Multigene Combined Detection by RT-qPCR Using Cytological Specimens

2021 ◽  
pp. 1-10
Author(s):  
Yang Ma ◽  
Jingxia Zhao ◽  
Yun Du ◽  
Rui Wang ◽  
Xiaokun Ji ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Smoking Status ◽  
Single Gene ◽  
Rna Extraction ◽  
Pcr Amplification ◽  
Driver Mutations ◽  
Amplification Refractory Mutation System ◽  
Driver Genes ◽  
Mutation Status ◽  
Pik3ca Mutations

<b><i>Objective:</i></b> The aim of the study was to investigate the mutation status of multiple driver genes by RT-qPCR and their significance in advanced lung adenocarcinoma using cytological specimens. <b><i>Materials and Methods:</i></b> 155 cytological specimens that had been diagnosed with lung adenocarcinoma in the Fourth Hospital of Hebei Medical University were selected from April to November 2019. The cytological specimens included serous cavity effusion and fine-needle aspiration biopsies. Among cytological specimens, 108 cases were processed by using the cell block method (CBM), and 47 cases were processed by the disposable membrane cell collector method (MCM) before DNA/RNA extraction. Ten drive genes of EGFR, ALK, ROS1, BRAF, KRAS, NRAS, HER2, RET, PIK3CA, and MET were combined detected at one step by the amplification refractory mutation system and ABI 7500 RT-qPCR. <b><i>Results:</i></b> The purity of RNA (<i>p</i> = 0.005) and DNA (<i>p</i> = 0.001) extracted by using the MCM was both significantly higher than that extracted by using the CBM. Forty-seven cases of fresh cell specimens processed by the MCM all succeeded in multigene detections, while of 108 specimens processed by the CBM, 6 cases failed in multigene detections. Among 149 specimens, single-gene mutation rates of EGFR, ALK, ROS1, RET, HER2, MET, KRAS, NRAS, BRAF, and PIK3CA mutations were 57.71%, 6.04%, 3.36%, 2.68%, 2.01%, 2.01%, 1.34%, 0.67%, 0% and 0% respectively, and 6 cases including 2 coexistence mutations. We found that mutation status was correlated with gender (<i>p</i> = 0.047), but not correlated with age (<i>p</i> = 0.141) and smoking status (<i>p</i> = 0.083). We found that the EGFR mutation status was correlated with gender (<i>p</i> = 0.003), age (<i>p</i> = 0.015) and smoking habits (<i>p</i> = 0.007), and ALK mutation status was correlated with age (<i>p</i> = 0.002). <b><i>Conclusion:</i></b> Compared with the CBM, the MCM can improve the efficiency of DNA/RNA extraction and PCR amplification by removing impurities and enriching tumor cells. And we speculate that the successful detection rate of fresh cytological specimens was higher than that of paraffin-embedded specimens. EGFR, ALK, and ROS1 mutations were the main driver mutations in patients with advanced lung adenocarcinoma. We speculate that EGFR and ALK are more prone to concomitant mutations, respectively. Targeted therapies for patients with coexisting mutations need further study.

scholarly journals Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing

2018 ◽  
Vol 64 (11) ◽  
pp. 1626-1635 ◽  
Author(s):  
Sonia Mansukhani ◽  
Louise J Barber ◽  
Dimitrios Kleftogiannis ◽  
Sing Yu Moorcraft ◽  
Michael Davidson ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Error Correction ◽  
Copy Number ◽  
Point Mutations ◽  
Driver Mutations ◽  
Single Nucleotide Variants ◽  
Driver Genes ◽  
Target Region ◽  
Pik3ca Mutations ◽  
Genome Wide

Abstract BACKGROUND Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies. METHODS We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes and a novel duplex DNA molecule identification tool for enhanced error correction. RESULTS Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied to &gt;95% of patients with metastatic colorectal cancer (mCRC). cfDNA-Seq of a 32-gene, 163.3-kbp target region detected 100% of single-nucleotide variants, with 0.15% variant frequency in spike-in experiments. Molecular barcode error correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed patients with mCRC, 80 out of 91 mutations previously detected by tumor tissue sequencing were called in the cfDNA. Call rates were similar for point mutations and indels. cfDNA-Seq identified typical mCRC driver mutations in patients in whom biopsy sequencing had failed or did not include key mCRC driver genes. Mutations only called in cfDNA but undetectable in matched biopsies included a subclonal resistance driver mutation to anti-EGFR antibodies in KRAS, parallel evolution of multiple PIK3CA mutations in 2 cases, and TP53 mutations originating from clonal hematopoiesis. Furthermore, cfDNA-Seq off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction in 20 of 28 cases. Copy number profiles were validated by low-coverage whole-genome sequencing. CONCLUSIONS This error-corrected, ultradeep cfDNA-Seq technology with a customizable target region and publicly available bioinformatics tools enables broad insights into cancer genomes and evolution. ClinicalTrials.gov Identifier NCT02112357

