RESUMO
Targeted next-generation sequencing is becoming increasingly common as a clinical diagnostic and prognostic test for patient- and tumor-specific genetic profiles as well as to optimally select targeted therapies. Here, we describe a custom-developed, next-generation sequencing test for detecting single-nucleotide variants (SNVs) and short insertions and deletions (indels) in 93 genes related to gastrointestinal cancer from routine formalin-fixed, paraffin-embedded clinical specimens. We implemented a validation strategy, based on the College of American Pathologists requirements, using reference DNA mixtures from cell lines with known genetic variants, which model a broad range of allele frequencies. Test sensitivity achieved >99% for both SNVs and indels, with allele frequencies >10%, with high specificity (97.4% for SNVs and 93.6% for indels). We further confirmed test accuracies using primary formalin-fixed, paraffin-embedded colorectal cancer specimens characterized by alternative and conventional clinical diagnostic technologies. Robust performance was observed on the formalin-fixed, paraffin-embedded specimens: sensitivity was 97.2% and specificity was 99.2%. We also observed high intrarun and inter-run reproducibility, as well as a low cross-contamination rate. Overall assessment using cell line samples and formalin-fixed, paraffin-embedded samples showed that our custom next-generation sequencing assay has consistent detection sensitivity down to 10% variant frequency.
Assuntos
Biomarcadores Tumorais , Neoplasias Gastrointestinais/diagnóstico , Neoplasias Gastrointestinais/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normas , Humanos , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Gastric cancer is a major cause of global cancer mortality. We surveyed the spectrum of somatic alterations in gastric cancer by sequencing the exomes of 15 gastric adenocarcinomas and their matched normal DNAs. Frequently mutated genes in the adenocarcinomas included TP53 (11/15 tumors), PIK3CA (3/15) and ARID1A (3/15). Cell adhesion was the most enriched biological pathway among the frequently mutated genes. A prevalence screening confirmed mutations in FAT4, a cadherin family gene, in 5% of gastric cancers (6/110) and FAT4 genomic deletions in 4% (3/83) of gastric tumors. Frequent mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) also occurred in 47% of the gastric cancers. We detected ARID1A mutations in 8% of tumors (9/110), which were associated with concurrent PIK3CA mutations and microsatellite instability. In functional assays, we observed both FAT4 and ARID1A to exert tumor-suppressor activity. Somatic inactivation of FAT4 and ARID1A may thus be key tumorigenic events in a subset of gastric cancers.
Assuntos
Adenocarcinoma/genética , Adesão Celular/genética , Montagem e Desmontagem da Cromatina/genética , Exoma/genética , Genes Supressores de Tumor , Mutação/genética , Neoplasias Gástricas/genética , Estudos de Casos e Controles , DNA/genética , Mucosa Gástrica/metabolismo , Humanos , Instabilidade de Microssatélites , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNARESUMO
Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Neoplasias Pulmonares/genética , Proteínas de Membrana/genética , Polimorfismo Genético/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Deleção de Sequência/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Anexinas/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína 11 Semelhante a Bcl-2 , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Estudos de Coortes , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Ensaio de Imunoadsorção Enzimática/métodos , Receptores ErbB/genética , Éxons/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Frequência do Gene , Genótipo , Humanos , Cooperação Internacional , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Estatísticas não Paramétricas , TransfecçãoRESUMO
OBJECTIVE: Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. DESIGN: Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. RESULTS: 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets (FGFR2, ERBB2) and also novel genes in gastric cancer (KLF5, GATA6). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2-amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2-amplified gastric cancers. CONCLUSION: The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.