Integrative DNA Methylation and Gene Expression Analysis Reveals Candidate Biomarkers Associated with Dichotomized Response to Chemoimmunotherapy in Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. While 60% of DLBCL patients achieve complete remission with frontline therapy, relapsed/refractory (R/R) DLBCL patients have a poor prognosis with median overall survival below one year, necessitating investigation into the biological principles that distinguish cured from R/R DLBCL. Recent analyses have identified unfavorable molecular signatures when accounting for gene expression, copy number alterations and mutational profiles in R/R DLBCL. However, an integrative analysis of the relationship between epigenetic and transcriptomic changes has yet to be described. In this study, we compared baseline methylation and gene expression profiles of DLBCL patients with dichotomized clinical outcomes.

Methods: Diagnostic DLBCL biopsies were obtained from two patient cohorts: patients who relapsed or were refractory following chemoimmunotherapy ("R/R"), and patients who entered durable clinical remission following therapy ("cured"). The median age for R/R and cured cohorts were 62 (range 35-86) years vs. 64 (range 28-83) years (P= 0.27). High-intermediate or high IPI scores were present in 14 vs. 6 patients (P= 0.08) in the R/R and cured cohorts, respectively. All patients were treated with frontline R-CHOP or R-EPOCH. DNA and RNA were extracted simultaneously from formalin-fixed, paraffin embedded biopsy samples. An Illumina 850k Methylation Array was used to identify DNA methylation levels in 29 R/R patients and 20 cured patients. RNA sequencing was performed on 9 R/R patients and 7 cured patients at diagnosis using Illumina HiSeq4000. Differentially methylated probes were identified using the DMRcate package, and differentially expressed genes were identified using the DESeq2 package. Gene set enrichment analysis was performed using canonical pathway gene sets from MSigDB.

Results: At the time of diagnosis, we found significant epigenetic and transcriptomic differences between cured and R/R patients. Comparing cured to R/R samples, there were 8,159 differentially methylated probes (FDR<0.05). Differentially methylated regions between R/R and cured cohorts overlap with genes previously identified as mutation hotspots in DLBCL. Upon comparing transcriptomic profiles between R/R and cured, 267 genes were found to be differentially expressed (Log2FC>|1| and FDR<0.05). Gene set enrichment analysis revealed gene sets related to cell cycle, membrane trafficking, Rho and Rab family GTPase function, and transcriptional regulation were upregulated in the R/R samples. Gene sets related to innate immune signaling, Type I and II interferon signaling, fatty acid and carbohydrate metabolism were upregulated in the cured samples. To identify genes likely to be regulated by specific changes in methylation, we selected genes that were both differentially expressed and differentially methylated between the R/R and cured cohorts. In the R/R samples, 13 genes (ARMC5, ARRDC1, C12orf57, CCSER1, D2HGDH, DUOX2, FAM189B, FKBP2, KLF5, MFSD10, NEK8, NT5C, and WDR18) were significantly hypermethylated and underexpressed when compared to cured specimens, suggesting that epigenetic silencing of these genes is associated with lack of response to chemoimmunotherapy. In contrast, 12 genes (ATP2B1, C15orf41, FAM102B, FAM3C, FHOD3, FYTTD1, GPR180, KIAA1841, LRMP, MEF2A, RRAS2, and TPD52) were significantly hypermethylated and underexpressed in cured patients, suggesting that epigenetic silencing of these genes is favorable for treatment response. Many of these epigenetically modified genes have been previously implicated in cancer biology, including roles in NOTCH signaling, chromosomal instability, and biomarkers of prognosis.

Conclusions: This is the first integrative epigenetic and transcriptomic analysis of diagnostic biopsies from cured and R/R DLBCL patients following chemoimmunotherapy. At the time of diagnosis, both the methylation and gene expression profiles significantly differ between patients that enter durable remission as opposed to those who are R/R to therapy. Soon, the hypomethylating agent CC-486 (i.e. oral azacitidine) will be explored in combination with mini-R-CHOP for older DLBCL patients in whom DNA methylation is likely increased. These data support the use of hypomethylating agents to potentially restore sensitivity of DLBCL to chemoimmunotherapy.