Prevalence and clinical association of gene mutations through multiplex mutation testing in patients with NSCLC: results from the ETOP Lungscape Project.

Background: Reported prevalence of driver gene mutations in non-small-cell lung cancer (NSCLC) is highly variable and clinical correlations are emerging. Using NSCLC biomaterial and clinical data from the European Thoracic Oncology Platform Lungscape iBiobank, we explore the epidemiology of mutations and association to clinicopathologic features and patient outcome (relapse-free survival, time-to-relapse, overall survival). Methods: Clinically annotated, resected stage I-III NSCLC FFPE tissue was assessed for gene mutation using a microfluidics-based multiplex PCR platform. Mutant-allele detection sensitivity is >1% for most of the ∼150 (13 genes) mutations covered in the multiplex test. Results: Multiplex testing has been carried out in 2063 (76.2%) of the 2709 Lungscape cases (median follow-up 4.8 years). FFPE samples mostly date from 2005 to 2008, yet recently extracted DNA quality and quantity was generally good. Average DNA yield/case was 2.63 µg; 38 cases (1.4%) failed QC and were excluded from study; 95.1% of included cases allowed the complete panel of mutations to be tested. Most common were KRAS, MET, EGFR and PIK3CA mutations with overall prevalence of 23.0%, 6.8%, 5.4% and 4.9%, respectively. KRAS and EGFR mutations were significantly more frequent in adenocarcinomas: PIK3CA in squamous cell carcinomas. MET mutation prevalence did not differ between histology groups. EGFR mutations were found predominantly in never smokers; KRAS in current/former smokers. For all the above mutations, there was no difference in outcome between mutated and non-mutated cases. Conclusion: Archival FFPE NSCLC material is adequate for multiplex mutation analysis. In this large, predominantly European, clinically annotated stage I-III NSCLC cohort, none of the mutations characterized showed prognostic significance.

Large scale, prospective screening of EGFR mutations in the blood of advanced NSCLC patients to guide treatment decisions.

BACKGROUND: In a significant percentage of advanced non-small-cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses. We prospectively analyzed if circulating-free DNA (cfDNA) purified from blood can be used as a surrogate in this setting to select patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). PATIENTS AND METHODS: Blood samples were collected in 119 hospitals from 1138 advanced NSCLC patients at presentation (n = 1033) or at progression to EGFR-TKIs (n = 105) with no biopsy or insufficient tumor tissue. Serum and plasma were sent to a central laboratory, cfDNA purified and EGFR mutations analyzed and quantified using a real-time PCR assay. Response data from a subset of patients (n = 18) were retrospectively collected. RESULTS: Of 1033 NSCLC patients at presentation, 1026 were assessable; with a prevalence of males and former or current smokers. Sensitizing mutations were found in the cfDNA of 113 patients (11%); with a majority of females, never smokers and exon 19 deletions. Thirty-one patients were positive only in plasma and 11 in serum alone and mutation load was higher in plasma and in cases with exon 19 deletions. More than 50% of samples had <10 pg mutated genomes/µl with allelic fractions below 0.25%. Patients treated first line with TKIs based exclusively on EGFR positivity in blood had an ORR of 72% and a median PFS of 11 months. Of 105 patients screened after progression to EGFR-TKIs, sensitizing mutations were found in 56.2% and the p.T790M resistance mutation in 35.2%. CONCLUSIONS: Large-scale EGFR testing in the blood of unselected advanced NSCLC patients is feasible and can be used to select patients for targeted therapy when testing cannot be done in tissue. The characteristics and clinical outcomes to TKI treatment of the EGFR-mutated patients identified are undistinguishable from those positive in tumor.

Endobronchial ultrasound-guided transbronchial needle aspiration for identifying EGFR mutations.

The presence of somatic mutations of the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene in patients with advanced nonsmall cell lung cancer (NSCLC) correlates with a good response to tyrosine kinase inhibitors. The usefulness of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for the detection of EGFR mutations in cells recovered from malignant mediastinal nodes in patients with NSCLC was assessed. All patients with lung adenocarcinoma or unspecified NSCLC referred for staging with EBUS-TBNA were included. Nodes with a short-axis diameter of >5 mm were sampled, and genomic DNA from metastatic tumour cells was obtained for analysis of exons 19 and 21. The impact of sampling on management was assessed. EGFR gene analysis of the EBUS-TBNA sample was feasible in 26 (72.2%) out of the 36 patients with lymph node metastasis. Somatic mutations of the EGFR gene were detected in tissue obtained through EBUS-TBNA in two (10%) out of 20 patients with metastasic lung adenocarcinoma. Malignant tissue samples obtained by EBUS-TBNA from patients with nodal metastasis of NSCLC are suitable for the detection of EGFR mutations in most cases, and this technique demonstrates mutated neoplastic cells in a tenth of patients with adenocarcinoma.