Quantitative and Qualtitative Evaluation Of Circulating Cell-Free DNA As a Prognostic Biomarker In Hodgkin Lymphoma

Tumor tissue is known to release cell-free DNA, that can be detected in peripheral blood. Cell-free DNA (cfDNA) is significantly higher and more fragmented in plasma of cancer patients and levels have been reported to associate with prognosis. The aim of our study was to further explore cfDNA as a biomarker in Hodgkin lymphoma (HL) at diagnosis, during treatment and at follow-up. We studied cfDNA levels in pre-treatment plasma samples of 163 consecutive HL patients diagnosed at our Institution and 64 healthy volunteers. cfDNA was analyzed by quantitative PCR for the beta-globin gene. cfDNA at diagnosis was significantly increased in patients with HL when compared to controls (median, 21.83 vs 12.18 ng/ml; p<0.0001). Using the cfDNA levels of normal volunteers to determine the cut-off point for pathologically elevated cfDNA levels, cfDNA levels were elevated in 62 (38%) patients. Increased cfDNA at diagnosis was associated to parameters indicating an unfavorable prognosis as an IPS score>2 (p=0.0008). In a group of 31 patients and 19 controls, we also evaluated cfDNA fragmentation, amplifying fragments of different length. DNA integrity indices were calculated according to Mouliere et al, PLoS One 2011. At diagnosis, cfDNA was more fragmented in HL patients than in controls resulting in a lower DNA integrity index (median: 0.15 vs 0.22; p=0.016). However, cfDNA in HL patients resulted less fragmented when compared to cfDNA form patients with aggressive tumors like Burkitt lymphoma or solid tumors. Patients received treatment with ABVD (101 patients), BEACOPP (42 young patients with advanced HL) and other types of chemotherapy (17 patients). Seventy patients were also evaluated after two cycles of chemotherapy. cfDNA levels significantly decreased after 2 cycles of chemotherapy in patients with increased pre-treatment levels, while they remained in the normal range in patients with low cfDNA at diagnosis (ratio 0.32 and 1.44, respectively; p<0.0001). Patients with elevated cfDNA levels at diagnosis had an inferior outcome compared to patients with cfDNA levels within the normal range both in univariate and in a multivariate analysis that included IPS and was adjusted for the type of chemotherapy (EFS, p<0.03). In conclusion, our data suggest that cfDNA could serve as a biomarker in HL, that it is frequently elevated at diagnosis of HL, tends to normalize during treatment, and is associated to an inferior outcome. The study of DNA fragmentation suggests that most of the increase of cfDNA in HL is probably due to the tumor microenvironment rather than to the neoplastic cells.