scholarly journals Liquid Biopsy to Identify Actionable Genomic Alterations

2018 ◽  
pp. 978-997 ◽  
Author(s):  
Sai-Hong Ignatius Ou ◽  
Misako Nagasaka ◽  
Viola W. Zhu
Keyword(s):  
Liquid Biopsy ◽  
Residual Disease ◽  
Single Gene ◽  
Prognostic Significance ◽  
Circulating Tumor Dna ◽  
Resistance Mechanisms ◽  
Driver Mutations ◽  
Braf Mutations ◽  
Clinicopathologic Characteristics ◽  
Pik3ca Mutations

Liquid biopsy has been used extensively in solid malignancies to detect actionable driver mutations, to monitor treatment response, to detect recurrence, to identify resistance mechanisms, and to prognosticate outcome. Although many liquid biopsy sequencing platforms are being used, only five test kits have received government approval. We review representative literature on these government-approved liquid biopsy kits, which are primarily used to detect EGFR mutation in lung cancer and RAS ( KRAS, NRAS, BRAF) mutations in colorectal carcinoma. Another emerging use of single-gene liquid biopsy is to detect PIK3CA mutations and to understand resistance to hormonal blockade in breast and prostate cancers. The two most commonly used next-generation sequencing (NGS) liquid biopsy tests (Guardant 360, Guardant Health; FoundationACT, Foundation Medicine Inc.) are discussed. The ability and the applicability of NGS platform to detect tumor mutation burden are also addressed. Finally, the use of circulating tumor DNA (ctDNA) to detect minimal residual disease may be the most important use of ctDNA in the setting of tumor heterogeneity. The ability to identify “shedders” and “nonshedders” of ctDNA may provide important insight into the clinicopathologic characteristics of the tumor and portend important prognostic significance regarding survival.

scholarly journals CYFRA 21-1 predicts the efficacy of nivolumab in patients with advanced lung adenocarcinoma

Tumor Biology ◽  
2018 ◽  
Vol 40 (2) ◽  
pp. 101042831876042 ◽  
Author(s):  
Hiromichi Shirasu ◽  
Akira Ono ◽  
Katsuhiro Omae ◽  
Kazuhisa Nakashima ◽  
Shota Omori ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Independent Predictor ◽  
Smoking Status ◽  
Performance Status ◽  
Growth Factor Receptor ◽  
Progression Free Survival ◽  
Mutation Status ◽  
Free Survival ◽  
Median Progression Free Survival ◽  
Epidermal Growth

CYFRA 21-1 is a prognostic marker for non–small cell lung cancer. The serum CYFRA 21-1 level is also known as an adjunct for the diagnosis of lung squamous cell carcinoma. This study aimed to examine whether CYFRA 21-1 has predictive implications for nivolumab therapy in patients with advanced lung adenocarcinoma. Of the 79 patients who were treated with nivolumab therapy at the Shizuoka Cancer Center between December 2015 and September 2016, we retrospectively reviewed the data of 50 patients. The patient characteristics were as follows: age <70/≥70 years: 43 (86%)/7; male/female: 31 (62.0%)/19; Eastern Cooperative Oncology Group performance status 0–1/2: 43 (86%)/7; smoking status: no/yes: 18 (36%)/32; epidermal growth factor receptor mutation status negative/positive: 36 (72%)/14; CYFRA 21-1 ≥2.2/<2.2 ng/mL: 28 (56%)/22; carcinoembryonic antigen ≥5/<5 ng/mL: 29 (58%)/21; and number of prior regimens 2–3/≥4: 16 (32%)/34. With a median follow-up of 263.5 (range, 64–352) days, the median progression-free survival was 70 days. The clinical variables investigated using univariate analysis were as follows: age (p = 0.423), carcinoembryonic antigen (p = 0.888), epidermal growth factor receptor mutation status (p = 0.105), performance status (p = 0.968), sex (p = 0.210), number of prior regimens (p = 0.146), CYFRA 21-1 (p = 0.026), and smoking status (p = 0.041). A multivariate analysis identified a serum CYFRA 21-1 level ≥2.2 ng/mL as an independent predictor of a favorable outcome (hazard ratio, 0.44; 95% confidence interval, 0.23–0.85; p = 0.015; median progression-free survival, 155 vs 51.5 days). In conclusion, CYFRA 21-1 might be an independent predictor of outcome for patients with advanced lung adenocarcinoma treated with nivolumab.

