High-throughput parallel amplicon sequencing of common driver mutations from FFPE lung cancer samples in molecular pathologic routine diagnostics for a regional health care provider network.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e12517-e12517
Author(s):  
Reinhard Buettner ◽  
Katharina Koenig ◽  
Martin Peifer ◽  
Katrin Stamm ◽  
Marc Christiaan Allardt Bos ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Multiplex Pcr ◽  
Sanger Sequencing ◽  
Mutation Screening ◽  
Genomic Medicine ◽  
Genetic Alterations ◽  
Driver Mutations ◽  
Minimal Amount ◽  
Parallel Sequencing ◽  
Regional Health Care

e12517 Background: Treatment paradigms for non–small-cell lung cancer have shifted from histology based towards incorporation of molecular subtypes involving particular genetic alterations such as mutations in EGFR or translocations of ALK. The list of targetable lesions is rapidly increasing including mutations in genes such as EGFR, HER2, KRAS, ALK, BRAF, PIK3CA, AKT1, ROS1, NRAS, FGFR1 and MAP2K1. Analysis of these potential targets is becoming a challenge in terms of work load, tissue availability as well as cost. Within the Network Genomic Medicine Lung Cancer (NGM), a regional molecular screening network of the Center for Integrated Oncology Köln Bonn, we aimed to improve the sequential analysis of a set of 9 target amplicons by Sanger sequencing using bench top ultra-deep parallel sequencing platforms. We aimed to reduce 1) the time requirement for comprehensive molecular diagnostics, 2) the minimal amount of formalin fixed paraffin embedded (FFPE) derived input DNA, 3) while at the same time increasing the number of target regions analysed. Methods: We established a multiplex PCR to amplify up to 640 lung cancer relevant target regions from at least 20ng of FFPE derived tumor DNA. The amplicon libraries were ligated to adapters encompassing medical identifier sequences that allowed multiplexing of up to 48 patients. The resulting libraries were sequenced on a benchtop Illumina platform (MiSeq). Mutations identified by parallel sequencing were confirmed by Sanger sequencing. Results: 330 patients were analyzed by traditional Sanger sequencing of 9 amplicons and the newly established parallel sequencing protocol. The time needed to complete the mutation screening was significantly reduced to 7 working days from previously 21 days. A total of at least 300ng of DNA was needed to complete the analysis of 9 amplicons by Sanger sequencing compared to 20 to 100ng of DNA needed for up to 640 amplicons analyzed by parallel sequencing. Conclusions: Newly multiplex PCR based parallel sequencing allows rapid comprehensive mutation testing in routine molecular pathological diagnostics even on small transbronchial biopsies.

scholarly journals Paired analysis of tumor mutation burden for lung adenocarcinoma and associated idiopathic pulmonary fibrosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasuto Yoneshima ◽  
Eiji Iwama ◽  
Shingo Matsumoto ◽  
Taichi Matsubara ◽  
Testuzo Tagawa ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Lung Adenocarcinoma ◽  
Genetic Alterations ◽  
Driver Mutations ◽  
Somatic Variant ◽  
Tumor Mutation Burden ◽  
Mutation Burden ◽  
And Oxidative Stress

AbstractGenetic alterations underlying the development of lung cancer in individuals with idiopathic pulmonary fibrosis (IPF) have remained unclear. To explore whether genetic alterations in IPF tissue contribute to the development of IPF-associated lung cancer, we here evaluated tumor mutation burden (TMB) and somatic variants in 14 paired IPF and tumor samples from patients with IPF-associated lung adenocarcinoma. We also determined TMB for 22 samples of lung adenocarcinoma from patients without IPF. TMB for IPF-associated lung adenocarcinoma was significantly higher than that for matched IPF tissue (median of 2.94 vs. 1.26 mutations/Mb, P = 0.002). Three and 102 somatic variants were detected in IPF and matched lung adenocarcinoma samples, respectively, with only one pair of specimens sharing one somatic variant. TMB for IPF-associated lung adenocarcinoma was similar to that for lung adenocarcinoma samples with driver mutations (median of 2.94 vs. 2.51 mutations/Mb) and lower than that for lung adenocarcinoma samples without known driver mutations (median of 2.94 vs. 5.03 mutations/Mb, P = 0.130) from patients without IPF. Our findings suggest that not only the accumulation of somatic mutations but other factors such as inflammation and oxidative stress might contribute to the development and progression of lung cancer in patients with IPF.

