A Multiplex PCR-Based Assay for Detection of Plasma Cell Free DNA Integrity as a Marker of Bladder Cancer

Abstract

Background: Cell free DNA (cfDNA) analysis in patient plasma has been suggested for screening of different cancers. Healthy cells may shed more degraded DNA than non-apoptotic tumor cells. Objectives: Our goal was to check whether DNA fragments extracted from patient’s plasma with bladder cancer (BC) had higher integrity than DNA purified from plasma of healthy controls. Methods: cfDNA was extracted from the plasma of a healthy control group and patients with BC. The association between clinical status and length of DNA fragments was examined. Plasma cfDNA integrity was examined using multiplex PCR with specific primers producing progressively long PCR fragments (200, 400 & 800-bp). Length and concentration of PCR amplicons obtained from BC patients and healthy individuals were compared. Results: Using a multiplex PCR assay, the used p53 primers efficiently amplified the increasingly long fragments. High molecular weight DNA fragments (400 & 800-bp) in the plasma cfDNA are associated with the presence of cancer. Moreover, elevation of the 200-bp fragment concentration in the plasma cfDNA is significantly (p < 0.002) associated with the presence of cancer. Conclusions: The detected long DNA fragments in plasma cfDNA is associated with BC indicating that the multiplex PCR assay of plasma cfDNA integrity could be a useful future marker for the detection of BC.