Cell-free DNA (cfDNA) is considered as non-invasive method for cancer detection with high specificity and variable sensitivity. Mainstream cfDNA studies are based on qualified samples which is plasma kept at -80, with no cycle of re-thawing, and minimum volume of 1 ml. We aim to investigate feasibility of cfDNA detection in archival serum samples kept at -20 with several cycles of re-thawing and volume less than or equal to 0.7 ml.

Leftover sera from a population-based study of esophageal squamous cell carcinoma (ESCC) in Northern Iran- conducted 14 years ago- were used. Sera were applied for several serology assays and underwent between 4 and 6 cycles of re-thawing. TP53 mutation data from formalin-fixed paraffin-embedded ESCC tissues was available for one-third of the cases. We extracted cfDNA from 44 ESCC and 39 controls (matched for storage condition). 27 TP53 amplicons were deep sequenced on ion-torrent platform. Needlestack, a pipeline for calling variants with extremely low allelic fraction (AF) was applied. RVSB >0.85, Q-value<30, and minimum distance form highest AF <10 nt. were filtered.

We detected 20% of tumor tissue mutations concordantly in CfDNA of archival sera. After applying filtrations, 59 unique mutations were detected in all cfDNAs, out of them 24 were exclusively detected in ESCC cases and 29 found just in controls. None of the tumor mutations was detected among controls. non-germline cfDNA mutation was detected in both case and control which had significantly higher AF in case than control.

Applying our method, detection rate of concordant TP53 mutations in archived low- volume sera and cancerous tissue is comparable to results of the same gene in well-kept plasma. These results suggest the crucial role of bioinformatics pipeline in detectability of variants in cfDNA comparing to quality of sample. TP53 variants in healthy resident of ESCC endemic area warrants further investigation.