scholarly journals Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing

2009 ◽  
Vol 3 (1-4) ◽  
pp. 41-49 ◽  
Author(s):  
Hao Hu ◽  
Klaus Wrogemann ◽  
Vera Kalscheuer ◽  
Andreas Tzschach ◽  
Hugues Richard ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Multiplex Pcr ◽  
Mutation Screening ◽  
Massive Parallel Sequencing ◽  
Parallel Sequencing

Expanded molecular routine testing for targetable mutations in non-small cell lung cancer to reveal frequent co-occuring mutations.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e20596-e20596
Author(s):  
Anna Kostenko ◽  
Jana Fassunke ◽  
Susanne Steinhauser ◽  
Matthias Scheffler ◽  
Sabine Merkelbach-Bruse ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Overall Survival ◽  
Molecular Diagnostics ◽  
Clinical Data ◽  
Single Gene ◽  
Genomic Medicine ◽  
Genetic Alterations ◽  
Clinical Diagnostics ◽  
Central Platform ◽  
The Impact

e20596 Background: Using next-gen sequencing of predefined gene panels in routine clinical diagnostics of lung cancer allows, in contrast to single-gene assays, assessment of co-occuring mutations, which might underly heterogeneity of response to targeted drugs and survival. The Network Genomic Medicine (NGM) performs high sensitive next generation sequencing (NGS) based routine molecular diagnostics on a central platform for about 5000 inoperable lung cancer patients (pts) annually in Germany. Methods: NGS panel used in NGM consists of 17 genes to cover potentially targetable aberrations. Mutation analyses were run on an Illumina (MySeq) platform, while FISH analyses were performed separately. In 2016, we have started the evaluation of all NGM pts with available clinical data who had received NGS-based molecular diagnostics. In particular, we have focused on non-squamous (non-sq) and squamous (sq) NSCLC pts with co-occurring mutations: their frequency, significance and impact on overall survival. Results: From 2014 molecular genotyping was performed for 7,893 NGM pts (n = 7,246 NSCLC (5,667 non-sq and 1,487 sq pts) and n = 489 SCLC) with eligible clinical data. Genetic alterations in transformation-associated pathways were found in 79 % of all NSCLC pts. Furthermore, co-occurring mutations were detected in 39 % of these pts: 40 % in non-sq and 37 % in sq NSCLC. 11 % of pts had more than 2 co-occurring mutations. 1 % of all pts had 5 co-occurring mutations. The most frequent paired mutations were KRAS, EGFR and MET each with TP53 in non-sq and FRGF1 and TP53 in sq NSCLC. The incidences and significance of 3, 4 and 5 co-mutations as well as the impact of these co-occurring mutations on overall survival will be presented. Conclusions: Frequent occurrence of co-occuring mutations in transformation – associated pathways underlines the genetic heterogeneity also of lung cancer with classical driver mutation and the impact of co-occurring mutations on survival. This work confirms the use of molecular multiplex testing in routine molecular diagnostics of NSCLC. Assessment of co-occuring mutations will help to further specify genetically defined subgroups of lung cancer with therapeutic relevance.

scholarly journals Genomic Characterization of Non–Small-Cell Lung Cancer in African Americans by Targeted Massively Parallel Sequencing

2015 ◽  
Vol 33 (17) ◽  
pp. 1966-1973 ◽  
Author(s):  
Luiz H. Araujo ◽  
Cynthia Timmers ◽  
Erica Hlavin Bell ◽  
Konstantin Shilo ◽  
Philip E. Lammers ◽  
...  
Keyword(s):  
Lung Cancer ◽  
African Americans ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Massively Parallel Sequencing ◽  
Small Cell ◽  
Massively Parallel ◽  
Driver Mutations ◽  
Small Cell Lung ◽  
Parallel Sequencing