scholarly journals A comprehensive study on the oncogenic mutation and molecular pathology in Chinese lung adenocarcinoma patients

2020 ◽  
Author(s):  
Xilin Zhang ◽  
Yan Jiang ◽  
Huanming Yu ◽  
Hui Xia ◽  
Xiang Wang
Keyword(s):  
Lung Adenocarcinoma ◽  
Critical Role ◽  
Evaluation Criteria ◽  
Gene Mutations ◽  
Egfr Mutations ◽  
Driver Mutations ◽  
Amplification Refractory Mutation System ◽  
Clinicopathologic Characteristics ◽  
Oncogenic Mutation ◽  
Formalin Fixed Paraffin Embedded

Abstract Background: Several genetic driver alterations have been identified in micropapillary lung adenocarcinoma (MPA). However, the frequency of co-alteration of ROS1, EGFR and EML4-ALK is yet unclear. Herein, we investigated the relationship between clinicopathologic characteristics and well-identified driver mutations of MPA compared with non-micropapillary lung adenocarcinoma (LA). Methods: Formalin fixed paraffin-embedded (FFPE) sections derived from lung adenocarcinoma patients who never received adjuvant chemotherapy or radiation therapy prior to surgical resection were collected from October 2016 to June 2019. EGFR mutations, ROS1 rearrangements and EML4-ALK fusion were identified in a set of 131 MPA and LA cases by using the Amplification Refractory Mutation System (ARMS). The response rate and duration of response were assessed using Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). Results: EGFR mutations had occurred in 42 (76.4%) MPA patients and 42 (55.3%) LA patients. Interestingly, ROS1 rearrangements were highly enriched only in the MPA cases (6/55, 10.9%) but rarely in the LA cases (1/76, 1.3%). Furthermore, 7.3% (4/55) MPA samples had double gene mutations, while only 1.3% (1/76) LA cases had double gene alterations. Of 5 patients with harboring two driver oncogenes mutations, four patients (80%) obtained partial response and one patient (20%) suffered recurrence. Conclusions: A higher prevalence of ROS1 rearrangement or combined mutations of ROS1, EGFR and EML4-ALK may play a critical role in the tumorigenesis of MPA. These findings provide a novel therapeutic strategy for patients with malignant MPA through combining TKIs than one TKI.

Genomic characterization of sub-centimeter pulmonary nodules.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13530-e13530
Author(s):  
Muyun Peng ◽  
Guanlan Xing ◽  
Bingyu Zhang ◽  
Yaping Xu ◽  
Fenglei Yu
Keyword(s):  
Lung Adenocarcinoma ◽  
Pulmonary Nodules ◽  
Driver Mutations ◽  
Adenocarcinoma In Situ ◽  
Lung Carcinogenesis ◽  
Braf Mutations ◽  
Invasive Adenocarcinoma ◽  
Driver Genes ◽  
Invasive Group ◽  
Genomic Landscape

e13530 Background: There has been a dramatic increase in the detection of indeterminate pulmonary nodules (IPNs), many of which are 10 mm or less in diameter. The management of subcentimeter pulmonary nodules remains controversial. Deciphering the genomic landscape of subcentimeter pulmonary nodules will provide critical insights to the mechanisms of carcinogenesis and pave the way for the early prevention and interception of lung cancer. Methods: We subjected 439 IPN samples including 249 subcentimeter pulmonary nodules (≤10 mm in diameter) and 190 larger pulmonary nodules (>10 and ≤30mm in diameter) to deep next generation sequencing by using customized panels of 1021 genes. Clinical parameters of these IPNs were collected. The genomic landscape of subcentimeter pulmonary nodules and differences from that of larger pulmonary nodules were defined. Results: The proportions of atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and invasive adenocarcinoma (ADC) in the subcentimeter group and the larger nodule group were 2.81% vs 0.00%, 13.25% vs 0.80%, 28.92% vs 4.82% and 55.02% vs 70.68%, respectively. The most commonly mutated genes in subcentimeter group were in EGFR, ERBB2, BRAF, MED12 and MAP2K1. Compared with the larger nodule group, the subcentimeter nodule group had lower TMB ( p< 0.0001), lower mutation frequencies in EGFR, TP53, RBM10, and SMARCA4 ( p< 0.05), but higher mutation frequencies in ERBB2, BRAF, MAP2K1, and TSC1 ( p< 0.05). In the subset of ADC, the subcentimeter nodule group had lower TMB ( p< 0.0001), lower mutation frequencies in EGFR, TP53, RBM10, and PIK3CA ( p< 0.05), but higher mutation frequencies in ERBB2, BRAF ( p< 0.05). Clonal index analysis of driver genes in lung adenocarcinoma showed that the larger nodule group had more driver mutations, but the clonality index of BRAF was higher in the subcentimeter group ( p= 0.0014). Furthermore, in the subcentimeter nodule group, the mutation frequency of BRAF was significantly higher in pre-invasive group (AAH/AIS) than that in the invasive group (IAC). And BRAF and EGFR were significantly mutually exclusive ( q< 0.001) in subcentimeter nodules, which showed the subgroup with BRAF mutations was a special subtype. Conclusions: Our results provided the distinct mutation pattern of subcentimeter nodules and showed that BRAF could play different roles in early phase of lung carcinogenesis. It would be helpful for understanding the genomic evolution mechanisms underlying the progression and invasiveness of early lung adenocarcinoma.

