Richter Syndrome (RS): Genome-Wide Promoter Methylation Profile Differs From De Novo Diffuse Large B-Cell Lymphoma (DLBCL) and Affects Genes Involved in Stem-Cell Maintenance and TP53 Pathway
Authors: Rossi Davide , Rinaldi Andrea , Kwee Ivo , Arcaini Luca , Lucioni Marco , Gattei Valter , Marasca Roberto , Forconi Francesco , Berger Francoise , Cogliatti Sergio , Zucca Emanuele , Gaidano Gianluca , Bertoni Francesco 
Published: October 2019

Abstract
Abstract 1359
RS represents the development of DLBCL in the context of chronic lymphocytic leukemia (CLL). The pathogenesis of RS is still largely unknown. We have previously shown that RS lacks many of the typical genetic lesions recurrently observed in de novo DLBCL (Rossi et al, Blood 2011). Here, we report genome-wide promoter methylation profiling of RS and compare it with the methylation profile of de novo DLBCL, and non-transformed CLL.

Methods.
The study included 21 RS, 10 clonally related CLL phases (seven paired with RS samples), 9 de novo non-GC DLBCL, 6 non-transformed CLL and 6 normal peripheral blood CD19+ B-cells. All RS were classified as DLBCL, showed a non-germinal center (GC) phenotype, and lacked EBV infection. Both RS and DLBCL DNA was extracted from diagnostic frozen lymph node biopsies. DNA samples underwent bisulfite treatment with the EZ DNA methylation kit and were hybridized on Illumina Infinium HumanMethylation27 arrays, that enables the interrogation of more than 27,000 CpG island over the entire human genome. Probes were discarded if they: i) mapped outside CpG islands; ii) presented a standard deviation <0.10 across all the samples; or iii) were always non-methylated, always fully methylated, or always partially methylated. For clustering and for unpaired t-tests, probes mapped on sex chromosomes were excluded. Supervised analysis of differential methylation between groups was performed using a t-test on the continuous beta values, followed by multiple test correction (MTC). A paired t-test was utilized for comparing the methylation profile between the RS phase and the matched CLL-phase. Probes showing a q-value <0.1 were defined differentially methylated.

Results.
By unsupervised clustering, RS samples separated from both de novo DLBCL, that instead clustered in a single group, and non-transformed CLL.
There were over 3,000 probes differentially methylated between RS and de novo DLBCL. The 443 probes showing higher methylation in RS were significantly enriched of promoter regions of genes involved in TP53 signaling (P=6E-03) and cell cycle regulation (P=5E-02) pathways. The 2,687 probes showing higher methylation in de novo DLBCL were significantly enriched of promoter regions of genes involved in histone modification, including genes harboring the H3K27me3 mark (P < 1.0E–16), and of genes that are targets of the EED and SUZ12 Polycomb proteins (P < 1.0E–16).
Analysis of paired CLL/RS sequential samples revealed an extensive overlap between the methylation profile of the CLL phase and the transformed RS phase, suggesting that changes in the methylation pattern are an early event in RS pathogenesis that occurred already at the time of the initial CLL clone. Two single probes in the promoter regions of the OSM and S1PR4/EDG6 genes were differentially methylated in CLL/RS sequential samples and, therefore, might have contributed to the acquisition of the aggressive clinico-pathologic phenotype of RS. OSM codes for oncostatin M, which inhibits cell proliferation and induces cell differentiation and apoptosis. S1PR4/EDG6 is a gene specifically expressed in the lymphoid tissue and involved in cell migration. Both OSM and S1PR4/EDG6 promoter regions were unmethylated in the chronic CLL phase, and became methylated in the paired aggressive RS phase, suggesting the acquisition of methylation during transformation.
CLL that subsequently transformed to RS differed from CLL that never transformed to RS for 2 probes showing a higher and 2 a lower degree of methylation. Since changes in the methylation pattern are an early event in RS, this observation prompts the investigation of the methylation status of DLX5 and GBGT1, the two genes with promoter regions showing a higher degree of methylation, as biomarkers allowing the early identification of CLL that will transform to RS.

Conclusions.
RS clearly differ from de novo DLBCL in terms of methylation profile. The overrepresentation of epigenetic changes affecting genes of the TP53 pathway, along with the significantly higher prevalence of TP53 structural abnormalities, might explain the differences in chemosensitivity between RS and de novo DLBCL. Epigenetic changes are an early events in RS pathogenesis, suggesting that, similarly to what shown in transformed follicular lymphoma, lesions initiating transformation are acquired by a cell belonging to the initial tumor clone, rather being progressively accumulated during disease course.

Disclosures:
No relevant conflicts of interest to declare.