Hepatic Carcinoma—Associated Fibroblasts Promote an Adaptative Response in Colorectal Cancer Cells That Inhibit Proliferation and Apoptosis: Nonresistant Cells Die by Nonapoptotic Cell Death

AbstractCarcinoma-associated fibroblasts (CAFs) are important contributors of microenvironment in determining the tumor’s fate. This study aimed to compare the influence of liver microenvironment and primary tumor microenvironment on the behavior of colorectal carcinoma. Conditioned medium (CM) from normal colonic fibroblasts (NCFs), CAFs from primary tumor (CAF-PT) or liver metastasis (CAF-LM) were obtained. We performed functional assays to test the influence of each CM on colorectal cell lines. Microarray and gene set enrichment analysis (GSEA) were performed in DLD1 cells cultured in matched CM. In DLD1 cells, CAF-LM CM compared with CAF-PT CM and NCF led to a more aggressive phenotype, induced the features of an epithelial-to-mesenchymal transition more efficiently, and stimulated migration and invasion to a greater extent. Sustained stimulation with CAF-LM CM evoked a transient G2/M cell cycle arrest accompanied by a reduction of apoptosis, inhibition of proliferation, and decreased viability of SW1116, SW620, SW480, DLD1, HT-29, and Caco-2 cells and provoked nonapoptotic cell death in those cells carrying KRAS mutations. Cells resistant to CAF-LM CM completely changed their morphology in an extracellular signal-regulated protein kinase-dependent process and depicted an increased stemness capacity alongside the Wnt pathway stimulation. The transcriptomic profile of DLD1 cells treated with CAF-LM CM was associated with Wnt and mitogen-activated protein kinase pathways activation in GSEA. Therefore, the liver microenvironment induces more efficiently the aggressiveness of colorectal cancer cells than other matched microenvironments do but secondarily evokes cell death. Resistant cells displayed higher stemness capacity

Comprehensive establishment and characterization of orthoxenograft mouse models of malignant peripheral nerve sheath tumors for personalized medicine

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas that can arise either sporadically or in association with neurofibromatosis type 1 (NF1). These aggressive malignancies confer poor survival, with no effective therapy available. We present the generation and characterization of five distinct MPNST orthoxenograft models for preclinical testing and personalized medicine. Four of the models are patient-derived tumor xenografts (PDTX), two independent MPNSTs from the same NF1 patient and two from different sporadic patients. The fifth model is an orthoxenograft derived from an NF1-related MPNST cell line. All MPNST orthoxenografts were generated by tumor implantation, or cell line injection, next to the sciatic nerve of nude mice, and were perpetuated by 7-10 mouse-to-mouse passages. The models reliably recapitulate the histopathological properties of their parental primary tumors. They also mimic distal dissemination properties in mice. Human stroma was rapidly lost after MPNST engraftment and replaced by murine stroma, which facilitated genomic tumor characterization. Compatible with an origin in a catastrophic event and subsequent genome stabilization, MPNST contained highly altered genomes that remained remarkably stable in orthoxenograft establishment and along passages. Mutational frequency and type of somatic point mutations were highly variable among the different MPNSTs modeled, but very consistent when comparing primary tumors with matched orthoxenografts generated. Unsupervised cluster analysis and principal component analysis (PCA) using an MPNST expression signature of ~1,000 genes grouped together all primary tumor-orthoxenograft pairs. Our work points to differences in the engraftment process of primary tumors compared with the engraftment of established cell lines. Following standardization and extensive characterization and validation, the orthoxenograft models were used for initial preclinical drug testing. Sorafenib (a BRAF inhibitor), in combination with doxorubicin or rapamycin, was found to be the most effective treatment for reducing MPNST growth. The development of genomically well-characterized preclinical models for MPNST allowed the evaluation of novel therapeutic strategies for personalized medicine

BARD1 Pathogenic Variants are Associated with Triple-Negative Breast Cancer in a Spanish Hereditary Breast and Ovarian Cancer Cohort

