Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma.
The prognostic value of aberrant DNA methylation of cell-free circulating DNA in plasma has not previously been evaluated in diffuse large B cell lymphoma (DLBCL). The aim of this study was to investigate if aberrant promoter DNA methylation can be detected in plasma from DLBCL patients and to evaluate this as a prognostic marker. Furthermore, we wanted to follow possible changes in methylation levels during treatment. Seventy-four patients were enrolled in the study, of which 59 received rituximab and CHOP-like chemotherapy. Plasma samples were collected from all patients at the time of diagnosis and from 14 healthy individuals used as controls. In addition, plasma samples were collected during and after treatment for surviving patients. In total, 158 plasma samples were analyzed for DNA methylation in the promoter regions of DAPK (DAPK1), DBC1, MIR34A, and MIR34B/C using pyrosequencing.



Aberrant methylation levels at the time of diagnosis were detected in 19, 16, 8, and 10 % of the DLBCL plasma samples for DAPK1, DBC1, MIR34A, and MIR34B/C, respectively. DAPK1 methylation levels were significantly correlated with DBC1 and MIR34B/C methylation levels (P < 0.001). For the entire cohort, 5-year overall survival (OS) rates were significantly lower in the groups carrying aberrant DAPK1 (P = 0.004) and DBC1 (P = 0.044) methylation, respectively. DAPK1 methylation status were significantly correlated with stage (P = 0.015), as all patients with aberrant DAPK1 methylation were stages III and IV. Multivariate analysis identified DAPK1 as an independent prognostic factor for OS with a hazard ratio of 8.9 (95 % CI 2.7-29.3, P < 0.0007). Patients with DAPK1 methylated cell-free circulating DNA at time of diagnosis, who became long-term survivors, lost the aberrant methylation after treatment initiation. Conversely, patients that maintained or regained aberrant DAPK1 methylation died soon thereafter.



Aberrant promoter methylation of cell-free circulating DNA can be detected in plasma from DLBCL patients and hold promise as an easily accessible marker for evaluating response to treatment and for prognostication. In particular, aberrant DAPK1 methylation in plasma was an independent prognostic marker that may also be used to assess treatment response.
Department of Haematology, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark.
Lasse Sommer
LS
Kristensen
Department of Haematology, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark.
Jakob Werner
JW
Hansen
Department of Haematology, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark.
Søren Sommer
SS
Kristensen
Department of Haematology, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark.
Dorte
D
Tholstrup
Department of Haematology, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark.
Laurine Bente Schram
LB
Harsløf
Department of Clinical Immunology, Næstved Sygehus, Ringstedgade 77A, Næstved, Denmark.
Ole Birger
OB
Pedersen
Department of Haematology, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark.
Peter
P
De Nully Brown
Department of Haematology, Rigshospitalet, Blegdamsvej 9, Copenhagen, Denmark.
Kirsten
K
Grønbæk
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D058846:Antibodies, Monoclonal, Murine-Derived / Q000627:therapeutic use; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D002474:Cell-Free System; D003520:Cyclophosphamide / Q000627:therapeutic use; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000097:blood*; D004317:Doxorubicin / Q000627:therapeutic use; D044127:Epigenesis, Genetic; D005260:Female; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D008297:Male; D008875:Middle Aged; D011241:Prednisone / Q000627:therapeutic use; D011401:Promoter Regions, Genetic; D012189:Retrospective Studies; D000069283:Rituximab / Q000627:therapeutic use; D017422:Sequence Analysis, DNA / Q000379:methods; D016019:Survival Analysis; D016896:Treatment Outcome; D014750:Vincristine / Q000627:therapeutic use
D058846:Antibodies, Monoclonal, Murine-Derived; D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; C571759:R-CHOP protocol; D000069283:Rituximab; D014750:Vincristine; D004317:Doxorubicin; D003520:Cyclophosphamide; D011241:Prednisone
Biomarker; Circulating DNA; DNA methylation; Diffuse large B cell lymphoma; Epigenetics; Liquid biopsy; Plasma; Prognostic markers