Aberrant DNA Methylation Patterns in Peripheral Blood Are Detectable Years before the Diagnosis of Mature B Cell Neoplasms
Authors: Wong Doo Nicole , Makalic Enes , Schmidt Daniel , Joo Jihoon E , Jung Chol-Hee , Giles Graham G 
Published: October 2019

Abstract
Background
Aberrant DNA methylation is a feature of mature B cell neoplasms (MBCN) however it is not clear whether this is an early or late event in the development of MBCN. We have previously reported that aberrant global methylation patterns are detectable in peripheral blood sampled years prior to diagnosis and now hypothesize that abnormal methylation at specific genomic regions precedes the diagnosis of MBCN.
Methods:
In our prospective cohort study peripheral blood was collected from healthy participants in the Melbourne Collaborative Cohort Study, (41,514 adult volunteers, recruited from 1990-94). Blood was stored as either dried blood spots, mononuclear cells or buffy coat. New cases of MBCN (including low grade non Hodgkin lymphoma [NHL], high grade NHL, chronic lymphocytic leukaemia and multiple myeloma) diagnosed during follow up until December 2011 were identified by cancer registry linkage. Cases were matched to controls at a 1:1 ratio based on age, gender, ethnicity and DNA source. Genome-wide DNA methylation was measured using the Infinium®HumanMethylation450 BeadChip and M values were derived after background correction, normalization and probe bias correction methods. Differentially methylated probes (DMPs) were identified by conditional logistic regression using case-control status as the outcome to compute odds ratios and p values for the association between methylation difference and occurrence of MBCN. Bonferroni adjustment was used for multiple testing correction. Adjusted conditional logistic regression models were created to control for the white blood cell (WBC) content of peripheral blood samples. Differentially methylated regions (DMRs) were identified using the DMRcate package. DMPs and DMRs were cross-checked against genes associated with MBCN pathogenesis by a review of current literature.
Results
We identified 438 cases of MBCN, with a median time between blood collection and MBCN diagnosis of 10.6 years. Following Bonferroni p value adjustment, there were 1,215 DMPs of which 81 were more methylated in cases compared to controls. There were 74 promoter-associated DMPs, with increased methylation in: HOXA9 (cg07778029, OR, 1.58; 95% CI, 1.36-1.83[punadj 2.4x10-9]), HOXA11 (cg05977669, OR, 1.62; 95% CI, 1.37-1.92 [punadj 2.0x10-8], cg24446586, OR, 1.57; 95% CI, 1.34-1.85 [punadj 2.4x10-8]), MYADM (cg25693289, OR, 1.64; 95% CI, 1.39-1.92 [punadj 1.59x10-9]). Decreased methylation of DMPs was noted in: ROBO1 (cg24512093, OR, 1.67; 95% CI, 1.39-1.96 [punadj 2.83x10-8]) and IKBKB (cg11283404, OR, 1.54; 95% CI, 1.32-1.82 [punadj 8.66x10-8]).Using a more relaxed p value cut-off (p < 1x10-5 before Bonferroni adjustment), we identified 10,792 DMPs with 17 probes corresponding to nine genes involved in MBCN pathogenesis: CARD11, CD79B, TNFAIP3, NR2F2, NPTX2, NOTCH1, NOTCH2, EP300 and DTX1. Adjustment for cell content did not significantly change the results.
Analysis of differentially methylated regions (clusters of differentially methlyated probes) revealed 4,629 DMRs with p <1x10-7. Of these, 1,824 regions were more methylated in cases and included several genes of interest in MBCN pathogenesis (e.g. GATA3, HOXA9, HOXA11, EBF3, SOX11, SOX9).
Conclusions
This is the first study to report differential DNA methylation patterns detectable in the peripheral blood many years prior to MBCN diagnosis. Increased methylation of promoter-associated probes in HOXA9, HOXA11 and MYADM and decreased methylation of probes in ROBO1 and IKBKB were associated with an increased risk of developing MBCN. These findings suggest that aberrant methylation is a very early event in the pathogenesis of MBCN.

Disclosures
No relevant conflicts of interest to declare.