The feasibility of using small biopsy samples from lung cancer for targeted next-generation sequencing.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e20584-e20584
Author(s):  
Hee Kyung Kim ◽  
Bo Mi Ku ◽  
Hansang Lee ◽  
Mi Hwa Heo ◽  
Joo Hyun Hong ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Endobronchial Ultrasound ◽  
Next Generation ◽  
Tissue Samples ◽  
Targeted Next Generation Sequencing ◽  
Biopsy Specimens ◽  
Small Tissue ◽  
Targeted Ngs ◽  
Generation Sequencing

e20584 Background: As the genomic based personalized medicine has been advanced, biomarker analysis using targeted next-generation sequencing (NGS) becomes crucial diagnostic approach for treatment of lung cancer. Most patients of lung cancer are diagnosed on small biopsy specimen, but the feasibility of the small tissue samples for targeted NGS has not been evaluated. We aimed to investigate the feasibility of using small tissue samples of lung cancer for targeted NGS. Methods: The medical records of the patients who underwent biopsy and targeted NGS were reviewed. Two types of panel for targeted NGS were used which were AmpliSeq Cancer Hotspot panel v2 and Oncomine Focus Assay. Results: A total of 603 biopsy specimens of lung cancer were analyzed from April 2014 to October 2016. The sites of biopsy were lung or bronchus (51.2%), lymph node (26.2%), Pleura (2.3%), liver (2.5%), Brain (1.7%) and the others (adrenal gland, bone, muscle, spleen, stomach, thymus). Percutaneous transthoracic core needle biopsy (PTCNB) was most frequently used method for acquisition of tissue (24.9%). The other methods of biopsy were bronchoscopy (14.4%), endobronchial ultrasound (EBUS, 23.8%), Video-assisted transthoracic surgery (VATS, 9.9%) and so on. The types of tissue samples were formalin-fixed, paraffin-embedded (FFPE, 80.6%) and fresh tissue (19.4%). All of these samples, 398 cases (66.0%) were successfully analyzed using targeted, NGS. In 34% of analysis failure cases, 23.5% were not adequate for DNA or RNA extraction and 10.5% had amount not to be enough. On multivariate analysis, PTCNB was more likely to fail than other methods of biopsy such as bronchoscopy, EBUS (HR 2.31, 95% CI 1.4-3.8, p = 0.001). Conclusions: The biopsy of lung cancer for targeted NGS was taken from a variety of sites via different methods, and the type of tissue samples was mostly FFPE. Considering the yield of our study, small biopsy specimens could be used to investigate genomic characteristics of lung cancer via targeted NGS.

scholarly journals Targeted Next-Generation Sequencing of Cancer-Related Genes in a Norwegian Patient Cohort With Head and Neck Squamous Cell Carcinoma Reveals Novel Actionable Mutations and Correlations With Pathological Parameters

2021 ◽  
Vol 11 ◽  
Author(s):  
Harsh N. Dongre ◽  
Hilde Haave ◽  
Siren Fromreide ◽  
Fredrik A. Erland ◽  
Svein Erik Emblem Moe ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Head And Neck ◽  
Squamous Cell ◽  
Ffpe Tissue ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Mutational Burden ◽  
Custom Made ◽  
Targeted Ngs ◽  
Generation Sequencing

BackgroundTargeted next-generation sequencing (NGS) is increasingly applied in clinical oncology to advance personalized treatment. Despite success in many other tumour types, use of targeted NGS panels for assisting diagnosis and treatment of head and neck squamous cell carcinomas (HNSCC) is still limited.AimThe focus of this study was to establish a robust NGS panel targeting most frequent cancer mutations in long-term preserved formalin-fixed paraffin-embedded (FFPE) tissue samples of HNSCC from routine diagnostics.Materials and MethodsTumour DNA obtained from archival FFPE tissue blocks of HNSCC patients treated at Haukeland University Hospital between 2003-2016 (n=111) was subjected to mutational analysis using a custom made AmpliSeq Library PLUS panel targeting 31 genes (Illumina). Associations between mutational burden and clinical and pathological parameters were investigated. Mutation and corresponding clinicopathological data from HNSCC were extracted for selected genes from the Cancer Genome Atlas (TCGA) and used for Chi-square and Kaplan-Meier analysis.ResultsThe threshold for sufficient number of reads was attained in 104 (93.7%) cases. Although the specific number of PCR amplified reads detected decreased, the number of NGS-annotated mutations did not significantly change with increased tissue preservation time. In HPV-negative carcinomas, mutations were detected mainly in TP53 (73.3%), FAT1 (26.7%) and FLG (16.7%) whereas in HPV-positive, the common mutations were in FLG (24.3%) FAT1 (17%) and FGFR3 (14.6%) genes. Other less common pathogenic mutations, including well reported SNPs were reproducibly identified. Presence of at least one cancer-specific mutations was found to be positively associated with an extensive desmoplastic stroma (p=0.019), and an aggressive type of invasive front (p=0.035), and negatively associated with the degree of differentiation (p=0.041). Analysis of TCGA data corroborated the association between cancer-specific mutations and tumour differentiation and survival analysis showed that tumours with at least one mutation had shorter disease-free and overall survival (p=0.005).ConclusionsA custom made targeted NGS panel could reliably detect several specific mutations in archival samples of HNSCCs preserved up to 17 years. Using this method novel associations between mutational burden and clinical and pathological parameters were detected and actionable mutations in HPV-positive HNSCC were discovered.

