Enhanced detection of EGFR mutations in plasma from non-small cell lung cancer (NSCLC) patients using Scorpion primers

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 7682-7682
Author(s):  
W. Holland ◽  
A. M. Davies ◽  
N. C. Farneth ◽  
O. Gautschi ◽  
P. N. Lara ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Nsclc Patient ◽  
Detection Methods ◽  
Egfr Mutations ◽  
Wild Type ◽  
Allele Specific ◽  
Nsclc Patients ◽  
Tumor Dna

7682 Background: Activating epidermal growth factor receptor (EGFR) mutations identified in NSCLC patient tumors are often associated with rapid and profound response to EGFR tyrosine kinase inhibitors (TKIs). Conventional detection methods are cumbersome and may underestimate mutations frequencies due to limited sensitivity. We and others have shown that tumor-specific mutations such as KRAS can be detected in tumor DNA shed into patient plasma (Kimura, Ann N Y Acad Sci, 2004). Here we describe the performance of an allele- specific real-time PCR system utilizing Scorpion primers (kindly provided by DxS, UK) to detect EGFR mutations in plasma. Methods: DNA was extracted from archival tumor blocks, slides and plasma obtained from consenting patients. In order to determine the sensitivity of this technique both in terms of the ratio of mutant-to-wild-type genomic DNA as well as the minimum amount of DNA required for detection, a dilution experiment was conducted. Genomic DNA from cell lines containing either the exon 21 L858R point mutation or the exon 21 E746–750 deletion was diluted with wild-type genomic DNA at ratios ranging from 1:2 to 1:10,000. Clinical specimens including plasma and/or tissue from 35 advanced stage NSCLC patients treated with EGFR-TKIs were examined. Results: Mutant DNA was successfully detected when it comprised as little as 0.1% of the total sample or as low as 25 pg of mutant-positive genomic DNA in a pool of 2.5 μg of total DNA. EGFR mutations were identified by this approach in both plasma and tissue of 2 patients who were complete responders to EGFR-TKI therapy, only one of which was detectable by direct sequencing. For the 7 patients where only tissue was available, two were positive both with the Scorpion primers and direct sequencing while the rest were wild-type. Of 21 patients where only plasma was available, 6 mutations were detected using the Scorpion primers. Conclusions: Allele-specific Scorpion technology is 1) highly specific and sensitive for EGFR mutation analysis, 2) able to detect mutations that were not observable by direct sequencing in plasma and tissue, 3) capable of detecting mutations in shed tumor DNA in plasma of advanced NSCLC patients and 4) may be suitable for monitoring response or detecting recurrence. No significant financial relationships to disclose.

Routine detection of EGFR mutations in patients with NSCLC: A comparative study of three alternative methods in 105 patients

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7184-7184
Author(s):  
G. Zalcman ◽  
N. Richard ◽  
A. Hardouin ◽  
R. Gervais ◽  
M. Antoine ◽  
...  
Keyword(s):  
High Performance ◽  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Tumor Resection ◽  
Growth Factor Receptor ◽  
Alternative Methods ◽  
Egfr Mutations ◽  
Genomic Sequencing ◽  
Surgical Biopsy ◽  
Nsclc Patients

