based on highly sensitive mutation detection methods using was likely to offer novel insights into the dynamics of tumor cell populations in early and advanced tumorigenesis

MLH1 promoter hypermethylation in the analytical algorithm of Lynch syndrome: a cost-effectiveness study

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

MLH1 promoter hypermethylation in the analytical algorithm of Lynch syndrome: a cost-effectiveness study

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

Benchmarking of Whole Exome Sequencing and Ad Hoc Designed Panels for Genetic Testing of Hereditary Cancer

Acknowledgements: We thank all patients who contributed to this study. The work was supported by grants from the Instituto de Salud Carlos III (ISCIII, MINECO) (operating grants: PI13/00285 and RD12/0036/0008 awarded to C.L. and PIE13/00022 and RD12/0036/0031 awarded to G.C.) and confunded by FEDER funds/European Regional Development Fund (ERDF) - a way to Build Europe-"// FONDOS FEDER "una manera de hacer Europa", the Generalitat de Catalunya (Government of Catalonia) (operating grant 2014SGR338, awarded to G.C.) and the Asociación Española Contra el Cáncer (operating grants, 2010 Grupos Estables, awarded to G.C.). J.B. received a Spanish Society of Medical Oncology grant. This activity is sponsored by the ISCIII Ministerio de Economía y Competitividad (PT13/0001/0044).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. Eightythree 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

Benchmarking of Whole Exome Sequencing and Ad Hoc Designed Panels for Genetic Testing of Hereditary Cancer

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. Eightythree 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

Benchmarking of Whole Exome Sequencing and Ad Hoc Designed Panels for Genetic Testing of Hereditary Cancer

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. Eightythree 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