Abstract 1522: Direct quantitative PCR of LINE repeats to quantify CFC-DNA in serum

Background: Cell free circulating (cfc)-HMW DNA is a proposed diagnostic and prognostic biomarker for malignant tumors. Apoptotic DNA (less than 200 bp) is present in the circulation of normal healthy individuals whereas DNA released from dead cancer cells produces HMW DNA (greater than 400 bp). We developed LINE assay for direct serum/plasma quantitation of cfc-DNA concentration and integrity. LINE (Long Interspersed Elements) is highly conserved repetitive element in human genome with ∼500,000 copies/g.e. These large numbers of copies allow cfc DNA quantitation from small sample volume. Methods: Sera of 15-30 CRC and Lung cancer patients (early and late stage) from retrospective studies, 5 CRC patients (late stage) from prospective studies along with healthy normals were characterized for cfc-DNA quantity and integrity by LINE Assay. All the samples were prescreened for hemolysis by Potassium assay. Quantitative real-time PCR of LINE repeats was performed with 2 sets of primers (less than 200 bp and greater than 400 bp) amplifying different lengths of DNA. We used 10ul serum directly as a template for qPCR without DNA purification. DNA integrity was determined as a ratio of concentration of longer over shorter LINE fragments. Results: LINE Assay has a detection limit of 0.08 pg of DNA. Both CRC and lung cancer patients from Retrospective studies have significantly high HMW DNA concentration and DNA integrity as compared to healthy normals (statistically significant at p-value less than 0.05). ROC curves of HMW DNA for detecting early and late stage CRC and Lung have areas under the curves of greater than 0.80. High concentration of HMW DNA along with high DNA integrity also follows the trend of treatment of CRC patients from prospective studies. Conclusions: LINE Assay is a robust, highly sensitive, and high-throughput method to measure different sizes of cfc-DNA. Our experiments demonstrate that this sizing capability helps to calculate DNA integrity which is a strong indicator for treatment monitoring in prospective studies and cancer staging in solid tumors. The high sensitivity of the LINE assay helps in direct DNA quantitation in serum. Citation Format: David Jean Charlot, Megha Bhalla, Raj Krishnan, Gene Tu. Direct quantitative PCR of LINE repeats to quantify CFC-DNA in serum. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1522. doi:10.1158/1538-7445.AM2014-1522