7184 Background: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) has been reported in lung adenocarcinoma patients with tumoral EGFR mutations. Those mutations were found mainly by direct genomic sequencing on snap-frozen surgical specimens. Conversely, TKIs are used in metastatic patients who do not undergo tumor resection. In these patients, there is a need for routine sensitive molecular procedures, to overcome the small size of non-surgical bronchoscopy paraffin-embedded biopsy samples. Methods: Patients were selected on clinical and pathological characteristics: never (n = 43) or former smokers, patients with non-squamous NSCLC (n = 105) or patients with bronchioloalveolar adenocarcinoma (n = 34). Direct genomic sequencing assay was performed as reported elsewhere. Denaturing, high-performance, liquid chromatography (DHPLC) assay was performed with EGFR-specific primers that amplify exons 18, 19, and 21. A multiplex, allele-specific, oligonucleotide PCR (MASO-PCR) assay was carried out with a set of primers that identify the 14 most frequent molecular events described for the EGFR gene, which covers 90% of EGFR gain-of-function mutations described to date. Results: 123 samples were screened from 105 non-squamous NSCLC patients (female/male ratio = 0.84). Non-surgical biopsy specimens were available in 38 patients. EGFR mutations were detected by at least two of three procedures in 18/105 patients (17%). In paraffin-embedded specimens with low tumor content, EGFR heterozygous mutations were found either by MASO-PCR alone (n = 2, confirmed in the matched surgical sample by another procedure), or both by MASO-PCR and DHPLC (n = 16); they were missed by nucleotidic sequencing in 6 samples. 18 patients received TKI. 6 dramatic responses were achieved in patients with EGFR mutation, while no mutation could be found in non-responsive patients. Overall disease control was obtained in 8/18 patients (44%). Conclusions: MASO-PCR and DHPLC appear more sensitive than direct sequencing in non-surgical paraffin-embedded biopsies, which represent the bulk of samples in lung cancer patients. We propose that the cost-effective MASO-PCR be used for routine screening of EGFR mutations in NSCLC patients. No significant financial relationships to disclose.

scholarly journals Detection of EGFR Mutations in Plasma Cell-Free Tumor DNA of TKI-Treated Advanced-NSCLC Patients by Three Methodologies: Scorpion-ARMS, PNAClamp, and Digital PCR

Diagnostics ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1062
Author(s):  
Annamaria Siggillino ◽  
Paola Ulivi ◽  
Luigi Pasini ◽  
Maria Sole Reda ◽  
Elisa Chiadini ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Growth Factor Receptor ◽  
Digital Pcr ◽  
Egfr Mutations ◽  
Amplification Refractory Mutation System ◽  
Clinical Method ◽  
Thermo Fisher Scientific ◽  
Exon 19 Deletion ◽  
Nsclc Patients ◽  
Tumor Dna

Analysis of circulating cell-free tumor DNA (cftDNA) has emerged as a specific and sensitive blood-based approach to detect epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC) patients. Still, there is some debate on what should be the preferential clinical method for plasma-derived cftDNA analysis. We tested 31 NSCLC patients treated with anti-EGFR tyrosine kinase inhibitors (TKIs), at baseline and serially during therapy, by comparing three methodologies in detecting EGFR mutations (L858R, exon 19 deletion, and T790M) from plasma: scorpions-amplification refractory mutation system (ARMS) methodology by using EGFR Plasma RGQ PCR Kit-QIAGEN, peptide nucleic acid (PNA) clamp and PANA RealTyper integration by using PNAClamp EGFR-PANAGENE, and digital real time PCR by using QuantStudio 3D Digital PCR System-Thermo Fisher Scientific. Specificity was 100% for all three mutations, independently from the platform used. The sensitivity for L858R (42.86%) and T790M (100%) did not change based on the method, while the sensitivity for Del 19 differed markedly (Scorpion-ARMS 45%, PNAClamp 75%, and Digital PCR 85%). The detection rate was also higher (94.23%) as measured by Digital PCR, and when we monitored the evolution of EGFR mutations over time, it evidenced the extreme inter-patient heterogeneity in terms of levels of circulating mutated copies. In our study, Digital PCR showed the best correlation with tissue biopsy and the highest sensitivity to attain the potential clinical utility of monitoring plasma levels of EGFR mutations.

Sensitivity of TargetSelector in clinical experience in ctDNA profiling of NSCLC 2000 cases.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23033-e23033
Author(s):  
Veena M. Singh ◽  
Anthony J. Daher ◽  
Jeffery J. Chen ◽  
Lyle Arnold ◽  
Cecile Rose T. Vibat
Keyword(s):  
Kinase Inhibitors ◽  
Acquired Resistance ◽  
Growth Factor Receptor ◽  
Egfr Mutations ◽  
Wild Type ◽  
T790m Mutation ◽  
Blood Samples ◽  
Copy Numbers ◽  
Activating Mutation ◽  
Nsclc Patients

