Heparinase enables reliable quantification of circulating tumor DNA from heparinized plasma samples by droplet digital PCR

Abstract

Background: Heparin is often used as a blood anticoagulant for tumor marker analysis but results in the inhibition of PCR detection of circulating tumor DNA (ctDNA), which has been deemed a potential "liquid biopsy". We aimed to evaluate the impact of heparinase addition on heparinized plasma samples to allow ctDNA analysis.

Methods: Plasma samples were collected in heparinized (n=194) and EDTA (n=8) tubes from hormone receptor-positive metastatic breast cancer (HR+MBC) (n=144) and pancreatic adenocarcinoma (PA) patients (n=50). Circulating ESR1 and KRAS mutations were detected with or without heparinase by digital PCR in HR+MBC and PA patients, respectively. Patients were classified into 2 subgroups i) inhibition, I+ and ii) no inhibition, I- based on a threshold of 200copies/μL for PCR inhibition by heparin.

Results: In the I+ subgroup (91/144 HR+MBC and 26/50 PA), heparinase treatment significantly improved PCR efficacy, enabling ctDNA detection in 22/91 and 13/26 patients. Moreover, comparable results for ctDNA detection (4/8) were obtained with heparinized and EDTA PA samples. In the I- subgroup, heparinase addition did not quantitatively and qualitatively alter ctDNA detection.

Conclusion: Heparinase addition removes the heparin inhibition and allows accurate ctDNA detection in heparinized samples. These findings could make the samples from heparinized blood suitable for ctDNA analysis.

Keywords: Cell-free DNA; Circulating tumor DNA; Droplet digital PCR; ESR1; Heparin; Heparinase; KRAS.