-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

4371 Methylated Whole Genome Cell-Free DNA Sequencing before Chimeric Antigen Receptor T-Cell Therapy for Large B-Cell Lymphoma Predicts Treatment Outcomes

Program: Oral and Poster Abstracts
Session: 621. Lymphomas: Translational—Molecular and Genetic: Poster III
Hematology Disease Topics & Pathways:
Research, Biological therapies, assays, Lymphomas, Translational Research, non-Hodgkin lymphoma, B Cell lymphoma, Chimeric Antigen Receptor (CAR)-T Cell Therapies, genomics, Diseases, immune mechanism, aggressive lymphoma, Therapies, Lymphoid Malignancies, emerging technologies, Biological Processes, Technology and Procedures, omics technologies
Monday, December 11, 2023, 6:00 PM-8:00 PM

Hua-Jay Jeffery Cherng, MD1, Xiaojing Yang, PhD2*, Ryan Yancey, MS2*, Christian A Gordillo, BSc3*, Ted B. Piorczynski, PhD4*, Hei T. Chan, RN5*, Kenneth N. Ofori, MHS, MD6,7*, Govind Bhagat, MD7, Andrea Califano, PhD8*, Barbara Pro, MD9, Keith Booher, PhD2*, Jennifer E. Amengual, MD1, Ran Reshef, MD, MSc10 and Abu A. I. Sina, PhD8*

1Lymphoma Program, Division of Hematology & Oncology, Columbia University Irving Medical Center, New York
2Zymo Research, Irvine
3Blood and Marrow Transplantation and Cell Therapy Program, Columbia University Irving Medical Center, New York, NY
4Division of Hematology and Oncology, Columbia University Irving Medical Center, New York, NY
5Blood and Marrow Transplantation and Cell Therapy Program, Columbia University Irving Medical Center, New York
6Indiana University Health, Indianapolis
7Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York
8Department of Systems Biology, Columbia University Irving Medical Center, New York
9Lymphoma Program, Division of Hematology & Oncology, Columbia University, New York, NY
10Division of Hematology & Oncology, Columbia University Medical Center, New York


More than half of patients with relapsed or refractory large B-cell lymphoma (R/R LBCL) will experience progressive disease after CD19 chimeric antigen receptor T-cell therapy (CART). Circulating tumor DNA profiling has been used to prognosticate patients before CART, but mainly has focused on identification of somatic genomic aberrations. Epigenetic changes, specifically aberrant DNA methylation, can alter transcriptional regulation of genes important for treatment resistance. Altered methylation features can be detected from cell-free DNA (cfDNA) in patients with LBCL and are associated with outcomes after frontline immunochemotherapy. In the current study, we used whole genome bisulfite sequencing (WGBS) to analyze the methylation landscape in cfDNA from patient plasma before CART and identified novel methylation signatures associated with poor treatment outcomes.


Patients with R/R LBCL who received standard-of-care CART with plasma samples drawn before lymphodepletion (LD) or at cell infusion were identified. The primary endpoint was 3-month overall response after CART. cfDNA was extracted from 3-5 ml of plasma and cfDNA libraries were prepared using a Zymo-Seq cell free WGBS kit. Each library was sequenced to 300 – 600 million read pairs at a read length of 150 bp, enabling an average CpG coverage of 7-13X per detected CpG. Unique alignment rate among all libraries were about or above 82%. Reads were adapter trimmed with Trim Galore and aligned to reference genome hg19 using Bismark. Methylation calling was performed by MethylDackel. Differentially methylated cytosines (DMC, FDR ≤0.05) and regions (DMR) from WGBS data with an absolute methylation difference of ≥ 0.1 between 3-month responders and non-responders were identified using DSS. Functional enrichment analysis of genes overlapped by DMRs was performed by gprofiler2.


A total of 22 patients with plasma collected before LD (n=21) or at cell infusion (n=1) who received CART were identified. Median follow-up was 21.5 months (95% CI 12.5-NR). Three-month response was evaluable in 21 patients (1 pending evaluation). Overall response rate (ORR) was 77% (8 CR, 9 PR) and 55% (9 CR, 2 PR) at one- and 3-months post-CART, respectively. Median PFS was 6.5 months (95% CI 3.0-NR), and median OS was 57.7 months (95% CI 7.7-NR). Patient and disease characteristics are described in Table 1. Differential methylation analysis identified 22,815 DMCs, of which 1,043 were hypomethylated and 21,772 were hypermethylated in non-responders. The global average methylation values across 25 million cytosines were 0.807 and 0.786 for non-responders and responders, respectively (p=0.265). A total of 582 DMRs were identified, of which 34 were hypomethylated and 548 were hypermethylated in non-responders (Figure 1). Of these, 281 (48%) overlapped intergenic regions, 265 (46%) gene bodies, and 33 (6%) promoter regions. DMRs overlapping DNA repair gene PARP1 (areaStat 88.2), B-cell receptor signaling genes SYK (27.2), FYN (21.4), and PTPN6 (20.9), polycomb repressor complex genes JARID2 (25.2) and BCOR (21.4), tumor suppressor WT1 (47.1), immune mediators NRP1 (80.0), TRAF3 (56.7), PAK3 (28.4), IRAK2 (28.7), and TNFS14 (23.6), and non-coding RNAs MIR4520 (95.8) and VTRNA-2 (23.1) were hypermethylated in non-responders and IL17RA (-33.2) hypomethylated. Functional enrichment analysis on overlapped genes also identified common molecular functions involving guanyl nucleotide exchange factors (p=0.0007), GTPase regulation (p=0.001), and cGMP binding (p=0.007).


This proof-of-concept study is the first to perform WGBS on pretreatment plasma from patients with R/R LBCL to associate methylation signatures with post-CART outcomes. We identified that non-responders were enriched in hypermethylated DMRs compared to responders, overlapping with genes important to lymphomagenesis, immune function, and GTP energy metabolism. Further work will expand this cohort, compare with matched-tissue to find tumor-specific markers, and identify candidate DMR targets for targeted bisulfite sequencing from pre- and post-treatment plasma samples to assess response. By identifying high-risk methylation signatures in the plasma of CART recipients, we may be able to identify patients to be prioritized for novel treatment strategies such as combination with hypomethylating agents to improve outcomes.

Disclosures: Yang: Zymo Research: Current Employment. Yancey: Zymo Research: Current Employment. Califano: DarwinHealth: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Pro: Seattle genetics: Honoraria; Bio Secura: Honoraria. Booher: Zymo Research: Current Employment. Amengual: Epizyme: Honoraria; Incyte: Consultancy; Astrazeneca: Consultancy. Reshef: TScan Therapeutics: Consultancy.

*signifies non-member of ASH