e23033 Background: Targeted cancer therapy relies on identifying specific DNA mutations from a patient’s tumor. Tyrosine kinase inhibitors (TKIs) tend to be effective for non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) activating mutations, of which exon 19 deletions (Del19) and L858R are most common. Acquired resistance to TKI therapy is associated with a T790M mutation. Standard biomarker analyses may not reflect tumor heterogeneity; they entail tissue biopsies often with surgical complications. To address these limitations, Biocept developed a minimally invasive method to characterize cancer biomarkers in blood. Biocept's proprietary TargetSelector assays selectively amplify relevant mutations from circulating tumor DNA (ctDNA). Clinical validations demonstrated high concordances between molecular tests in blood vs tissue. As further validation, EGFR mutation detection frequencies were compared to US averages (mycancergenome.org). Here we analyze 2000 blood samples received at Biocept from 1Mar 2016 to 4Jan 2017 from late stage NSCLC patients. Methods: Blood was collected in Biocept OncoCEE BCT validated to preserve DNA ≤ 8 days. TargetSelector was used to detect ctDNA L858R, Del19 and T790M.EGFR allele copy numbers for wild type and each mutant were calculated. The prevalence of each mutation was compared to US averages. Results: Del19, L858R, and T790M mutations were detected in 12.9%, 8.5%, and 9.9% of the analyzed blood samples, respectively. This is concordant with US averages, which are 10% for each mutation. Median copy numbers/ml of blood were 30 for Del19 (range: 1 – 91974), 15 for L858R (range: 1 – 91200), and 10 for T790M (range: 1 – 137360). The median wild type EGFR copy number detected/ml blood was 2304 (range: 8 – 2498725). In ~80% of T790M cases, ≥ 1 concomitant activating mutation was detected. Conclusions: Biocept's TargetSelector detects EGFR mutations (Del19, L585R, and T790M) at a very high level of sensitivity down to 1 mutant copy/ml in advanced NSCLC patients at frequencies consistent with cited US rates. Moreover, the underlying activating mutation was detected in ~80% of T790M cases.

A novel mutant-enriched liquidchip for detection of circulating EGFR mutations in advanced non-small cell lung cancer patients

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14526-e14526
Author(s):  
S. Lu ◽  
H. Yang ◽  
X. Ye ◽  
X. Xu ◽  
Z. Li ◽  
...  
Keyword(s):  
Egfr Mutation ◽  
Direct Sequencing ◽  
Small Cell ◽  
Egfr Mutations ◽  
Wild Type ◽  
Small Cell Lung ◽  
Exon 19 Deletion ◽  
L858r Mutation ◽  
Nsclc Patients ◽  
Exon 19

e14526 Background: We developed a novel technology, Mutant-enriched liquidchip (MEL), which integrates the sensitive mutant enriched PCR and quantitative high throughput liquidchip (suspension array), to detect circulating EGFR mutations (Exon 19 deletion and exon 21 L858R mutation) in patients with advanced non-small cell lung cancer (NSCLC). Methods: To enrich mutant EGFR, a unique restriction site is introduced into the mutation alleles so that the wild type sequence can be selectively removed by restriction digestion, and the undigested mutated DNA is amplified by PCR. The product is then hybridized to complementary probes (including 15 types of exon 19 deletion and exon 21 L858R mutation) which had been conjugated to beads coding with different fluorescent dye, followed by measuring through Luminex 200 system. Plasmid DNA mixture with different EGFR genotypes was applied to determine the sensitivity and accuracy of MEL. Afterwards, the MEL was validated in 49 patients whose EGFR genotypes of tissue specimen had been tested with direct sequencing The circulating genomic DNA was obtained from serum sample of other 201 Chinese stage IIIB or IV NSCLC patients without EGFR-TKI administration, and the EGFR mutation status was analyzed by using of MEL. Results: The results shows that MEL is capable of detecting as few as 20 copies of mutant EGFR alleles with a sensitivity limit of at least mutant/wild-type ratio of 0.1%. It also shows that MEL can not only confirm EGFR mutations status in tissue specimens already known by direct sequencing (13/49), but also detect mutations in some of those showing wild type by sequencing (16/49). Overall, 54% of patients had circulating EGFR mutation. 34% of patients had an exon 19 deletion and 29.6% had L858R. 63.1% of mutations were found in females and 67.6% in never-smokers. Conclusions: This novel MEL method allows for highly sensitive and reproducible detection of human somatic mutations in heterogeneous specimens, and could be applicable to test EGFR mutations non-invasively in advanced NSCLC patients for predicting response to targeted therapy. No significant financial relationships to disclose.