Purpose Technologic advances have enabled the comprehensive analysis of genetic perturbations in non–small-cell lung cancer (NSCLC); however, African Americans have often been underrepresented in these studies. This ethnic group has higher lung cancer incidence and mortality rates, and some studies have suggested a lower incidence of epidermal growth factor receptor mutations. Herein, we report the most in-depth molecular profile of NSCLC in African Americans to date. Methods A custom panel was designed to cover the coding regions of 81 NSCLC-related genes and 40 ancestry-informative markers. Clinical samples were sequenced on a massively parallel sequencing instrument, and anaplastic lymphoma kinase translocation was evaluated by fluorescent in situ hybridization. Results The study cohort included 99 patients (61% males, 94% smokers) comprising 31 squamous and 68 nonsquamous cell carcinomas. We detected 227 nonsilent variants in the coding sequence, including 24 samples with nonoverlapping, classic driver alterations. The frequency of driver mutations was not significantly different from that of whites, and no association was found between genetic ancestry and the presence of somatic mutations. Copy number alteration analysis disclosed distinguishable amplifications in the 3q chromosome arm in squamous cell carcinomas and pointed toward a handful of targetable alterations. We also found frequent SMARCA4 mutations and protein loss, mostly in driver-negative tumors. Conclusion Our data suggest that African American ancestry may not be significantly different from European/white background for the presence of somatic driver mutations in NSCLC. Furthermore, we demonstrated that using a comprehensive genotyping approach could identify numerous targetable alterations, with potential impact on therapeutic decisions.

scholarly journals Assessment of the evolutionary consequence of putative driver mutations in colorectal cancer with spatial multiomic data

2021 ◽  
Author(s):  
Andrea Sottoriva ◽  
Trevor A Graham ◽  
Timon Heide ◽  
Jacob Househam ◽  
George D Cresswell ◽  
...  
Keyword(s):  
Genomic Medicine ◽  
Genetic Alterations ◽  
Neutral Evolution ◽  
Driver Mutations ◽  
Sufficient Data ◽  
Driver Genes ◽  
Phenotypic Properties ◽  
Evolutionary Consequence ◽  
Selective Evolution

Cancer genomic medicine relies on targeting driver genes. However, current catalogues of cancer drivers are mostly based on indirect measurements of mutation frequencies, positions or types, rather than their effect on clonal expansions in vivo. Moreover, nongenetic drivers are largely unknown, as are the epigenetic and transcriptomic effects of genetic drivers. Here we perform spatial computational inference on multiomic data with matched whole-genome sequencing, ATAC-seq and RNA-seq. Using 436 samples, we directly quantify the contribution, or lack thereof, of putative driver genes to subclonal expansions in vivo in 30 colorectal carcinomas (4-33 samples per patient, median=15). Although subclonal neutral evolution was widespread (13/26 cases with sufficient data), there were cases with clear evidence of subclonal selection (6/26) in which we measured epigenetic and transcriptomic differences between subclones in vivo. In 7/26 cases we could not distinguish between neutral or selective evolution with the available data. We identified expanding subclones that were not driven by known genetic alterations, and propose candidate epigenetic drivers. We identified the distinguishing patterns of genomic heterogeneity produced in fast, exponentially growing tumours (7/26) versus neoplasms growing only at the periphery (19/26), as well as identifying clonally intermixed (16/28 cases with sufficient data) versus segregated malignancies (10/28). Our model-based approach measures genetic and non-genetic subclonal selection, or lack thereof, in space and time and allows in vivo comparisons of the emergent phenotypic properties of subclones within human tumours.

