Development CRISPR-Cas12-based rapid EGFR mutation detection kit.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13669-e13669
Author(s):  
Kei Kunimasa ◽  
Yuya Bando ◽  
Miyako Shiraishi ◽  
Emi Aizawa ◽  
Kazumi Nishino ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Egfr Mutation ◽  
Low Cost ◽  
Lateral Flow ◽  
Detection Methods ◽  
Egfr Mutations ◽  
Driver Mutations ◽  
Guide Rna ◽  
Testing Method ◽  
Nsclc Patients

e13669 Background: Recently, several cancer gene panel tests have been developed to determine many driver mutations including lung cancer simultaneously. However, the current next-sequencing based platform is not ideal testing method in the clinical practice due to the high examination cost and the time required for the result to reach the field. By refining recently reported CRISPR-Cas-based nucleic acid detection methods, we developed a novel CRISPR-Cas12 family based rapid-detection kit for targetable EGFR mutations in non-small lung cancer (NSCLC) patients. Methods: To develop a powerful CRISPR-based testing method, we focused on unique Cas12 family proteins which contain a single RuvC nuclease domain that cleave target double-stranded DNA adjacent to protospacer adjacent motif (PAM) sequences as well as non-target single-stranded DNA (ssDNA) collaterally. We purified 6 Cas12 family proteins (Cas12a, Cas12c1, Cas12c2, Cas12g, Cas12i1, and Cas12i2) as well as two types of target-defined guide RNA for EGFR Ex.19 del(E746-A750) and L858R mutations. The target sequences including EGFR Ex.19(E746-A750) and L858R mutations were amplified from EGFR mutated cell lines, FFPE lung cancer tissues or cell free DNA (cfDNA) of NSCLC patients using PCR with PrimeSTAR GXL DNA polymerase (Takara, Shiga, Japan) or isothermal recombinase polymerase amplification (RPA) with TwistAmp Basic (TwistDx, Cambridge, UK). CRISPR-Cas12-based cleavage assay was conducted in a reaction buffer consisting of 10 nM Cas12 proteins, 10 nM purified guide RNA, 0.4nM target DNA, and 50 nM collateral ssDNA (quenched fluorescent DNA reporter) in a 20 μl volume at 37 °C for 1 h. The excited fluorescence, which is an indicator of the presence of target cancer mutation, was measured by Synergy H1 hybrid Multi-Mode Reader (BioTek, Vermont, USA). For simpler instrument-free and portable detection lateral flow readouts were also developed and tested using Milenia HybriDetect1 kit (TwistDx). Results: Cas12a, Cas12i1, Cas12i2-based assays successfully detected the EGFR Ex.19 del(E746-A750) and L858R mutations from PCR and RPA products. Lateral flow readout kit could also detect EGFR Ex.19 del (E746-A750) mutation from cfDNA derived from NSCLC patients. It took about 3 hours from plasma collection to DNA extraction, RPA, and measurement with the kit. Conclusions: CRISPR-Cas based lateral flow readout kit can detect EGFR mutation at an unprecedented pace with low cost. Our developed system will be an innovative testing tool for lung cancer patients with low cost, rapid, multiplexable, and noninvasive procedure.

Assessment of EGFR mutations in patients diagnosed of squamous non-small cell lung cancer (NSCLC).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e18106-e18106
Author(s):  
Francisco Lobo ◽  
Manuel Domine ◽  
Federico Rojo ◽  
Yann Izarzugaza ◽  
Ana Leon ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Squamous Cell ◽  
Cell Lung Cancer ◽  
Egfr Mutation ◽  
Small Cell ◽  
Egfr Mutations ◽  
Small Cell Lung ◽  
Exon 20 ◽  
Nsclc Patients