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.

Detection of the JAK2 V617F Mutation by Real Time PCR in Granulocytes and Platelets of ET Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4877-4877
Author(s):  
Beatriz Bellosillo ◽  
Eva Gimeno ◽  
Raquel Longaron ◽  
Lourdes Florensa ◽  
Antonio Salar ◽  
...  
Keyword(s):  
Real Time ◽  
Direct Sequencing ◽  
Specific Treatment ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Wild Type ◽  
Rt Pcr ◽  
Jak2 Mutation ◽  
Allele Specific ◽  
V617f Mutation

Abstract Introduction. The JAK2 V617F mutation has been detected in 23%–57% of ET patients by direct sequencing or allele-specific (AS) PCR. It remains unknown, however, if the mutation detected in the granulocyte population, may be equally detected in platelets from these patients. Objective. To compare the detection of the JAK2V617F mutation in granulocytes and platelets from ET patients by real time AS RT-PCR. Patients and methods. Platelets and granulocytes from 50 ET patients from a single institution were studied. Patients were diagnosed according to the WHO criteria. At the time when JAK2 mutation was analyzed 16/50 patients were receiving platelet-lowering therapy ± ASA, 14/50 patients only received ASA and 20/50 received no specific treatment. JAK2 mutation was analyzed by real-time AS RT-PCR with probes specific for the mutated and the wild type form. Results. The V617F JAK2 mutation was detected in 18 out of 50 patients in both granulocytes and platelets by real time AS RT-PCR, and was negative in both cell populations in the remaining 32 patients. In the V617F JAK2 positive cases, the mean Ct(V617FJAK2)/Ct(wild type JAK2) ratio was 1.074±0.062 for granulocytes and 1.038±0.039 for platelets (p=0.048). These values corresponded to a 17.79 ±7.4% of mutated population when granulocytes were analyzed, whereas, a significantly higher percentage of mutated population was observed, 23.45±7.78 %, when platelets were analyzed (p=0.032). Conclusions. The results of V617FJAK2 mutation detection by AS RT-PCR were the same in granulocytes and platelets (either positive or negative). The percentage of clonal population detected in ET patients was significantly higher in platelets than in granulocytes.

Mutational analysis of EGFR and KIT in thymic epithelial tumor

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 18049-18049
Author(s):  
K. Yoh ◽  
Y. Nishiwaki ◽  
G. Ishii ◽  
K. Goto ◽  
K. Kubota ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Thymic Carcinoma ◽  
Kinase Inhibitors ◽  
Mutational Analysis ◽  
Direct Sequencing ◽  
Egfr Mutations ◽  
Epithelial Tumor ◽  
Thymic Epithelial Tumor ◽  
Number Of Patients

18049 Background: Thymic epithelial tumor has been reported to express the epidermal growth factor receptor (EGFR), and thymic carcinoma has recently been described to have overexpression of KIT. We investigated the prevalence of EGFR and KIT mutations in patients with thymoma and thymic carcinoma to explore the potential for a targeted therapy with tyrosine kinase inhibitors. Methods: Genomic DNA was isolated from 41 paraffin-embedded tumor samples including 24 thymoma and 17 thymic carcinoma. EGFR mutations in exons 18, 19 and 21, and KIT mutations in exons 9, 11, 13 and 17, were analyzed by PCR and direct sequencing. Protein expressions of EGFR and KIT were also evaluated by immunohistochemistry. Results: We detected the EGFR mutation in 2 of 20 thymoma, but none of thymic carcinoma had mutation. All of the detected EGFR mutations were missense mutations in exon 21 (L858R and G863D, respectively). Expression of EGFR was seen in 71% of thymoma and 53% of thymic carcinoma. On mutational analysis of KIT, only one thymic carcinoma displayed a missense mutation in exon 11 (L576P). Expression of KIT was observed in 88% of thymic carcinoma and 0% of thymoma. Conclusions: Our findings indicate that a small number of patients with thymic epithelial tumor exhibit somatic mutations of EGFR or KIT although expressions of EGFR or KIT are present frequently in thymic epithelial tumor. Further investigations are warranted to determine their susceptibility to tyrosine kinase inhibitors in the treatment of thymoma and thymic carcinoma with EGFR or KIT mutations. No significant financial relationships to disclose.