Characteristics of advanced non-small cell lung cancer (aNSCLC) patients (pts) receiving molecular diagnostic (MD) testing in U.S. routine clinical practice.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e20627-e20627 ◽  
Author(s):  
Lisa I Wang ◽  
Ashwini Shewade ◽  
Peter Lambert ◽  
Brandon Arnieri ◽  
William Capra ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Real World ◽  
Genetic Alterations ◽  
Driver Mutations ◽  
Molecular Diagnostic ◽  
Large Network ◽  
The Us ◽  
Molecular Interpretation ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

e20627 Background: Hybrid capture (HC)-based next generation sequencing (NGS) combined with extensive molecular interpretation, such as Foundation Medicine (FMI) testing, is increasingly important for a comprehensive molecular diagnosis in the routine clinical management of lung cancer (LC) pts. FMI and other NGS platforms (including HC alone) support optimal treatment (trt) decisions by assessing multiple genetic alterations that drive LC progression. Our aim was to characterize aNSCLC pts receiving FMI or other types of MD tests using a real world oncology electronic health record (EHR) database. Methods: Flatiron aggregates pt-level EHR from a large network of cancer clinics in the US. Inclusion criteria were aNSCLC diagnosis and ≥2 clinic visits within the Flatiron network on or after January 1, 2011. Presence of MD testing was based on 5 LC biomarkers ( EGFR, ALK, KRAS, ROS1, PD-L1). Demographic and tumor characteristics at the time of aNSCLC diagnosis and trts received were summarized across 3 mutually exclusive testing groups (grps): FMI, other NGS and non-NGS. Results: As of Sept 30, 2016, the aNSCLC cohort included 30,489 pts across 4 groups: FMI (1,019 pts); other NGS (1,327 pts); non-NGS (15,205); no tests (12,938). As expected, the number of pts with FMI or NGS testing has increased in recent years. Pts in the FMI grp tended to be younger (66 vs. 68-69 years), non-smokers (25% vs. 17-19%) and have squamous cell histology (13% vs. 8-10%) compared to other MD test grps. 30% of FMI pts received testing prior to initiating first trt, compared with 38% of other NGS and 53% of non-NGS pts. For 565 pts (of 1,019) with available data on first trt after FMI testing, 24% (136 pts) initiated a NCCN recommended targeted trt for LC. In the FMI grp, 528 pts (52%) received at least one other MD test: EGFR (89%), ALK (83%), ROS1 (39%), KRAS (31%), PD-L1 (18%) and test types were 67% FISH, 55% PCR, 17% other NGS and 15% IHC. Conclusions: Differences in age, smoking and histology exist between pts receiving FMI vs. other MD tests. This analysis of a large real world pt cohort tested with FMI further supports the value of broad genomic profiling to identify rare driver mutations and ensure optimal trt.

scholarly journals Erratum to: Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing

2009 ◽  
Vol 3 (1-4) ◽  
pp. 83-83 ◽  
Author(s):  
Hao Hu ◽  
Klaus Wrogemann ◽  
Vera Kalscheuer ◽  
Andreas Tzschach ◽  
Hugues Richard ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Multiplex Pcr ◽  
Mutation Screening ◽  
Massive Parallel Sequencing ◽  
Parallel Sequencing

scholarly journals Plasma circulating tumor DNA-based genetic profiling of lung cancer patients in Vietnam using ultra-deep massive parallel sequencing with unique identifier tagging.

2019 ◽  
Vol 5 (suppl) ◽  
pp. 58-58
Author(s):  
Huy Phuoc Do ◽  
Thao Thanh Tran Nguyen ◽  
Uyen Vu Tran ◽  
Thanh-Truong Tran ◽  
Anh-Thu Huynh Dang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Liquid Biopsy ◽  
Circulating Tumor Dna ◽  
Massive Parallel Sequencing ◽  
Driver Mutations ◽  
Unique Identifier ◽  
Parallel Sequencing ◽  
Cancer Driver ◽  
Lung Cancer Patients ◽  
Tumor Dna