e18106 Background: Mutations in the Epidermal Growth Factor Receptor (EGFR) predict a better outcome to EGFR tyrosine kinase inhibitors than platinum-based chemotherapy in advanced non-small cell lung cancer (NSCLC). Previous studies with Caucasian patients have shown a prevalence of EGFR mutation of 10-15%. The aim of this study is to analyze the prevalence of EGFR mutations in squamous-cell NSCLC patients from an area of influence of 500,000 habitants. Methods: Prospective mutation testing in NSCLC patients included in our institution since October 2010 to January 2012 was performed on DNA obtained from available tumor tissue and cytologic samples, using ARMS-scorpion TheraScreen EGFR 29 Mutation Test Kit (Qiagen). Results: From 218 consecutive NSCLC diagnoses, 18 (8.25%) patients showed EGFR mutations: 6 (33.3%) exon 19 deletion, 9 (50%) exon 21 mutations (7 L858R and 2 L861Q) and 3 (16.6%) cases exon 20 insertion. In the EGFR mutated population, 16 (88.88%) patients were diagnosed as adenocarcinoma and 2 (11.11%) as squamous cell carcinoma. The characteristics of these squamous cell cancer patients were: 2 male; 1 non-smoker, 1 former-smoker; 1 stage IV and 1 stage IB at diagnosis; one case exon 20 insertion and one exon 21 mutation (L858R). Conclusions: The EGFR mutation rate in squamous-cell NSCLC patients in our referral area is superior (11.17%) than previously reported, reinforcing the importance of including EGFR mutation testing in squamous-cell NSCLC population for selecting optimal therapy for these patients.

scholarly journals Incorporation of EGFR mutation status into M descriptor of new TNM classification influences survival curves in non-small cell lung cancer patients

2019 ◽  
Vol 53 (4) ◽  
pp. 453-458
Author(s):  
Karmen Stanic ◽  
Nina Turnsek ◽  
Martina Vrankar
Keyword(s):  
Lung Cancer ◽  
Medical Records ◽  
Disease Burden ◽  
Egfr Mutation ◽  
Tnm Classification ◽  
Small Cell ◽  
Egfr Mutations ◽  
Small Cell Lung ◽  
Mutation Status ◽  
Nsclc Patients

Abstract Background The 8th edition of tumor node metastasis (TNM) staging system for lung cancer introduced a revision of M descriptor. The limitation of new classification to predict prognosis is its focus on anatomical extent of the disease only. Information on molecular status of the tumor significantly influences treatment response and survival; however, data addressing this issue is scarce. This report points to the impact of epidermal growth factor receptor (EGFR) mutation in non-small cell lung cancer (NSCLC) patients on survival in view of new M descriptors of TNM classification system. Patients and methods Medical records of 479 consecutive metastatic NSCLC patients treated between 2009 and 2011, all tested for EGFR mutations, were retrospectively reviewed. For 355 patients medical records included sufficient information to be appropriately categorized into one of the new subgroups according to the M descriptor in 8th TNM classification, of those 89 (25.1%) patients harboured EGFR mutations (EGFR-m). Results Median overall survival (mOS) of EGFR-m patients was significantly longer than mOS of patients without EGFR mutations (20.6 months vs. 8.3 months, p < 0.001). Patients with limited disease burden (M1b sub-group) had the longest mOS among EGFR wild type patients (EGFR-wt) and also among EGFR-m patients, 14.4 months and 39.2 month, respectively. In spite of widespread metastatic disease of M1c EGFR-m patients, their mOS (18.8 months) was longer than mOS of oligometastatic EGFR-wt patients (M1b), who had the lowest disease burden (14.4 months). Median follow up was 53.9 months. Conclusions Incorporation of EGFR mutation status in advanced NSCLC further differentiates survival curves of M categories in 8th TNM classification and more precisely predicts survival compared to number of metastasis or number of metastatic sites alone.

HSR19-082: Epidemiological Findings and Outcomes in Non-Small Cell Lung Cancer Patients with Exon 20 Insertion Mutations: A Meta-Analysis

2019 ◽  
Vol 17 (3.5) ◽  
pp. HSR19-082
Author(s):  
Victoria Crossland ◽  
Aaron Galaznik ◽  
Huamao M. Lin ◽  
Dimitrios Tomaras ◽  
Shan Ashton Garib ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Egfr Mutation ◽  
Meta Analysis ◽  
Small Cell ◽  
Egfr Mutations ◽  
Small Cell Lung ◽  
Exon 20 ◽  
Meta Analyses ◽  
Nsclc Patients ◽  
Insertion Mutations

