Dataset Information

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

ABSTRACT: MET inhibitors have shown activity in non-small cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (MET?ex14). However, patient stratification is imperfect and thus response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with Next Generation Sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with MET?ex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally co-exist with other drivers. For MET?ex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

SUBMITTER: Aguado C

PROVIDER: S-EPMC7858100 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

Aguado Cristina C Teixido Cristina C Román Ruth R Reyes Roxana R Giménez-Capitán Ana A Marin Elba E Cabrera Carlos C Viñolas Nuria N Castillo Sergi S Muñoz Silvia S Arcocha Ainara A López-Vilaró Laura L Sullivan Ivana I Aldeguer Erika E Rodríguez Sonia S Moya Irene I Viteri Santiago S Cardona Andrés Felipe AF Palmero Ramon R Sainz Cristina C Mesa-Guzmán Miguel M Lozano Maria D MD Aguilar-Hernández Andrés A Martínez-Bueno Alejandro A González-Cao María M Gonzalvo Elena E Leenders William P J WPJ Rosell Rafael R Montuenga Luis M LM Prat Aleix A Molina-Vila Miguel A MA Reguart Noemi N

Molecular oncology 20201207 2

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction ...[more]

PMID: 33236532

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Project description:The genus Anaplasma (Rickettsiales: Anaplasmataceae), which includes the species Anaplasma capra, Anaplasma bovis, Anaplasma ovis, and Anaplasma phagocytophilum, is responsible for a wide variety of infections in both human and veterinary health worldwide. Multiple infections with these four Anaplasma pathogens have been reported in many cases. We introduce a novel multiplex PCR for the simultaneous detection of A. capra, A. bovis, A. ovis, and A. phagocytophilum, based on species-specific primers against the groEL (A. capra and A. bovis), msp4 (A. ovis), and 16S rRNA (A. phagocytophilum) genes. To verify the specificity of the PCR reactions, we evaluated four sets of primers to analyze samples containing different blood pathogens. The sensitivity of the multiplex PCR was evaluated by amplifying 10-fold dilutions of total genomic DNA extracted from sheep blood infected with A. capra, A. bovis, A. ovis, or A. phagocytophilum. The reproducibility of the assay was evaluated by testing 10-fold dilutions of total genomic DNA extracted from sheep blood infected with these pathogens from 100 to 10-3 ng/μL per reaction in triplicate on three different days. A total of 175 field blood DNA samples were used to evaluate the reproducibility of multiplex PCR compared with the simplex PCRs. PCR primers used in this study were confirmed to be 100% species-specific using blood pathogens previously identified by other methods. The lower limit of detection of the multiplex PCR with good repeatability enabled the detection of A. capra, A. bovis, A. ovis and A. phagocytophilum at concentrations of 3 × 10-5, 5 × 10-7, 2 × 10-5, and 7 × 10-7 ng/μL, respectively. There was no significant difference between conventional and multiplex PCR protocols used to detect the four Anaplasma species (P > 0.05). The results of the multiplex PCR revealed that the A. capra groEL gene, the A. bovis groEL gene, the A. ovis msp4 gene, and the A. phagocytophilum 16S rRNA gene were reliable target genes for species identification in clinical isolates, being specific for each of the four target Anaplasma species. Our study provides an effective, sensitive, specific, and accurate tool for the rapid differential clinical diagnosis and epidemiological surveillance of Anaplasma pathogens in sheep and goats.

Project description:PURPOSE: Non-small cell lung cancers (NSCLC) comprise multiple distinct biological groups with different prognoses. For example, patients with epithelial-like (EL) tumors have a better prognosis and exhibit greater sensitivity to inhibitors of the epidermal growth factor receptor (EGFR) pathway than patients with mesenchymal-like (ML) tumors. Here we test the hypothesis that EL NSCLCs can be distinguished from ML NSCLCs on the basis of global DNA methylation patterns. EXPERIMENTAL DESIGN: To determine whether phenotypic subsets of NSCLC can be defined based on their DNA methylation patterns, we combined microfluidics-based gene expression analysis and genome-wide methylation profiling. We derived robust classifiers for both gene expression and methylation in cell lines and tested these classifiers in surgically resected NSCLC tumors. We validate our approach using quantitative RT-PCR and methylation specific PCR in formalin-fixed biopsies from NSCLC patients who went on to fail front-line chemotherapy. RESULTS: We show that patterns of methylation divide NSCLCs into EL and ML subsets as defined by gene expression and that these signatures are similarly correlated in NSCLC cell lines and tumors. We identify multiple DMRs, including ERBB2 and ZEB2, whose methylation status is strongly associated with an epithelial phenotype in NSCLC cell lines, surgically resected tumors, and formalin-fixed biopsies from NSCLC patients who went on to fail front-line chemotherapy. CONCLUSIONS: Our data demonstrate that patterns of DNA methylation can divide NSCLCs into two phenotypically distinct subtypes of tumors and provide proof of principle that differences in DNA methylation can be used for predictive biomarker discovery and development. To determine whether phenotypic subsets of NSCLC can be defined based on their DNA methylation patterns, we combined microfluidics-based gene expression analysis and genome-wide methylation profiling. We derived robust classifiers for both gene expression and methylation in cell lines and tested these classifiers in surgically resected NSCLC tumors. We validate our approach using quantitative RT-PCR and methylation specific PCR in formalin-fixed biopsies from NSCLC patients who went on to fail front-line chemotherapy.

Dataset Information

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

Publications

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

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