scholarly journals The Story of a Ship Journey, Malaria, and the HBB Gene IVS-II-745 Mutation: Circassian İmmigration to Cyprus

2021 ◽  
Author(s):  
Mahmut C. Ergoren ◽  
Sehime G. Temel ◽  
Gamze Mocan ◽  
Munis Dundar
Keyword(s):  
Autosomal Recessive ◽  
Single Gene ◽  
Pcr Amplification ◽  
Health Condition ◽  
Amplification Refractory Mutation System ◽  
Chain Reaction ◽  
Analysis Technique ◽  
Endemic Malaria ◽  
Mutation System ◽  
Polymerase Chain

Abstract Background During 19th century, the Circassians were secluded from their lands and forced to migrate to Ottoman Empire properties. Approximately 2,346 Circassians were exiled from Istanbul to Cyprus Island. During the deportation journey, many of Circassian were passed away in consequence of malaria and unknown reasons. Overall, 1,351 survivor Circassians managed to reach the island, however, many of them had faced with endemic malaria again in Cyprus. An autosomal recessive hematological disorder thalassemia was the second endemic health condition after malaria, whereas thalassemia carriers show resistance to malaria infections. Materials and Methods A large Cypriot family with 57 members whose grandparents were supposed to be in that ship journey has been investigated in this study. Polymerase chain reaction (PCR)–amplification refractory mutation system (ARMS) analysis technique was used for genotyping the HHB gene. Results The human β-globin (HBB) gene c.316–106C > G (IVS-II-745) (II-745) heterozygous variation have been detected. Conclusion Overall, this study is a very good example for a typical natural selection. In this case, one single gene point mutation did not limit survival in the society; natively, it increased their survival changes to form new colonization and the inheritance of the mutation to the next generations.

scholarly journals Identifying new variation at the J locus, previously identified as e6, in long juvenile ‘Paranagoiana’ soybean

2021 ◽  
Author(s):  
Nour Nissan ◽  
Elroy R. Cober ◽  
Michael Sadowski ◽  
Martin Charette ◽  
Ashkan Golshani ◽  
...  
Keyword(s):  
Single Gene ◽  
Pcr Amplification ◽  
Gene Control ◽  
Grain Yields ◽  
Short Day ◽  
Exon 4 ◽  
Short Days ◽  
Kasp Markers

Abstract Key message A previously identified soybean maturity locus, E6, is discovered to be J, with the long juvenile allele in Paranagoiana now deemed j−x. Abstract Soybean grown at latitudes of ~20° or lower can produce lower grain yields due to the short days. This limitation can be overcome by using the long juvenile trait (LJ) which delays flowering under short day conditions. Two LJ loci have been mapped to the same location on Gm04, J and E6. The objective of this research was to investigate the e6 allele in ‘Paranagoiana’ and determine if E6 and J are the same locus or linked loci. KASP markers showed that e6 lines did not have the j−1 allele of LJ PI 159925. A population fixed for E1 but segregating for E6, with e6 introgressed from Paranagoiana, showed single gene control for flowering and maturity under short days. Sequencing Glyma.04G050200, the J gene, with long amplification Taq found that the e6 line ‘Paranagoiana’ contains a Ty1-copia retrotransposon of ~10,000 bp, inserted within exon 4. PCR amplification of the cDNA of Glyma.04G050200 also showed differences between the mRNA sequences (presence of insertion in j−x). Hence, we conclude that the loci E6 and J are one locus and deem this new variation found in Paranagoiana as j−x.

