A Pilot Study to Evaluate Early Predictive Value of Thorax Perfusion-CT in Advanced NSCLC.

BACKGROUND: The role of perfusion computed tomography (pCT) in detecting changes in tumor vascularization as part of a response to antiangiogenic therapy in non-small cell lung cancer (NSCLC) remains unclear. METHODS: In this prospective pilot study (IMPACT trial, NCT02316327), we aimed to determine the ability of pCT to detect early changes in blood flow (BF), blood volume (BV), and permeability (PMB), and to explore whether these changes could predict the response at day +42 in patients with advanced, treatment-naive, non-squamous NSCLC treated with cisplatin and gemcitabine plus bevacizumab. RESULTS: All of the perfusion parameters showed a consistent decrease during the course of treatment. The BV difference between baseline and early assessment was significant (p = 0.013), whereas all perfusion parameters showed significant differences between baseline and day +42 (p = 0.003, p = 0.049, and p = 0.002, respectively). Among the 16 patients evaluable for efficacy, a significant decline in BV at day +7 from baseline was observed in tumors with no response (p = 0.0418). CONCLUSIONS: Our results confirm that pCT can capture early changes in tumor vasculature. A substantial early decline of BV from baseline might identify tumors less likely responsive to antiangiogenic-drugs.

Technical Evaluation of the COBAS EGFR Semiquantitative Index (SQI) for Plasma cfDNA Testing in NSCLC Patients with EGFR Exon 19 Deletions.

The cobas® EGFR Test provides a semiquantitative index (SQI) that reflects the proportion of mutated versus wild-type copies of the EGFR gene in plasma. The significance of SQI as an indirect measure of the variant allele frequency (VAF) or mutated copies/mL remains unclear. The aim of this study was to evaluate the correlation of SQI with the VAF and the number of mutated copies/mL obtained by a digital droplet PCR (ddPCR) test in NSCLC samples. The study included 118 plasma samples from a retrospective cohort of 25 stage IV adenocarcinoma patients with EGFR exon 19 deletions (Ex19Del), obtained before and during tyrosine kinase inhibitor (TKI) treatment. Both SQI and VAF and SQI and mutated copies/mL showed the same significant correlation (r2 = 0.79, p < 0.00001) across the whole study cohort. We found better correlation in samples collected at the baseline between SQI and VAF (r2 = 0.94, p < 0.00001) and SQI and mutated copies/mL (r2 = 0.97, p < 0.00001) compared to samples collected during TKI treatment: r2 = 0.76; p < 0.00001 for SQI and VAF and r2 = 0.75; p < 0.00001 for SQI and mutated copies/mL. The study indicates that SQI is a robust quantitative indirect measure of VAF and the number of mutated copies/mL in plasma from patients with an EGFR Ex19Del mutation. Further studies are desirable to assess the SQI cut-off values related to the clinical status of the patient.

Molecular characterization of advanced non-small cell lung cancer patients by cfDNA analysis: experience from routine laboratory practice.

BACKGROUND: Analysis of circulating free DNA (cfDNA) by the real-time PCR cobas® EGFR Mutation Test v2 (cobas® EGFR Test) is a diagnostic approach used in clinical practice for the characterization of advanced non-small cell lung cancer (NSCLC) patients. The test additionally outputs a semiquantitative index (SQI) which reflects the proportion of mutated versus wild-type copies of the EGFR gene in cfDNA with potential use as a biomarker. CfDNA concentration and cfDNA fragmentation pattern have also shown potential utility as biomarkers for cancer patients. We evaluated the implementation of EGFR testing and cfDNA related parameters in NSCLC patients in routine clinical setting as biomarkers for disease stage and diagnosis. METHODS: A prospective cohort of 173 locally advanced or metastatic NSCLC TKI-naïve patients analyzed by the cobas® EGFR Test were included in the study. Reproducibility of the test was assessed in 56 patients. The concentration of cfDNA and fragment size pattern was measured using fluorometry and microchip electrophoresis respectively. RESULTS: The test showed high diagnostic accuracy when compared to the gold standard of biopsy tumor tissue testing. The SQI value showed a moderate reproducibility (r2=0.70) and did not correlate with cfDNA concentration (r2=0.17, P=0.28) or disease stage (stage III patients SQI =9.1±3.1 and stage IV patients SQI =11.5±4.8, P=0.41). We found differences in SQI values according to the type of EGFR mutation (Ex19Del mutations, SQI =13.6; p.L858R, SQI =8.88; P=0.001). Stage IV patients had higher concentrations of cfDNA (P<0.0001) and higher fractions of cfDNA 100-250 base pairs (bp) fragments (P=0.01) compared to stage III patients. From the ROC curve analysis, cfDNA concentration showed higher AUC compared to cfDNA 100-250 bp fragments (0.86 vs. 0.71). We obtained a cut-off value for cfDNA concentration of 20.3 ng/mL with 72.3% sensitivity and 95% specificity for predicting disease stage in TKI-naïve advanced NSCLC patients. CONCLUSIONS: The study indicates that cfDNA analysis in plasma for EGFR testing by RT-PCR is an accurate and fast method to initially stratify NSCLC patients in a real-world clinical setting. However, the SQI has limited clinical value. The cfDNA concentration and fragmentation pattern have clear potential clinical utility for tumor staging in NSCLC patients.