Validation study of direct sequencing and PCR-invader for detecting EGFR mutations in non-small cell lung cancer (NSCLC)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 8094-8094
Author(s):  
Y. Naito ◽  
K. Goto ◽  
H. Kenmotsu ◽  
Y. Nishiwaki ◽  
K. Kubota ◽  
...  
Keyword(s):  
Egfr Mutation ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
The Other ◽  
Egfr Mutations ◽  
Wild Type ◽  
Highly Sensitive ◽  
Formalin Fixed Paraffin Embedded ◽  
Exon 19 Deletion ◽  
Exon 19

8094 Background: EGFR mutation is both a predictive and prognostic factor for NSCLC treated with EGFR-TKI. Although new, highly sensitive methods for detecting EGFR mutations are currently available, these methods have not been validated. Methods: To validate direct sequencing and PCR-invader for detecting EGFR mutation, we analyzed 124 NSCLC by both methods concomitantly. Tumor tissues were obtained by surgical resection. Formalin-fixed paraffin-embedded specimens were prepared to analyze EGFR mutation. In direct sequencing, Exon 18, 19, and 21 of the EGFR gene were amplified, and PCR amplification products were sequenced directly (Mitsubishi Chemical Medience Corporation). PCR-invader was performed using the invasive cleavage of probe oligonucleotides to detect 10 mutations including Exon 18, 19, 20, 21 (BML incorporation). Results: EGFR mutations were detected in 51 patients (41%) by direct sequencing and 56 (45%) by PCR-invader. Discordance between two methods was found in 12 patients (10%). Exon 19 deletion was detected in 18 and 22 patients respectively. Exon 21 L858R was detected in 30 and 32 patients respectively. Each mutation in exon 19 deletions or L858R detected by direct sequencing could also be identified by PCR-invader. Overall 45 mutations were concordant by both methods. In two patients who received gefitinib, one patient with wild type by both methods did not respond to gefitinib. On the other hand, the other patient expressing Exon 19 deletion by PCR-invader but regarded as wild type by direct sequencing responded to gefitinib monotherapy. Conclusions: Discrepancy between two methods for detecting EGFR mutation was demonstrated and PCR-invader seems to be more sensitive. Further investigation including other highly sensitive methods is currently underway. No significant financial relationships to disclose.

Assessment of EGFR mutation status in matched plasma and tumor tissue of NSCLC patients.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e20032-e20032
Author(s):  
Qin Feng
Keyword(s):  
Tumor Tissue ◽  
Egfr Mutation ◽  
Circulating Tumor Dna ◽  
Egfr Mutations ◽  
Amplification Refractory Mutation System ◽  
Circulating Dna ◽  
Mutation Status ◽  
Nsclc Patients ◽  
Tumor Dna ◽  
Egfr Tkis