scholarly journals MBCL-08. INTEGRATIVE MOLECULAR ANALYSIS OF PATIENT-MATCHED DIAGNOSTIC AND RELAPSED MEDULLOBLASTOMAS

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii389-iii389
Author(s):  
Rahul Kumar ◽  
Maximilian Deng ◽  
Kyle Smith ◽  
Anthony Liu ◽  
Girish Dhall ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Copy Number ◽  
Driver Mutations ◽  
P Value ◽  
Next Generation ◽  
Driver Genes ◽  
Methylation Array ◽  
Primary Tumors ◽  
Group 4 ◽  
Group 3

Abstract INTRODUCTION The next generation of clinical trials for relapsed medulloblastoma demands a thorough understanding of the clinical behavior of relapsed tumors as well as the molecular relationship to their diagnostic counterparts. METHODS A multi-institutional molecular cohort of patient-matched (n=126 patients) diagnostic MBs and relapses/subsequent malignancies was profiled by DNA methylation array. Entity, subgroup classification, and genome-wide copy-number aberrations were assigned while parallel next-generation (whole-exome or targeted panel) sequencing on the majority of the cohort facilitated inference of somatic driver mutations. RESULTS Comprised of WNT (2%), SHH (41%), Group 3 (18%), Group 4 (39%), primary tumors retained subgroup affiliation at relapse with the notable exception of 10% of cases. The majority (8/13) of discrepant classifications were determined to be secondary glioblastomas. Additionally, rare (n=3) subgroup-switching events of Group 4 primary tumors to Group 3 relapses were identified coincident with MYC/MYCN pathway alterations. Amongst truly relapsing MBs, copy-number analyses suggest somatic clonal divergence between primary MBs and their respective relapses with Group 3 (55% of alterations shared) and Group 4 tumors (63% alterations shared) sharing a larger proportion of cytogenetic alterations compared to SHH tumors (42% alterations shared; Chi-square p-value &lt; 0.001). Subgroup- and gene-specific patterns of conservation and divergence amongst putative driver genes were also observed. CONCLUSION Integrated molecular analysis of relapsed MB discloses potential mechanisms underlying treatment failure and disease recurrence while motivating rational implementation of relapse-specific therapies. The degree of genetic divergence between primary and relapsed MBs varied by subgroup but suggested considerably higher conservation than prior estimates.

scholarly journals Lung Adenocarcinoma From East Asian Never-Smokers Is a Disease Largely Defined by Targetable Oncogenic Mutant Kinases

2010 ◽  
Vol 28 (30) ◽  
pp. 4616-4620 ◽  
Author(s):  
Yihua Sun ◽  
Yan Ren ◽  
Zhaoyuan Fang ◽  
Chenguang Li ◽  
Rong Fang ◽  
...  
Keyword(s):  
East Asian ◽  
Egfr Mutations ◽  
Driver Mutations ◽  
Single Institution ◽  
Pik3ca Mutations ◽  
Oncogenic Mutations ◽  
Oncogenic Driver ◽  
Never Smokers ◽  
Lung Adenocarcinomas ◽  
Concurrent Analysis

Purpose To determine the proportion of lung adenocarcinomas from East Asian never-smokers who harbor known oncogenic driver mutations. Patients and Methods In this surgical series, 52 resected lung adenocarcinomas from never-smokers (< 100 cigarettes in a lifetime) at a single institution (Fudan University, Shanghai, China) were analyzed concurrently for mutations in EGFR, KRAS, NRAS, HRAS, HER2, BRAF, ALK, PIK3CA, TP53 and LKB1. Results Forty-one tumors harbored EGFR mutations, three harbored EML4-ALK fusions, two harbored HER2 insertions, and one harbored a KRAS mutation. All mutations were mutually exclusive. Thus, 90% (47 of 52; 95% CI, 0.7896 to 0.9625) of lung adenocarcinomas from never-smokers were found to harbor well-known oncogenic mutations in just four genes. No BRAF, NRAS, HRAS, or LKB1 mutations were detected, while 15 had TP53 mutations. Four tumors contained PIK3CA mutations, always together with EGFR mutations. Conclusion To our knowledge, this study represents the first comprehensive and concurrent analysis of major recurrent oncogenic mutations found in a large cohort of lung adenocarcinomas from East Asian never-smokers. Since drugs are now available that target mutant EGFR, HER2, and ALK, respectively, this result indicates that prospective mutation testing in these patients should successfully assign a targeted therapy in the majority of cases.

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