Background: Epidermal growth factor receptor (EGFR) mutations are frequently found in non-small cell lung cancer (NSCLC) patients. Various EGFR mutations respond differently to EGFR tyrosine kinase inhibitors (TKIs), and several TKIs have been approved for use on common mutations but none have been approved for EGFR exon 20 insertions, indicating a need for targeted therapy for this subpopulation. A systematic literature review (SLR) and meta-analysis were conducted to synthesize epidemiological and outcome data for the uncommon EGFR exon 20 insertion mutation. Methods: An SLR was performed on August 7, 2018 following the Preferred Reporting Item for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, using the Population, Intervention Comparators, Outcomes and Study Design (PICOS) criteria. Studies were identified based on a systematic search using key biomedical literature databases: EMBASE, MEDLINE, and Cochrane. Relevant congress abstracts published between 2015–2018 were also identified. Two independent reviewers screened all citations and full-text articles using PICOS-based criteria; any discrepancies were resolved by a third independent reviewer. Data were extracted into a predefined template for meta-analysis and summarized using the PRISMA flow diagram. Results: A total of 61 studies reporting the number of EGFR mutation−positive patients and/or NSCLC patients were identified. A meta-analysis found that 3.7% of EGFR mutation−positive patients and 0.8% of NSCLC patients harbored the EGFR exon 20 insertion, with geographic variations in epidemiology. There were 12, 10, and 12 studies, respectively, that reported overall survival, progression-free survival, and overall response rates in 2 cohorts, patients with EGFR exon 20 insertions and patients without EGFR exon 20 mutations. A Most patient populations in these studies included a mixture of treatment at various lines. A meta-analysis of outcomes across these studies showed that patients with EGFR exon 20 insertions experienced worse outcomes compared with those without the mutation (Table 1). Meta-analyses were weighted based on each study’s relevant population. No economic or quality of life studies were identified. Conclusions: Exon 20 insertion mutations represent an important subgroup of EGFR mutations in patients with NSCLC, and current therapies have limited efficacy. These relatively poor outcomes indicate a need for novel treatment strategies.

Prospective analysis of the epidermal growth factor receptor gene mutations in non-small cell lung cancer in Japan

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7077-7077 ◽  
Author(s):  
N. Morikawa ◽  
A. Inoue ◽  
T. Suzuki ◽  
T. Fukuhara ◽  
S. Suzuki ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Epidermal Growth Factor Receptor ◽  
Egfr Mutation ◽  
Response Rate ◽  
Growth Factor Receptor ◽  
Small Cell ◽  
Egfr Mutations ◽  
Small Cell Lung ◽  
Epidermal Growth ◽  
Nsclc Patients

7077 Background: Previous clinical trials have revealed that gefitinib is more likely to be effective in non-small cell lung cancer (NSCLC) with activating somatic mutations of epidermal growth factor receptor (EGFR). Most of those reports evaluated NSCLC patients who had post-operative recurrence and then received gefitinib retrospectively using their surgical specimens. However, many NSCLC patients are inoperable at diagnosis. Thus we conducted this study to examine EGFR mutation status by diagnostic tumor samples before gefitinib treatment and investigate the correlation between EGFR mutation and the efficacy of gefitinib. Methods: We prospectively evaluated various tumor samples obtained from NSCLC patients who had never received gefitinib for EGFR mutations in exon 18–23. For patients treated with gefitinib after the examination of EGFR mutations, the response to gefitinib was also evaluated. Results: From June 2004 to November 2005, 91 patients with advanced or post-operative recurrent NSCLC enrolled onto this study and 104 tumor samples were obtained from transbronchial biopsies, effusions, as well as surgical specimens. Thirty-two mutations including deletions in exon 19 in 23 patients and L858R in 9 patients were detected among those 91 patients; 30 in 81 adenocaricinoma, 1 in 2 adenosquamous cell carcinoma, and 1 in 5 large cell carcinoma. The mutations were found more frequently in female (51.9%) than male (12.8%), in never smoker (52.0%) than smoker (14.6%). Response rate of gefitinib in patients with EGFR mutations was 65.0% (13 of 20) compared to 37.5% (3 of 8) in patients without mutations. Among 7 patients with EGFR mutations who were examined multiple tumor samples, 3 had the discrepancy of EGFR gene status between different samples obtained at different time points, suggesting genetic heterogeneity of their tumors. The EGFR status of the most recent samples is likely to be correlated to the response to gefitinib. Conclusions: The EGFR mutation analysis was possible not only from surgical specimens but also from daily available diagnostic samples. For patients with EGFR mutations, gefitinib could achieve a promising high response rate. We propose to examine the most recent tumor samples to predict the sensitivity to gefitinib reliably. No significant financial relationships to disclose.

