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Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma

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ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s13148-016-0261-y) contains supplementary material, which is available to authorized users.

No MeSH data available.


Methylation levels before and after treatment for surviving patients. Each solid line represents a sample pair. The dashed lines represent the individual cutoffs for aberrant methylation. aDAPK1. bDBC1. cMIR34A. dMIR34B/C
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Fig4: Methylation levels before and after treatment for surviving patients. Each solid line represents a sample pair. The dashed lines represent the individual cutoffs for aberrant methylation. aDAPK1. bDBC1. cMIR34A. dMIR34B/C

Mentions: Methylation levels for each of the markers were compared between the diagnostic sample and the sample collected 3 months after the end of treatment for each of the individual surviving patients. All patients with aberrant DAPK1 methylation at the time of diagnosis who were alive after the end of treatment showed a decrease in methylation levels, and all except two patients had methylation levels within the normal range after treatment (Fig. 4a). For DBC1, 11 of the patients showed a greater than 5 % decrease in methylation levels, while 10 of the patients showed a greater than 5 % increase. Altogether, aberrant methylation was detected in six of the patients after treatment (Fig. 4b). For MIR34A, one patient showed a greater than 5 % decrease in the methylation level, and one patient showed a greater than 5 % increase. Altogether, aberrant methylation was detected in four of the patients after treatment (Fig. 4c). For MIR34B/C, two patients showed a greater than 5 % decrease in methylation levels, and one patient showed a greater than 5 % increase. Altogether, aberrant methylation was detected in five of the patients after treatment (Fig. 4d).Fig. 4


Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma
Methylation levels before and after treatment for surviving patients. Each solid line represents a sample pair. The dashed lines represent the individual cutoffs for aberrant methylation. aDAPK1. bDBC1. cMIR34A. dMIR34B/C
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5015248&req=5

Fig4: Methylation levels before and after treatment for surviving patients. Each solid line represents a sample pair. The dashed lines represent the individual cutoffs for aberrant methylation. aDAPK1. bDBC1. cMIR34A. dMIR34B/C
Mentions: Methylation levels for each of the markers were compared between the diagnostic sample and the sample collected 3 months after the end of treatment for each of the individual surviving patients. All patients with aberrant DAPK1 methylation at the time of diagnosis who were alive after the end of treatment showed a decrease in methylation levels, and all except two patients had methylation levels within the normal range after treatment (Fig. 4a). For DBC1, 11 of the patients showed a greater than 5 % decrease in methylation levels, while 10 of the patients showed a greater than 5 % increase. Altogether, aberrant methylation was detected in six of the patients after treatment (Fig. 4b). For MIR34A, one patient showed a greater than 5 % decrease in the methylation level, and one patient showed a greater than 5 % increase. Altogether, aberrant methylation was detected in four of the patients after treatment (Fig. 4c). For MIR34B/C, two patients showed a greater than 5 % decrease in methylation levels, and one patient showed a greater than 5 % increase. Altogether, aberrant methylation was detected in five of the patients after treatment (Fig. 4d).Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s13148-016-0261-y) contains supplementary material, which is available to authorized users.

No MeSH data available.