58 Background: Lung cancer is by far the leading cause of cancer death worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. Genotype-directed therapy becomes a promising method for cancer treatment beside surgery and chemo-radiotherapy. Liquid biopsy using massive parallel sequencing has emerged as a non-invasive alternative procedure in profiling cancer driver mutations. In this study, we report the spectrum of clinically actionable mutations in plasma circulating tumor DNA of 299 non-small cell lung cancer patients using ultra-deep massive parallel sequencing with unique identifier tagging. Methods: Plasma circulating tumor DNA was extracted, ligated with unique identifier (Swift Bioscience), enriched of the target coding regions of EGFR, KRAS, NRAS, BRAF and the breakpoints of ALK, ROS1 (IDT) and sequenced using NextSeq 550 (Illumina) at mean coverage depth of 20,000X. Results: Out of 299 patients tested, 128 (42,8%) carried driver mutations. Genetic alterations were identified in EGFR (79 samples, 26,4%), KRAS (30 samples, 10%), ALK (7 samples, 2,34%), ROS1 (6 samples, 2%), BRAF (3 samples, 1%). There was no sample with NRAS mutation. In 79 EGFR-cases, there were 23 carry two pathogenic variants. 28 mutation types of EGFR were found including 19 indels and 9 missense variants L858R and T790M were the major ones. One case was found with concomitant EGFR and BRAF. Our study showed the spectrum and frequency of the cancer driver mutations detected in liquid biopsy was correlated to those detected in tissue biopsy samples. Conclusions: For the first time the spectrum of mutation types in liquid biopsy of Vietnamese NSCLC patients were investigated and showed the correlation with those detected in tissue biopsy samples.

Targeted DNA sequencing analysis to reveal genetic diversity and androgen-receptor alteration in advanced EGFR mutant lung adenocarcinoma.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9526-9526
Author(s):  
Wei Wu ◽  
Ross A. Okimoto ◽  
Collin Michael Blakely ◽  
James Fraser ◽  
Trever G. Bivona
Keyword(s):  
Lung Cancer ◽  
Androgen Receptor ◽  
Functional Annotation ◽  
Early Stage ◽  
Genetic Alterations ◽  
Egfr Mutations ◽  
Driver Mutations ◽  
Lung Cancers ◽  
Egfr Mutant ◽  
Lung Adenocarcinomas

9526 Background: Lung cancer remains the leading cause of death from cancer around the world. Several oncogenic drivers have been identified from large cancer genome projects focused mainly on profiling early-stage lung cancers. Targeted therapies have been developed for specific activated driver gene mutations and are used in advanced-stage patients. For instance, advanced EGFR mutant lung cancer is primarily treated with EGFR tyrosine receptor inhibitors (TKIs). However, resistance remains an obstacle to durable anti-tumor control. We hypothesize that concurrent genetic alterations co-exiting with EGFR driver mutations contribute to the failure of EGFR TKI therapy. Methods: To understand the complexity and diversity of genetic alterations present in EGFR mutant advanced lung cancers, we utilized 660 EGFR mutant advanced lung adenocarcinomas samples with targeted DNA sequencing from Foundation Medicine, 394 cases from MSK-IMPACT dataset, along with TCGA lung cancer data. We performed systematic co-mutation analysis, molecular simulation, functional annotation and pathway enrichment analysis. Results: We updated mutational profiling on EGFR gene with hotspots at exon 18, 19, 20 and 21. Among them, EGFR L858R, exon19 deletion, T790M and G719A are top ranking alleles among EGFR mutations. Interestingly, a subset (n = 26 cases) of EGFR T790M mutations parallel with other EGFR mutations, which could affect the TKI binding pocket as inferred by molecular simulations. Furthermore, in advanced lung cancer EGFR mutations co-occurred with known oncogenic mutations in KRAS, MET, NF-1, MAP2K1, ERBB2, and ALK/ROS-1/RET fusions. Functional annotation suggests that concurrent mutated genes and copy number alterations in advanced EGFR mutant lung cancer were enriched in signatures of epigenetic modifiers, genome instability, WNT signaling, and RNA splicing. Compared to early stage TCGA-lung adenocarcinomas, Cell cycle, DNA repair, WNT signaling and androgen receptor-mediated signaling pathways are predominantly altered in advanced EGFR mutant lung cancers. Conclusions: We characterized the genetic landscape of advanced EGFR-mutant lung adenocarcinomas and further dissected concurrent mutated genes with EGFR driver mutations. Our findings provide a rational for polytherapy roadmap for testing in advanced EGFR-mutant lung cancer.

Sign in / Sign up

Export Citation Format

Share Document