Nicht kleinzelliges Lungenkarzinom: Einfluss von Treibermutationen bei Immuncheckpoint-Inhibition erkennen

2020 ◽  
Vol 8 (6) ◽  
pp. 320-321
Author(s):  
Susanne Horter ◽  
Wolfgang Schütte
Keyword(s):  
Lung Cancer ◽  
Checkpoint Inhibitors ◽  
Tertiary Care ◽  
Treatment Decision ◽  
Kleinzelliges Lungenkarzinom ◽  
Egfr Mutations ◽  
Driver Mutations ◽  
Molecular Testing ◽  
Durable Response ◽  
Nsclc Patients

<b>Aims:</b> Non-small cell lung cancer (NSCLC) patients with EGFR mutations do not respond well to checkpoint inhibitors. However, little is known about the activity of immunotherapy in NSCLC with other driver mutations. The increasing use of next-generation sequencing (NGS) leads to molecular findings that face the clinician with problems while choosing the best treatment. This study aims at analyzing response of NSCLC with driver mutations to immunotherapy. <b>Patients and Methods:</b> We retrospectively included 84 NSCLC patients diagnosed and treated at 2 German tertiary-care lung cancer centers using NGS and treatment with immunotherapy. Response to immunotherapy was analyzed in correlation to molecular findings. <b>Results:</b> 51 patients harbored at least 1 driver mutation. PIK3CA, EGFR, and STK11 mutations did not respond to immunotherapy. KRAS, TP53, and MET exon 14 skipping mutations responded well. One patient with NF-1 mutation showed durable response. <b>Conclusions:</b> Molecular testing may be of use in guiding treatment decision making in NSCLC.

scholarly journals Iterative Upgrading of Small Molecular Tyrosine Kinase Inhibitors for EGFR Mutation in NSCLC: Necessity and Perspective

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1500
Author(s):  
Jing Zhu ◽  
Qian Yang ◽  
Weiguo Xu
Keyword(s):  
Lung Cancer ◽  
Kinase Inhibitors ◽  
Egfr Mutation ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
Egfr Mutations ◽  
Convenient Route ◽  
Epidermal Growth ◽  
Nsclc Patients ◽  
Genome Information

Molecular targeted therapy has been reported to have fewer adverse effects, and offer a more convenient route of administration, compared with conventional chemotherapy. With the development of sequencing technology, and research on the molecular biology of lung cancer, especially whole-genome information on non-small cell lung cancer (NSCLC), various therapeutic targets have been unveiled. Among the NSCLC-driving gene mutations, epidermal growth factor receptor (EGFR) mutations are the most common, and approximately 10% of Caucasian, and more than 50% of Asian, NSCLC patients have been found to have sensitive EGFR mutations. A variety of targeted therapeutic agents for EGFR mutations have been approved for clinical applications, or are undergoing clinical trials around the world. This review focuses on: the indications of approved small molecular kinase inhibitors for EGFR mutation-positive NSCLC; the mechanisms of drug resistance and the corresponding therapeutic strategies; the principles of reasonable and precision molecular structure; and the drug development discoveries of next-generation inhibitors for EGFR.

Prevalence and clinicopathological factors of the EGFR mutation status of Vietnamese non-small cell lung cancer patients.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e13120-e13120
Author(s):  
Khoa Trong Mai ◽  
Cam Phuong Pham ◽  
LUNG TIEN Nguyen ◽  
Quynh Thi Thuy Vo ◽  
Nguyen TUAN Anh ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Egfr Mutation ◽  
Small Cell ◽  
Disease Stage ◽  
Egfr Mutations ◽  
Small Cell Lung ◽  
Mutation Status ◽  
Nsclc Patients

e13120 Background: To determine the epidermal growth factor receptor ( EGFR) mutation status in non-small cell lung cancer (NSCLC) patients in Vietnamese population at Bach Mai Hospital, and relation of different variables to the frequency of EGFR mutations. Methods: The EGFR mutation status of 1451 tissue samples in NSCLC patients and correlation among different variables of age, gender, smoking habit and histology groups were evaluated. Results: A total of EGFR mutation analysis was performed, with an overall mutation rate of 40.7%. The most prevalent categories were in-frame deletions in exon 19 (57.2% of the overall mutations); followed by L858R and L861Q in exon 21 (35.7%); exon 18 (4,6%); double mutation: 2.9%; The T790M mutation in exon 20 represented approximately 2.5%; The pooled prevalence of EGFR mutation was higher in female (60.6%), non-smokers (61.1%), and patients with adenocarcinoma (42.2%). Conclusions: The prevalence of EGFR mutations of NSCLC in Vietnam is higher than that in the West, but comparably equal to that in some Asian countries. It is associated with female sex, non-smoker status, and adenocarcinoma, sample tissue type and age, but not depends on disease stage and tumor sampling.

