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Among advanced non-small cell lung cancer (NSCLC) patients with an acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI), about 50% carry the T790M mutation, but this frequency in EGFR-TKI-naïve patients and dynamic change during therapy remains unclear. This study investigated the quantification and dynamic change of T790M mutation in plasma cell-free DNA (cf-DNA) of advanced NSCLC patients to assess the clinical outcomes of EGFR-TKI therapy.We retrospectively investigated 135 patients with advanced NSCLC who obtained progression-free survival (PFS) after EGFR-TKI for >6 months for their EGFR sensitive mutations and T790M mutation in matched pre- and post-TKI plasma samples, using denaturing high-performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS), and digital-PCR (D-PCR). Real-time PCR was performed to measure c-MET amplification.Detection limit of D-PCR in assessing the T790M mutation was approximately 0.03%. D-PCR identified higher frequency of T790M than ARMS in pre-TKI (31.3% vs. 5.5%) and post-TKI (43.0% vs. 25.2%) plasma samples. Patients with pre-TKI T790M showed inferior PFS (8.9 vs. 12.1 months, p = 0.007) and overall survival (OS, 19.3 vs. 31.9 months, p = 0.001) compared with those without T790M. In patients harboring EGFR sensitive mutation, high quantities of pre-TKI T790M predicted poorer PFS (p = 0.001) on EGFR-TKI than low ones. Moreover, patients who experienced increased quantity of T790M during EGFR-TKI treatment showed superior PFS and OS compared with those with decreased changes (p = 0.044 and p = 0.015, respectively).Qualitative and quantitative T790M in plasma cf-DNA by D-PCR provided a non-invasive and sensitive assay to predict EGFR-TKI prognosis.

PURPOSE: This study evaluated occurrence and potential clinical significance of intratumoral EGFR mutational heterogeneity in Chinese patients with non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Eighty-five stage IIIa-IV NSCLC patients who had undergone palliative surgical resection were included in this study. Of these, 45 patients carried EGFR mutations (group-M) and 40 patients were wild-type (group-W). Each tumor sample was microdissected to yield 28-34 tumor foci and Intratumoral EGFR mutation were determined using Denaturing High Performance Liquid Chromatography (DHPLC) and Amplification Refractory Mutation System (ARMS). EGFR copy numbers were measured using fluorescence in situ hybridization (FISH). RESULTS: Microdissection yielded 1,431 tumor foci from EGFR mutant patients (group-M) and 1,238 foci from wild-type patients (group-W). The EGFR mutant frequencies in group-M were 80.6% (1,154/1,431) and 87.1% (1,247/1,431) using DHPLC and ARMS, respectively. A combination of EGFR-mutated and wild-type cells was detected in 32.9% (28/85) of samples by DHPLC and 28.2% (24/85) by ARMS, supporting the occurrence of intratumoral heterogeneity. Thirty-one patients (36.5%) were identified as EGFR FISH-positive. Patients harboring intratumoral mutational heterogeneity possessed lower EGFR copy numbers than those tumors contained mutant cells alone (16.7% vs. 71.0%, P

Genetic aberrancies within epidermal growth factor receptor (EGFR) pathway are associated with therapeutic outcomes of EGFR-tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). However, the impact of chemotherapy on EGFR-related genes alterations has not been defined in NSCLC. Our study aims to investigate the impact of neoadjuvant chemotherapy (Neoadj-Chemo) on EGFR activating mutations and associated EGFR-TKIs resistance-related genes.Matched tumor samples were obtained retrospectively from 66 NSCLC patients (stages IIb-IIIb) corresponding to pre- and post- Neoadj-Chemo. EGFR mutations were detected by denaturing high performance liquid chromatography (DHPLC) and confirmed by Amplification Refractory Mutation System technology (ARMS), KRAS mutations, T790M mutation and c-MET amplification were identified using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), ARMS, and real-time PCR, respectively.Before Neoadj-Chemo, EGFR mutations were identified in 33.3% (22/66) of NSCLC patients. Only 18.2% (12/66) of patients carried EGFR mutations after Neoadj-Chemo (p = 0.013). The median peak value of EGFR 19 exon mutations decreased non-significantly after Neoadj-Chemo. KRAS mutation rate decreased from 4.6% (3/66) to 3.0% (2/66) with Neoadj-Chemo. Although the overall percentage of patients exhibiting c-MET amplifications (6.1% [4/66]) did not change with Neoadj-Chemo, two patients transitioned from negative to positive c-MET amplification, and two patients reversed these changes post-Neoadj-Chemo. T790M mutations were absent from all samples.Neoadjuvant chemotherapy tends to decrease the mutation frequency of EGFR mutation and downstream genes, which suggests that real-time samples analysis for genetic aberrancies within EGFR pathways have important value to delineate specific patient populations and facilitate individualized treatment.