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

Prospective Evaluation of Single Nucleotide Variants by Two Different Technologies in Paraffin Samples of Advanced Non-Small Cell Lung Cancer Patients.

Targeted therapies are a new paradigm in lung cancer management. Next-generation sequencing (NGS) techniques have allowed for simultaneous testing of several genes in a rapid and efficient manner; however, there are other molecular diagnostic tools such as the nCounter® Vantage 3D single nucleotide variants (SNVs) solid tumour panel which also offer important benefits regarding sample input and time-to-response, making them very attractive for daily clinical use. This study aimed to test the performance of the Vantage panel in the routine workup of advanced non-squamous non-small cell lung cancer (NSCLC) patients and to validate and compare its outputs with the Oncomine Solid Tumor (OST) panel DNA kit, the standard technique in our institution. Two parallel multiplexed approaches were performed based on DNA NGS and direct digital detection of DNA with nCounter® technology to evaluate SNVs. A total of 42 advanced non-squamous NSCLC patients were prospectively included in the study. Overall, 95% of samples were successfully characterized by both technologies. The Vantage panel accounted for a sensitivity of 95% and a specificity of 82%. In terms of predictive values, the probability of truly presenting the SNV variant when it is detected by the nCounter panel was 82%, whereas the probability of not presenting the SNV variant when it is not detected by the platform was 95%. Finally, Cohen's Kappa coefficient was 0.76, indicating a substantial correlation grade between OST and Vantage panels. Our results make nCounter an analytically sensitive, practical and cost-effective tool.

Usefulness of Two Independent DNA and RNA Tissue-Based Multiplex Assays for the Routine Care of Advanced NSCLC Patients.

Personalized medicine is nowadays a paradigm in lung cancer management, offering important benefits to patients. This study aimed to test the feasibility and utility of embedding two multiplexed genomic platforms as the routine workup of advanced non-squamous non-small cell lung cancer (NSCLC) patients. Two parallel multiplexed approaches were performed based on DNA sequencing and direct digital detection of RNA with nCounter® technology to evaluate gene mutations and fusions. The results were used to guide genotype-directed therapies and patient outcomes were collected. A total of 224 advanced non-squamous NSCLC patients were prospectively included in the study. Overall, 85% of samples were successfully characterized at DNA and RNA levels and oncogenic drivers were found in 68% of patients, with KRAS, EGFR, METΔex14, BRAF, and ALK being the most frequent (31%, 19%, 5%, 4%, and 4%, respectively). Among all patients with complete genotyping results and follow-up data (n = 156), the median overall survival (OS) was 1.90 years (confidence interval (CI) 95% 1.69-2.10) for individuals harbouring an actionable driver treated with a matched therapy, compared with 0.59 years (CI 95% 0.39-0.79) in those not eligible for any targeted therapy and 0.61 years (CI 95% 0.12-1.10) in patients with no drivers identified (p < 0.001). Integrating DNA and RNA multiplexing technologies into the routine molecular testing of advanced NSCLC patients is feasible and useful and highlights the necessity of widespread integrating comprehensive molecular diagnosis into lung cancer care.