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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.
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Aminopiridinas/administración & dosificación , Bencimidazoles/administración & dosificación , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/genética , Mesotelioma Maligno/tratamiento farmacológico , Piperazinas/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridinas/administración & dosificación , Anciano , Aminopiridinas/farmacología , Animales , Bencimidazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Mesotelioma Maligno/genética , Mesotelioma Maligno/metabolismo , Ratones , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CHEK2 variants are associated with intermediate breast cancer risk, among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for their classification. First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next-generation sequencing in 1848 prospective patients with HC suspicion. We refined ACMG-AMP criteria and applied different combined rules to classify CHEK2 variants and define risk alleles. We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)-pathogenic; two can also be considered "established risk-alleles" and one as "likely risk-allele." The prevalence of (likely)-pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary nonpolyposis colorectal cancer patients). Here, we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this study would be useful for variant classification of other genes with low effect variants.
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Quinasa de Punto de Control 2/genética , Variación Genética , Neoplasias/genética , Sociedades Científicas , Secuencia de Bases , Estudios de Cohortes , Variaciones en el Número de Copia de ADN/genética , Familia , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Anotación de Secuencia Molecular , Mutación/genética , Neoplasias/patología , Linaje , Sitios de Empalme de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Purpose: The majority of genomic alterations causing intratumoral heterogeneity (ITH) in colorectal cancer are thought to arise during early stages of carcinogenesis as a burst but only after truncal mutations in APC have expanded a single founder clone. We have investigated if the initial source of ITH is consequent to multiple independent lineages derived from different crypts harboring distinct truncal APC and driver KRAS mutations, thus challenging the prevailing monoclonal monocryptal model.Experimental Design: High-depth next-generation sequencing and SNP arrays were performed in whole-lesion extracts of 37 familial adenomatous polyposis colorectal adenomas. Also, ultrasensitive genotyping of hotspot mutations of APC and KRAS was performed using nanofluidic PCRs in matched bulk biopsies (n = 59) and crypts (n = 591) from 18 adenomas and seven carcinomas and adjacent normal tissues.Results: Multiple co-occurring truncal APC and driver KRAS alterations were uncovered in whole-lesion extracts from adenomas and subsequently confirmed to belong to multiple clones. Ultrasensitive genotyping of bulk biopsies and crypts revealed novel undetected APC mutations that were prominent among carcinomas, whereas abundant wild-type APC crypts were detected in adenomas. KRAS mutational heterogeneity within crypts was evident in both adenomas and carcinomas with a higher degree of concordance between biopsy and crypt genotyping in carcinomas. Nonrandom heterogeneity among crypts was also observed.Conclusions: The striking degree of nonrandom intercrypt heterogeneity in truncal and driver gene mutations observed in adenomas and carcinomas is consistent with a polycryptal model derived from multiple independent initiation linages as the source of early ITH in colorectal carcinogenesis. Clin Cancer Res; 23(19); 5936-47. ©2017 AACR.
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Proteína de la Poliposis Adenomatosa del Colon/genética , Carcinogénesis/genética , Evolución Clonal/genética , Neoplasias Colorrectales/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Biopsia , Carcinoma , Neoplasias Colorrectales/patología , Femenino , Heterogeneidad Genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , MutaciónRESUMEN
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. Eighty-three 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.
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Predisposición Genética a la Enfermedad , Pruebas Genéticas/métodos , Síndromes Neoplásicos Hereditarios/diagnóstico , Benchmarking , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Síndromes Neoplásicos Hereditarios/genética , Secuenciación del Exoma/métodosRESUMEN
BACKGROUND: Concomitant quantification of multiple mutant KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) alleles may provide information in addition to that provided by standard mutation-detection procedures. We assessed the feasibility of a nanofluidic digital PCR array platform to detect and quantify KRAS mutations simultaneously in clinically relevant samples. METHODS: We assessed 2 groups of patients (colorectal and pancreatic disease): Group 1 consisted of 27 patients with colorectal carcinomas, 14 patients with adenomas, and 5 control individuals; group 2 consisted of 42 patients with pancreatic carcinoma, 4 with adenocarcinomas of the ampulla, and 6 with chronic pancreatitis). Digital PCR was performed with the Digital Array Chip (Fluidigm). RESULTS: Nanofluidic digital PCR detected mutant alleles at 0.05% to 0.1%, depending on the variant analyzed. For the colorectal disease group, conventional PCR detected 9 (64%) of 14 adenomas that were positive for KRAS mutants, whereas digital PCR increased this number to 11 (79%) of 14. Sixteen (59%) of 27 carcinomas showed KRAS mutation with conventional PCR. Two additional cases were detected with digital PCR. In 5 cases (3 adenomas, 2 carcinomas), the total number of mutant alleles changed. For the pancreatic disease group, digital PCR increased the number of positive cases from 26 to 34 (81%) and identified ≥ 2 mutant alleles in 25 cases, compared with conventional PCR, which identified multiple KRAS mutant alleles in only 12 cases. A good correlation was observed between results obtained with tumor biopsies and those obtained with pancreatic juice. CONCLUSIONS: Digital PCR provides a robust, quantitative measure of the proportion of KRAS mutant alleles in routinely obtained samples. It also allows a better classification of tumors, with potential clinical relevance.
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Neoplasias Gastrointestinales/genética , Genes ras , Microfluídica , Mutación , Nanotecnología , Adulto , Anciano , Estudios de Factibilidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la PolimerasaRESUMEN
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.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Algoritmos , Neoplasias Colorrectales Hereditarias sin Poliposis/diagnóstico , Metilación de ADN , Pruebas Genéticas/economía , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas Adaptadoras Transductoras de Señales/genética , Adulto , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Análisis Costo-Beneficio , Pruebas Genéticas/métodos , Mutación de Línea Germinal , Humanos , Pérdida de Heterocigocidad , Masculino , Inestabilidad de Microsatélites , Homólogo 1 de la Proteína MutL , Mutación , Proteínas Nucleares/genética , Linaje , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/economía , Análisis de Secuencia de ADN/métodos , Adulto JovenRESUMEN
Proteases have long been associated with tumor progression, given their ability to degrade extracellular matrix components and facilitate invasion and metastasis. However, recent findings indicate that different proteases can also act as tumor-suppressor enzymes. We have recently reported that lung carcinoma cells expressing the ADAMTS-12 metalloprotease show a remarkable impairment of growth in immunodeficient mice as compared with parental cells. Here, we show that ADAMTS12 promoter is hypermethylated in cancer cell lines and tumor tissues. Interestingly, ADAMTS12 expression in the stromal cells surrounding epithelial malignant cells is higher than in the paired normal tissues. Moreover, the expression of this metalloprotease in colon fibroblasts co-cultured with colon cancer cell lines is higher than in those cultured alone. Furthermore, the expression of ADAMTS-12 by these fibroblasts is linked with an anti-proliferative effect on tumor cells. Based on these findings, we hypothesize that ADAMTS-12 is a novel anti-tumor protease that can reduce the proliferative properties of tumor cells. This function is lost by epigenetic silencing in tumor cells, but concurrently induced in stromal cells, probably as part of a response of the normal tissue aimed at controlling the progression of cancer.