Measurements of total DNA and methylated tumor suppressor genes in the plasma of patients with small cell lung cancer (SCLC)

Abstract

7162 Background: Measuring DNA in plasma using quantitative RT-PCR is a promising method for the detection and surveillance of lung cancer. Methylated gene levels may be more specific for the detection of cancer. Data exploring these methods in SCLC are lacking. Methods: We plan to enroll 50 patients with SCLC in a prospective study during which blood is tested for levels of total DNA and selected methylated tumor suppressor genes before, and up to 3 times during therapy. Blood samples are coincident with radiologic response assessments by RECIST. Blood is collected in sodium citrate, gel density gradient tubes and separated by centrifugation. Plasma is transferred to cryovials without disturbing the mononuclear layer, and stored at -80°C until analysis. DNA is extracted from a set plasma volume and treated with sodium metabisulfite solution which converts unmethylated cytosines to uracil. For methylation-specific real-time PCR, primers and probes specific to bisulfite-converted DNA in the promoter region of the methylated gene of interest are used, assuming the sequence is fully methylated. Methylation values are normalized to the level of an internal reference gene (β-Actin) in modified DNA. Results: Data from the first 10 patients are available. All patients had partial radiographic response to therapy. Total DNA was detectable in all patients at diagnosis (median 16.7, range 0.9–118.7 ng/2ml). Two patients had detectable methylated APC (mAPC), one patient had methylated p16, and two patients had levels of both methylated genes. These levels became undetectable after treatment in all patients. One patient demonstrated recurrence of detectable mAPC and went on to demonstrate disease progression within two months. Total DNA levels varied widely over the course of therapy. Conclusions: Initial results suggest that methylated tumor suppressor genes are detectable in patients with SCLC, and these levels may correlate with disease status. A larger sample size is needed to verify these early observations. Patient enrollment continues, and analyses of additional methylated genes and correlation with survival are 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 Response Genetics