scholarly journals Characterizing the pharmacogenome using molecular inversion probes for targeted next-generation sequencing

2019 ◽  
Vol 20 (14) ◽  
pp. 1005-1020 ◽  
Author(s):  
Oscar Suzuki ◽  
Olivia M Dong ◽  
Rachel M Howard ◽  
Tim Wiltshire
Keyword(s):  
Next Generation Sequencing ◽  
Control Cell ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Technical Performance ◽  
Targeted Ngs ◽  
Molecular Inversion Probes ◽  
Next Generation Sequencing Ngs ◽  
Molecular Inversion Probe ◽  
Generation Sequencing

Aim: This study assesses the technical performance and cost of a targeted next-generation sequencing (NGS) multigene pharmacogenetic (PGx) test. Materials & methods: A genetic test was developed for 21 PGx genes using molecular inversion probes to generate library fragments for NGS. Performance of this test was assessed using 53 unique reference control cell lines from the Genetic Testing Reference Materials Coordination Program (GeT-RM). Results: 93.7% of variants were successfully called and the repeatability rate was 99.9%. Reference calls were available for 78.4% of diplotype calls resulting from PGx testing, and concordance for the test was 85.7%. Cost per sample was $32–$56. Conclusion: A targeted NGS assay using molecular inversion probe technology is able to characterize the pharmacogenome efficiently.

Identification of a Disease-Causing Mutation in a Chinese Patient with Retinitis Pigmentosa by Targeted Next-Generation Sequencing

2017 ◽  
Vol 27 (6) ◽  
pp. 791-796 ◽  
Author(s):  
Jianping Xiao ◽  
Xueqin Guo ◽  
Yong Wang ◽  
Mingkun Shao ◽  
Xiaoming Wei ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Retinitis Pigmentosa ◽  
Sanger Sequencing ◽  
Bioinformatics Analysis ◽  
Family Members ◽  
Chinese Patient ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Generation Sequencing

Purpose To identify disease-causing mutations in a Chinese patient with retinitis pigmentosa (RP). Methods A detailed clinical examination was performed on the proband. Targeted next-generation sequencing (NGS) combined with bioinformatics analysis was performed on the proband to detect candidate disease-causing mutations. Sanger sequencing was performed on all subjects to confirm the candidate mutations and assess cosegregation within the family. Results Clinical examinations of the proband showed typical characteristics of RP. Three candidate heterozygous mutations in 3 genes associated with RP were detected in the proband by targeted NGS. The 3 mutations were confirmed by Sanger sequencing and the deletion (c.357_358delAA) in PRPF31 was shown to cosegregate with RP phenotype in 7 affected family members, but not in 3 unaffected family members. Conclusions The deletion (c.357_358delAA) in PRPF31 was the disease-causing mutation for the proband and his affected family members with RP. To our knowledge, this is the second report of the deletion and the first report of the other 2 mutations in the Chinese population. Targeted NGS combined with bioinformatics analysis proved to be an effective molecular diagnostic tool for RP.

