Measurements of total DNA and methylated tumor suppressor genes in the plasma of patients with metastatic non-small cell lung cancer (NSCLC) before, and after chemotherapy, as potential biomarkers for response to treatment

Abstract

7203 Background: Measuring DNA in plasma using quantitative RT-PCR is a promising method for the detection and surveillance of NSCLC. Detection of methylated genes may be more specific than total DNA for the presence of cancer. Methods: We undertook a prospective trial measuring total DNA, and the levels of 5 commonly methylated tumor suppressor genes in the plasma of a planned 200 patients with metastatic NSCLC before, and up to 3 times during chemotherapy. Blood samples are coincident with radiologic response assessments by RECIST. Blood is collected in Na citrate, gel density gradient tubes spun at 1800 RCF for 20 min, plasma removed without disturbing the mononuclear layer, and frozen at -80°C. DNA is extracted from a fixed volume of plasma, and measured using quantitative PCR of the β-Actin gene. DNA is treated with sodium metabisulfite solution which converts unmethylated cytosines to uracil. For methylation-specific RT-PCR, primers and probes have been designed to amplify bisulfite-converted DNA in the promoter region of the methylated version of the gene of interest, assuming the DNA sequence is fully methylated, and values normalized to the level of β-Actin in modified DNA. Results: Data from the first 43 patients are available. In the initial experiment, methylated APC was detectable in 7 patients (16%, C.I. 7–31%), p16 in 6 (14%, 5–28%), and MGMT, DAPK, GSTP1 only rarely (1–2%). Total DNA was detectable in all patients at baseline (median 11.1, range 0.6 - 2030 ng/2ml). To date there are 33 patients with both DNA and radiologic response data (8 POD, 21 SD, 4 PR). To facilitate analysis, the RECIST category SD was subdivided into SD vs. MR (14 patients with ≥ 10% reduction in measurable disease). The median change in DNA concentration was -5 ng/2ml for PR/MR, + 45 for POD/SD (p= 0.04 by Krukal-Wallis test). Conclusions: Initial results find methylated tumor suppressor genes in plasma in the minority of patients. Total DNA was detectable in all patients, and changes appear to correlate with radiologic response. Enrollment in this trial is ongoing, with survival analysis planned. Supported by RO1-CA092315. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics (CEO), Response Genetics Response Genetics