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

\ 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 200 copies/pL 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.