Only a small fraction of hereditary breast and/or ovarian cancer (HBOC) cases are caused by germline variants in the high-penetrance breast cancer 1 and 2 genes (BRCA1 and BRCA2). BRCA1-associated ring domain 1 (BARD1), nuclear partner of BRCA1, has been suggested as a potential HBOC risk gene, although its prevalence and penetrance are variable according to populations and type of tumor. We aimed to investigate the prevalence of BARD1 truncating variants in a cohort of patients with clinical suspicion of HBOC. A comprehensive BARD1 screening by multigene panel analysis was performed in 4015 unrelated patients according to our regional guidelines for genetic testing in hereditary cancer. In addition, 51,202 Genome Aggregation Database (gnomAD) non-Finnish, non-cancer European individuals were used as a control population. In our patient cohort, we identified 19 patients with heterozygous BARD1 truncating variants (0.47%), whereas the frequency observed in the gnomAD controls was 0.12%. We found a statistically significant association of truncating BARD1 variants with overall risk (odds ratio (OR) = 3.78; CI = 2.10-6.48; p = 1.16 x 10(-5)). This association remained significant in the hereditary breast cancer (HBC) group (OR = 4.18; CI = 2.10-7.70; p = 5.45 x 10(-5)). Furthermore, deleterious BARD1 variants were enriched among triple-negative BC patients (OR = 5.40; CI = 1.77-18.15; p = 0.001) compared to other BC subtypes. Our results support the role of BARD1 as a moderate penetrance BC predisposing gene and highlight a stronger association with triple-negative tumors

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

Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair

Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk

ICO amplicon NGS data analysis: a web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS junior next-Generation Sequencing

Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.Peer ReviewedPostprint (published version

ICO amplicon NGS data analysis: a web tool for variant detection in common high-risk hereditary cancer genes analyzed by amplicon GS junior next-Generation Sequencing

Next-generation sequencing (NGS) has revolutionized genomic research and is set to have a major impact on genetic diagnostics thanks to the advent of benchtop sequencers and flexible kits for targeted libraries. Among the main hurdles in NGS are the difficulty of performing bioinformatic analysis of the huge volume of data generated and the high number of false positive calls that could be obtained, depending on the NGS technology and the analysis pipeline. Here, we present the development of a free and user-friendly Web data analysis tool that detects and filters sequence variants, provides coverage information, and allows the user to customize some basic parameters. The tool has been developed to provide accurate genetic analysis of targeted sequencing of common high-risk hereditary cancer genes using amplicon libraries run in a GS Junior System. The Web resource is linked to our own mutation database, to assist in the clinical classification of identified variants. We believe that this tool will greatly facilitate the use of the NGS approach in routine laboratories.Peer Reviewe

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

Comprehensive establishment and characterization of orthoxenograft mouse models of malignant peripheral nerve sheath tumors for personalized medicine

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas that can arise either sporadically or in association with neurofibromatosis type 1 (NF1). These aggressive malignancies confer poor survival, with no effective therapy available. We present the generation and characterization of five distinct MPNST orthoxenograft models for preclinical testing and personalized medicine. Four of the models are patient-derived tumor xenografts (PDTX), two independent MPNSTs from the same NF1 patient and two from different sporadic patients. The fifth model is an orthoxenograft derived from an NF1-related MPNST cell line. All MPNST orthoxenografts were generated by tumor implantation, or cell line injection, next to the sciatic nerve of nude mice, and were perpetuated by 7-10 mouse-to-mouse passages. The models reliably recapitulate the histopathological properties of their parental primary tumors. They also mimic distal dissemination properties in mice. Human stroma was rapidly lost after MPNST engraftment and replaced by murine stroma, which facilitated genomic tumor characterization. Compatible with an origin in a catastrophic event and subsequent genome stabilization, MPNST contained highly altered genomes that remained remarkably stable in orthoxenograft establishment and along passages. Mutational frequency and type of somatic point mutations were highly variable among the different MPNSTs modeled, but very consistent when comparing primary tumors with matched orthoxenografts generated. Unsupervised cluster analysis and principal component analysis (PCA) using an MPNST expression signature of ~1,000 genes grouped together all primary tumor-orthoxenograft pairs. Our work points to differences in the engraftment process of primary tumors compared with the engraftment of established cell lines. Following standardization and extensive characterization and validation, the orthoxenograft models were used for initial preclinical drug testing. Sorafenib (a BRAF inhibitor), in combination with doxorubicin or rapamycin, was found to be the most effective treatment for reducing MPNST growth. The development of genomically well-characterized preclinical models for MPNST allowed the evaluation of novel therapeutic strategies for personalized medicine