Next-generation sequencing for genetic analysis of endobronchial ultrasound transbronchial needle aspiration samples in advanced lung cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15586-e15586
Author(s):  
Xianjun Min ◽  
Mingwei Li ◽  
Qiang Liu ◽  
Yingshun Yang ◽  
Guotian Pei ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Squamous Cell ◽  
Needle Aspiration ◽  
Endobronchial Ultrasound ◽  
Advanced Lung Cancer ◽  
Genetic Mutations ◽  
Transbronchial Needle Aspiration ◽  
Tissue Samples ◽  
Generation Sequencing

e15586 Background: Next-generation sequencing (NGS) typically requires greater quantities of DNA than traditional molecular testing. Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) is a minimally invasive technique with high sensitivity in the mediastinal staging of lung cancer. This study aimed to evaluate the adequacy of EBUS-TBNA in providing adequate size specimens for genetic mutations and immunotherapy biomarkers analysis in patients with lung cancer. Methods: Tissue samples from patients with advanced lung cancer were collected by EBUS-TBNA and were formalin-fixed paraffin-embedded. NGS assay was carried on with acornmed panel including 808 genes. PD-L1 expression through immunohistochemistry was assessed. Results: A total of 98 patients was enrolled, of which 74 (76%) were adenocarcinoma and 20 (20%) were squamous cell carcinomas. Among the patients, 108 samples (including multipoint puncture of different location and retest of the same patient) were obtained. NGS assay was completed successfully on 106 of the 108 samples (98.14%), and 97.96% of patients had successful testing, identifying an average of 11.3 mutations. With a multi-gene panel comprising up to 808 tumor related genes, actionable variations were found in 93 (86%) samples. Of these, the positive rates of actionable alterations in lung adenocarcinoma samples and lung squamous cell carcinoma samples were 90% and 79%, respectively. The tumor mutation burden (TMB), Microsatellite instability (MSI) and PD-L1 expression were found High in 20 (41%), 1 (2%), and 37 (72%) patients respectively. Conclusions: NGS assay can be successfully conducted with tissue samples obtained from EBUS-TBNA. NGS assay provides more comprehensive information on genetic mutations in tumors, which greatly assists therapeutic decision making for advanced lung cancer.

scholarly journals Molecular diagnosis of inherited peripheral neuropathies by targeted next-generation sequencing: molecular spectrum delineation

BMJ Open ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. e021632 ◽  
Author(s):  
Juliette Bacquet ◽  
Tanya Stojkovic ◽  
Amandine Boyer ◽  
Nathalie Martini ◽  
Frédérique Audic ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Molecular Diagnosis ◽  
Sanger Sequencing ◽  
Diagnostic Yield ◽  
Motor Neuropathy ◽  
Peripheral Neuropathies ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Generation Sequencing

PurposeInherited peripheral neuropathies (IPN) represent a large heterogenous group of hereditary diseases with more than 100 causative genes reported to date. In this context, targeted next-generation sequencing (NGS) offers the opportunity to screen all these genes with high efficiency in order to unravel the genetic basis of the disease. Here, we compare the diagnostic yield of targeted NGS with our previous gene by gene Sanger sequencing strategy. We also describe several novel likely pathogenic variants.Design and participantsWe have completed the targeted NGS of 81 IPN genes in a cohort of 123 unrelated patients affected with diverse forms of IPNs, mostly Charcot-Marie-Tooth disease (CMT): 23% CMT1, 52% CMT2, 9% distal hereditary motor neuropathy, 7% hereditary sensory and autonomic neuropathy and 6.5% intermediate CMT.ResultsWe have solved the molecular diagnosis in 49 of 123 patients (~40%). Among the identified variants, 26 variants were already reported in the literature. In our cohort, the most frequently mutated genes are respectively:MFN2,SH3TC2,GDAP1,NEFL,GAN,KIF5AandAARS. Panel-based NGS was more efficient in familial cases than in sporadic cases (diagnostic yield 49%vs19%, respectively). NGS-based search for copy number variations, allowed the identification of three duplications in three patients and raised the diagnostic yield to 41%. This yield is two times higher than the one obtained previously by gene Sanger sequencing screening. The impact of panel-based NGS screening is particularly important for demyelinating CMT (CMT1) subtypes, for which the success rate reached 87% (36% only for axonal CMT2).ConclusionNGS allowed to identify causal mutations in a shorter and cost-effective time. Actually, targeted NGS is a well-suited strategy for efficient molecular diagnosis of IPNs. However, NGS leads to the identification of numerous variants of unknown significance, which interpretation requires interdisciplinary collaborations between molecular geneticists, clinicians and (neuro)pathologists.

