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Establishment of a Novel Method for Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance Mutations in Lung Cancer.

ABSTRACT: Drug resistance to targeted therapies occurs in lung cancer, and resistance mechanisms related to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are continuously being discovered. We aimed to establish a novel method for highly parallel multiplexed detection of genetic mutations related to EGFR TKI-resistant lung cancer using Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on the MassARRAY mass spectrometry platform.A review of the literature revealed 60 mutation hotspots in seven target genes (EGFR, KRAS, PIK3CA, BRAF, ERBB2, NRAS, and BIM) that are closely related to EGFR TKI resistance to lung cancer. A total of 183 primers comprised 61 paired forward and reverse amplification primers, and 61 matched extension primers were designed using Assay Design Software. The detection method was established by analyzing nine cell lines, and by comparison with LungCarta™ kit in ten lung cancer specimens. EGFR, KRAS, and BIM genes in all cell lines and clinical samples were subjected to Sanger sequencing for confirming reproducibility.Our data showed that designed panel was a high-throughput and robust tool, allowing genotyping for sixty hotspots in the same run. Moreover, it made efficient use of patient diagnostic samples for a more accurate EGFR TKIs resistance analysis. The proposed method could accurately detect mutations in lung cancer cell lines and clinical specimens, consistent with those obtained by the LungCarta™ kit and Sanger sequencing. We also established a method for detection of large-fragment deletions based on single-base extension technology of MassARRAY platform.We established an effective method for high-throughput detection of genetic mutations related to EGFR TKI resistance based on the MassARRAY platform, which could provide more accurate information for overcoming cancers with de novo or acquired resistance to EGFR-targeted therapies.

SUBMITTER: Tian HX

PROVIDER: S-EPMC5463475 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Establishment of a Novel Method for Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance Mutations in Lung Cancer.

Tian Hong-Xia HX Zhang Xu-Chao XC Wang Zhen Z Yang Jin-Ji JJ Guo Wei-Bang WB Chen Zhi-Hong ZH Wu Yi-Long YL

Chinese medical journal 20170601 12

<h4>Background</h4>Drug resistance to targeted therapies occurs in lung cancer, and resistance mechanisms related to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are continuously being discovered. We aimed to establish a novel method for highly parallel multiplexed detection of genetic mutations related to EGFR TKI-resistant lung cancer using Agena iPLEX chemistry and matrix-assisted laser desorption ionization time-of-flight analysis on the MassARRAY mass spectromet ...[more]

PMID: 28584208

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Project description:AimsEpidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.MethodsWe selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.ResultsIn the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC50 concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.ConclusionOur results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.

Project description:IntroductionTyrosine kinase inhibitors (TKIs) that target epidermal growth factor receptor (EGFR) mutations are commonly administered to EGFR-positive lung cancer patients. However, resistance to EGFR-TKIs (mostly gefitinib and erlotinib) is presently a significant problem. Limited studies have focused on an EGFR-TKI resistance-related gene signature (ERS) in lung adenocarcinoma (LUAD).MethodsGefitinib and erlotinib resistance-related genes were obtained through the differential analyses of three Gene Expression Omnibus datasets. These genes were investigated further in LUAD patients from The Cancer Genome Atlas (TCGA). Patients in the TCGA-LUAD cohort were split into two groups: one for training and one for testing. The training cohort was used to build the ERS, and the testing cohort was used to test it. GO and KEGG analyses were explored for the enriched pathways between the high-risk and low-risk groups. Various software, mainly CIBERSORT and ssGSEA, were used for immune infiltration profiles. Somatic mutation and drug sensitivity analyses were also explored.ResultsAn ERS based on five genes (FGD3, PCDH7, DEPDC1B, SATB2, and S100P) was constructed and validated using the TCGA-LUAD cohort, resulting in the significant stratification of LUAD patients into high-risk and low-risk groups. Multivariable Cox analyses confirmed that ERS had an independent prognostic value in LUAD. The pathway enrichment analyses showed that most of the genes that were different between the two risk groups were related to the immune system. Further immune infiltration results revealed that a lower immune infiltration score was observed in high-risk patients, and that various leukocytes were significantly related to the ERS. Importantly, samples from the high-risk group showed lower levels of PD-1, PD-L1, and CTLA-4, which are important biomarkers for immunotherapy responses. Patients in the high-risk group also had more gene mutation changes and were more sensitive to chemotherapy drugs like docetaxel and sorafenib. The ERS was also validated in the GSE30219, GSE11969 and GSE72094, and showed a favorable prognostic value for LUAD patients.DiscussionThe ERS established during this study was able to predict a poor prognosis for LUAD patients and had great potential for predicting drug responses.

Project description:The Lung Adjuvant Cisplatin Evaluation (LACE) meta-analysis and the meta-analysis of individual participant data reported by non-small cell lung cancer (NSCLC) Meta-analysis Collaborative Group in neo-adjuvant setting validated respectively that adjuvant and neoadjuvant chemotherapy would significantly improve overall survival (OS) and recurrence-free survival for resectable NSCLC. However, chemotherapy has reached a therapeutic plateau. It has been confirmed that epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeting therapy provides a dramatic response to patients with advanced EGFR-mutation positive NSCLC. Researchers have paid more attention to exploring applications of TKIs to early resectable NSCLCs. Several studies on adjuvant TKI treatment concluded its safety and feasibility. But there existed certain limitations of these studies as inference factors to interpret data accurately: the BR19 study recruited patients among which almost 52% had stage IB and only 15 (3.0%, 15/503) had been confirmed with EGFR-mutant type; retrospective studies performed at Memorial Sloan Kettering Cancer Center (MSKCC) selected EGFR mutant-type NSCLC patients but couldn't avoid inherent defects inside retrospective researches; the RADIANT study revised endpoints from targeting at EGFR immunohistochemistry (IHC)+ and/or fluorescence in situ hybridization (FISH)+ mutation to only EGFR IHC+ mutation, leading to selective bias; despite that the SELECT study validated efficacy of adjuvant TKI and second round of TKI after resistance occurred, a single-arm clinical trial is not that persuasive in the absence of comparison with chemotherapy. Taking all these limitations into account, CTONG1104 in China and IMPACT in Japan have been conducted and recruiting patients to offer higher level of evidences to explore efficacy of preoperative TKI therapy for early resectable EGFR mutation positive NSCLC patients (confirmed by pathological results of tumor tissue or lymph node biopsy). On the other hand, case reports and several phase II clinical trials with small sample size tried to elbow their way on respect of preoperative TKI treatment and advised that TKI tended to improve response rate. However, no data on survival rate was present. The first phase II study of biomarker-guided neoadjuvant therapy for stage IIIA-N2 NSCLC patients stratified by EGFR mutation status, sponsored by CSLC0702, showed erlotinib tended to improve response rate, but failed to show benefits of disease-free survival (DFS) or OS. Subsequently, CTONG1103 was designed to investigate efficacy of erlotinib vs. combination of gemcitabine/cisplatin (GC) as neoadjuvant treatment in stage IIIA-N2 NSCLC with sensitizing EGFR mutation in exon 19 or 21. All these ongoing trials should be worthy of our expect to provide convincing evidences for customized therapy for patients with resectable NSCLC.

Dataset Information

Establishment of a Novel Method for Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance Mutations in Lung Cancer.

Publications

Establishment of a Novel Method for Screening Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance Mutations in Lung Cancer.

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