e20032 Background: Tumor tissue is currently used for EGFR testing non-small cell lung cancer (NSCLC) patients, but the detection of circulating tumor DNA (ctDNA) is being actively investigated as a new method for the detection and longitudinal monitoring of actionable mutations in plasma samples. Around 30% patients with EGFR mutation presented inconsistent status of EGFR mutation between in tissues and plasma. We compared EGFR mutation detection in circulating tumor DNA from blood to that in matched tissue. Methods: EGFR mutation status were assessed by the Human EGFR Gene Mutations Detection Kit (Beijing ACCB Biotech Ltd.) both in tissue and plasma. Retrospective analysis to evaluate the concordance of tissue and plasma EGFR determination for assessing eligibility for EGFR-TKIs therapy in NSCLC patients. 10 mL tubes of blood were collected from patients who never had been treated by EGFR TKI, and plasma circulating tumor DNA were extracted from plasma by Biomark Circulating DNA Kit. Qubit2.0 Fluorometer was used to make plasma circulating DNA tumor quantitation. The concentration of final DNA sample is ≦2ng/μl. Results: A total of 224 NSCLC patients were detected by Amplification Refractory Mutation System (ARMS), with 92 tissue positive and 49 blood positive. Results showed 53.3% sensitivity in overall samples, but 81.4% sensitivity in ⅢB~Ⅲ patients. The specificity is 100%. Conclusions: The high sensitivity and specificity between tissue and plasma EGFR determination supports the blood-based EGFR mutation testing to determinate the eligibility of NSCLC patients for EGFR-TKIs treatment, especialy in ⅢB~Ⅲ NSCLC patients. Blood, in particular plasma, is a good screening substitute when tumor tissue is absent or insufficient for testing EGFR mutations to guide EGFR TKIs treatment in patients with NSCLC. EGFR mutation positivity in blood could be used to recommend EGFR TKIs treatment, but the blood negativity should be confirmed with other sample, biopsy tissue, pleural effusion, etc..

Prospective study for usefulness of circulating-free DNA on prediction of third generation EGFR tyrosine kinase inhibitors.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e21510-e21510
Author(s):  
Hironori Yoshida ◽  
Chiho Nakashima ◽  
Naohisa Matsumoto ◽  
Kentaro Iwanaga ◽  
Noriyuki Ebi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Disease Progression ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Egfr Mutations ◽  
Plasma Samples ◽  
Egfr Tyrosine Kinase ◽  
Egfr Tyrosine Kinase Inhibitors ◽  
Nsclc Patients ◽  
Egfr T790m

e21510 Background: Most non-small lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations develop resistance when exposed to EGFR-tyrosine kinase inhibitors (TKIs). T790M develops in about half of patients treated with TKI and can be detected by tumor tissue and cfDNA hotspot tests. However, co-occurring mutations at other loci may impact efficacy. We conducted a prospective, multi-center, observational study to assess the detection rates and predictive values of plasma-based EGFR T790M detection methods for Japanese NSCLC patients treated with osimertinib. Methods: NSCLC patients with tumor EGFR mutations and disease progression after treatment with 1st- or 2nd-generation EGFR-TKI were enrolled. Plasma was collected at the time of clinical disease progression, before osimertinib treatment. The collected plasma was tested for EGFR T90M by in-house plasma MBP-QP and ddPCR assays and compared to clinically tested cobas (Roche) results (including tissue, plasma). The primary endpoint was to demonstrate comparability of our MBP-QP system to cobas using plasma-based EGFR T790M detection to predict the therapeuitic effect of osimertinib via objective response rate (ORR) and disease control rate (DCR). As an exploratory analysis, we used Guardant360 to retrospectively test available banked plasma samples collected describe time points. Results: From Feb 2017 to Jan 2019, 145 patients enrolled. T790M was detected by cobas in 57 cases (44 tissue, 16 plasma, 3 both). ORR and DCR in plasma cobas-positive cases were 62.5% and 81.3%, respectively. MBP-QP found T790M in 9 patients with ORR and DCR 66.7% and 77.8%. ddPCR found 17 cases with ORR and DCR 70.6% and 82.4%. ORR was not correlated to AF. In plasma samples from 54 patients, Guardant360 detected T790M in 57%. Co-occurring alterations such as amplification or minor mutations in EGFR or other genes such as TP53 did not impact ORR, but in the group with poor response to osimertinib, the number of detected gene alterations tended to be large. Two patients with small cell carcinoma transformation had RB1 mutations and MYC amplification. Conclusions: Regardless of the test system, the detection of T790M could predict a good therapeutic effect of osimertinib, but there was no difference in response to osimertinib depending on EGFR T790M AF. Compared to single-gene assessment of EGFR, NGS of cfDNA may be useful for guiding treatment decisions for patients with TKI-resistant NSCLC. Clinical trial information: UMIN000025930.

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