scholarly journals Targeted Next-Generation Sequencing Analysis for Recurrence in Early-Stage Lung Adenocarcinoma

2020 ◽  
Author(s):  
In Ae Kim ◽  
Jae Young Hur ◽  
Hee Joung Kim ◽  
Jung Hoon Park ◽  
Jae Joon Hwang ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Lung Adenocarcinoma ◽  
Egfr Mutation ◽  
Early Stage ◽  
Genetic Alterations ◽  
Smoking History ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Targeted Ngs ◽  
Generation Sequencing

Abstract Background Despite surgical resection, early lung adenocarcinoma has a recurrence rate of 20–50%. No clear predictive markers for recurrence of early lung adenocarcinoma are available. Targeted next-generation sequencing (NGS) is rarely used to identify recurrence-related genes. We aimed to identify genetic alterations that can predict recurrence, by comparing the molecular profiles of patient groups with and without recurrence. Methods Tissues from 230 patients with resected stage I–II lung adenocarcinoma (median follow-up: 49 months) were analyzed via targeted NGS for 207 cancer-related genes. The recurrence-free survival according to the number and type of mutation was estimated using the Kaplan–Meier method. Independent predictive biomarkers related to recurrence were identified using the Cox proportional hazards model. Results Recurrence was observed in 64 patients (27.8%). In multivariate analysis adjusted for age, sex, smoking history, stage, surgical mode, and visceral pleural invasion, the CTNNB1 mutation and fusion genes (ALK, ROS1, RET) were negative prognostic factors for recurrence in early-stage lung adenocarcinoma (HR 4.47, p = 0.001; HR 2.73, p = 0.009). EGFR mutation was a favorable factor (HR 0.51, p = 0.016), but the CTNNB1/EGFR co-mutations were negative predictors (HR 19.2, p < 0.001). TP53 mutation was a negative predictor compared with EGFR mutation for recurrence (HR 5.24, p = 0.02). Conclusions: Targeted NGS can provide valuable information to predict recurrence and identify patients at high recurrence risk, facilitating selection of the treatment strategy among close monitoring and adjuvant-targeted therapy. Larger datasets are required to validate these findings.

The landscape of intergenic-breakpoint fusion in lung cancer.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15002-e15002
Author(s):  
Hua Liang ◽  
Yaqing Wu ◽  
Ningning Luo ◽  
Qin Zhang ◽  
Yingxue Qi ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Next Generation Sequencing ◽  
Drug Targets ◽  
Next Generation ◽  
Protein Assay ◽  
Targeted Ngs ◽  
Intergenic Regions ◽  
Functional Consequences ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

e15002 Background: Kinase fusions have been proved as driver factors and drug targets for lung cancer. DNA-based next-generation sequencing (NGS) has been widely used for gene fusion detection in patients with lung cancer and intergenic-breakpoint fusions can be detected, in which breakpoints localize to intergenic regions. Theoretically, intergenic-breakpoint fusions are unlikely to be functional, but recent studies have found that intergenic-breakpoint fusions are potentially activated. However, the proportion of intergene-breakpoint fusion occurrence in lung cancer is still unclear. Hereby, next generation sequencing was performed to describe lung intergenic-breakpoint fusions for further research. Methods: Tumor tissue, pleural effusion, or blood samples of 6798 patients with lung cancer from Simceredx were collected for targeted NGS panel sequencing from June 2020 to January 2021. Gene rearrangements of ALK, ROS1, RET, NTRK(1-3) were assessed. Results: We found gene fusions in 4.43% (301/6798) patients with lung cancer totally, of which 59.1% (178/301) ALK fusions, 18.9% (57/301) RET fusions, 15.9% (48/301) ROS1 fusions and 6.64% (20/301) NTRK(1-3) fusions. The intergenic-breakpoint fusions accounted for all fusions of ALK, RET and ROS1 genes were 24.2% (43/178), 22.8% (13 /57) and 18.8% (9/48), respectively. In patients with NTRK intergenic fusions (50.0%, 10/20), NTRK1, NTRK2 and NTRK3 was involved in 2, 1, 7, respectively. Co-occurrence of different fusions was rare. Conclusions: Our data firstly revealed the specific intergenic-breakpoint fusions ( ALK, RET, ROS1, NTRK1-3) in lung cancer. The functional consequences identifying by RNA or protein assay and clinical validation need to be carried out in the future.

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