scholarly journals Molecular profile of KRAS G12C-mutant colorectal and non-small-cell lung cancer

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Luiz Henrique Araujo ◽  
Bianca Mendes Souza ◽  
Laura Rabelo Leite ◽  
Sabrina A. F. Parma ◽  
Natália P. Lopes ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Small Cell ◽  
Driver Mutations ◽  
Molecular Profile ◽  
Common Mutation ◽  
Small Cell Lung ◽  
The South ◽  
Nsclc Patients

Abstract Background KRAS is the most frequently mutated oncogene in cancer, however efforts to develop targeted therapies have been largely unsuccessful. Recently, two small-molecule inhibitors, AMG 510 and MRTX849, have shown promising activity in KRAS G12C-mutant solid tumors. The current study aims to assess the molecular profile of KRAS G12C in colorectal (CRC) and non-small-cell lung cancer (NSCLC) tested in a clinical certified laboratory. Methods CRC and NSCLC samples submitted for KRAS testing between 2017 and 2019 were reviewed. CRC samples were tested for KRAS and NRAS by pyrosequencing, while NSCLC samples were submitted to next generation sequencing of KRAS, NRAS, EGFR, and BRAF. Results The dataset comprised 4897 CRC and 4686 NSCLC samples. Among CRC samples, KRAS was mutated in 2354 (48.1%). Most frequent codon 12 mutations were G12D in 731 samples (14.9%) and G12V in 522 (10.7%), followed by G12C in 167 (3.4%). KRAS mutations were more frequent in females than males (p = 0.003), however this difference was exclusive of non-G12C mutants (p < 0.001). KRAS mutation frequency was lower in the South and North regions (p = 0.003), but again KRAS G12C did not differ significantly (p = 0.80). In NSCLC, KRAS mutations were found in 1004 samples (21.4%). As opposed to CRC samples, G12C was the most common mutation in KRAS, in 346 cases (7.4%). The frequency of KRAS G12C was higher in the South and Southeast regions (p = 0.012), and lower in patients younger than 50 years (p < 0.001). KRAS G12C mutations were largely mutually exclusive with other driver mutations; only 11 NSCLC (3.2%) and 1 CRC (0.6%) cases had relevant co-mutations. Conclusions KRAS G12C presents in frequencies higher than several other driver mutations, and may represent a large volume of patients in absolute numbers. KRAS testing should be considered in all CRC and NSCLC patients, independently of clinical or demographic characteristics.

scholarly journals Peptide-Affinity Precipitation of Extracellular Vesicles and Cell-Free DNA Improves Sequencing Performance for the Detection of Pathogenic Mutations in Lung Cancer Patient Plasma

2020 ◽  
Vol 21 (23) ◽  
pp. 9083
Author(s):  
Catherine Taylor ◽  
Simi Chacko ◽  
Michelle Davey ◽  
Jacynthe Lacroix ◽  
Alexander MacPherson ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Nsclc Patient ◽  
Egfr Mutations ◽  
Single Nucleotide Variants ◽  
Cell Free Dna ◽  
Free Dna ◽  
Nsclc Patients ◽  
Peptide Affinity

Liquid biopsy is a minimally-invasive diagnostic method that may improve access to molecular profiling for non-small cell lung cancer (NSCLC) patients. Although cell-free DNA (cf-DNA) isolation from plasma is the standard liquid biopsy method for detecting DNA mutations in cancer patients, the sensitivity can be highly variable. Vn96 is a peptide with an affinity for both extracellular vesicles (EVs) and circulating cf-DNA. In this study, we evaluated whether peptide-affinity (PA) precipitation of EVs and cf-DNA from NSCLC patient plasma improves the sensitivity of single nucleotide variants (SNVs) detection and compared observed SNVs with those reported in the matched tissue biopsy. NSCLC patient plasma was subjected to either PA precipitation or cell-free methods and total nucleic acid (TNA) was extracted; SNVs were then detected by next-generation sequencing (NGS). PA led to increased recovery of DNA as well as an improvement in NGS sequencing parameters when compared to cf-TNA. Reduced concordance with tissue was observed in PA-TNA (62%) compared to cf-TNA (81%), mainly due to identification of SNVs in PA-TNA that were not observed in tissue. EGFR mutations were detected in PA-TNA with 83% sensitivity and 100% specificity. In conclusion, PA-TNA may improve the detection limits of low-abundance alleles using NGS.

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