Clinical and Molecular Significance of Peripheral Blood Cell-Free DNA in B-Cell Lymphomas for Detection of Genetic Mutations and Correlation with Disease Status
Authors: Suzuki Yasuhiro , Tomita Akihiro , Yoshida Kenichi , Shimada Kazuyuki , Iriyama Chisako , Sanada Masashi , Shiraishi Yuichi , Chiba Kenichi , Tanaka Hiroko , Miyano Satoru , Ogawa Seishi , Kiyoi Hitoshi 
Published: October 2019

Abstract
[Backgrounds]
Genetic abnormalities in B-cell lymphoma (BCL) have been analyzed by using tumor cell-derived DNA (tDNA) obtained from biopsy samples at diagnosis, while the amount of available samples is often limited due to the advance in diagnostic technique. Furthermore since repetitive biopsies during the clinical course are not generally acceptable, sequential analysis of tumor samples is difficult. The peripheral blood circulating cell-free DNA (PB-cfDNA), which is detected in human plasma or serum, has been shown to be a possible alternative source for DNA mutation analysis and an indicator for monitoring disease status especially in solid tumors. PB-cfDNA, if available, should be quite beneficial for lymphoma practice in terms of invasiveness and accessibility compared to the conventional biopsies, however the role of PB-cfDNA in BCL remains unknown. We thus investigate the feasibility of PB-cfDNA in diagnosing and monitoring disease status of BCLs.
[Aims]
To uncover the role of PB-cfDNA in BCLs, we evaluated the clinical and molecular features and the association with lymphoma status, and further investigated the usefulness for global genetic analyses.
[Patients and Methods]
The study population included 38 BCLs patients (diffuse large B-cell lymphoma (DLBCL), n=27; follicular lymphoma (FL), n=10; nodal marginal zone B-cell lymphoma (NMZL), n=1) diagnosed at Nagoya University Hospital and affiliated hospitals. Plasma separated from PB and tumor cells from biopsy samples were collected at diagnosis and during their clinical course with written informed consent. PB-cfDNA and tDNA were extracted from plasma (n=107) and tumor samples, respectively. The concentration and quality of PB-cfDNA were analyzed by the gel electrophoresis and BioAnalyzer (Agilent Technologies, Inc.). For genetic analyses, the Sanger sequencing and whole-exome sequencing (WES) using HiSeq 2000 (Illumina) were performed. PB mononuclear cells from each patient were used as the matched germline control. The profiles of genetic mutations of PB-cfDNA and tDNA were analyzed.
[Results]
In 31 of 38 (81%) patients (DLBCL, n=24; FL, n=6; NZML, n=1), PB-cfDNA was successfully obtained from plasma samples at diagnosis. The median PB-cfDNA concentrations in DLBCL and FL patients were 58.3 ng/mL (range, 1.4-8754.5) and 15.5 ng/mL (0.1-164.9), respectively (p=0.2127). Intriguingly, the concentration of PB-cfDNA in a specific subtype of DLBCL was much higher (median, 631.9 ng/mL; range, 70.4-1109.5). LDH and CRP were significantly correlated with the PB-cfDNA concentration (correlation coefficient, r = 0.9582 and 0.8023, respectively, n=38). The PB-cfDNA concentration in patients with B-symptom was significantly higher than that in patients without B-symptom (p=0.011) and tended to be higher in patients with advanced clinical stages and bone marrow invasion, although not statistically significant. In the time course analyses, the concentration of PB-cfDNA increased immediately after the commencement of chemotherapy and rapidly decreased within a week after the beginning of treatment, probably reflecting the release of PB-cfDNA after the collapse of tumor cells due to chemotherapy. When analyzed by WES, PB-cfDNA and tDNA were shown to have discrete sets of mutations; a set of mutations were shared by both samples and tended to have higher variant allele frequencies, whereas others were private to an either sample, suggestive of the presence of regional tumor heterogeneity. In a specific BCL subtype, WES easily detected mutations in PB-cfDNA, even though mutations were hardly detected in tDNA because of a relatively lower tumor content.
[Discussion and conclusions]
PB-cfDNA could be detected in high proportion of BCL patients. Considering the correlation coefficient with clinical indicators and detection of mutations, PB-cfDNA in BCL should be mainly originated from lymphoma cells and closely associated with tumor aggressiveness and disease progression. Moreover, we could successfully perform WES analyses using PB-cfDNA, indicating that PB-cfDNA was an alternate source for detecting lymphoma specific genetic abnormalities. PB-cfDNA might be a novel diagnostic tool for patients in whom sufficient tumor samples are not obtained by conventional diagnostic procedure.

Disclosures
Kiyoi: Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD.: Research Funding; Bristol-Myers Squibb: Research Funding; Zenyaku Kogyo: Research Funding; FUJIFILM Corporation: Research Funding.