Subtype Of Diffuse Large B-cell Lymphoma Research Articles

Longitudinal Changes in Immune Activation Serum Biomarkers Prior to Diagnosis and Risk of B-cell NHL Subtypes.

To examine the contribution of B-cell activation molecules to B-cell follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), a prospective study was conducted using pre-diagnosis serial serum samples from the US Department of Defense Serum Repository. Each case (n = 142 FL, n = 211 DLBCL) was matched to two controls on age, gender, race, military branch, and blood collection dates. Immune activation molecules (IL1β, IL2, IL4, IL5, IL6, IL10, IL12, CXCL13, IL8, TNFα, IFNγ, GM-CSF, VEGF, sCD30, IgE) were quantified using ELISA or multiplex immunometric (Luminex) assay. Longitudinal data were analyzed using linear mixed modeling. As serial specimens were collected over several years before diagnosis, we evaluated the temporal dynamics of these markers. Increased serum levels of sCD30, CXCL13, and to a lesser extent IL10, were associated with both FL and DLBCL in cases compared with controls, with a median follow-up of 5.5 years from the earliest specimen collection to diagnosis date. Significant increasing sCD30 and CXCL13 trajectories for FL and DLBCL subtypes were noted starting at the earliest time points and with IL10 levels increasing significantly at time points closer to diagnosis. These results suggest that sCD30, CXCL13, and IL10 may contribute to the etiology of FL and DLBCL and are potential biomarkers for these non-Hodgkin lymphoma subtypes. The increasing trajectories of the B-cell activation molecules, sCD30, CXCL13, and to a lesser extent IL10, may indicate early disease-induced effects or reflect the chronic stimulation of B-cells that promotes the development of FL and DLBCL subtypes.

CAR-T Therapy in Patients with Relapsed or Refractory Diffuse Large B Cell Lymphoma: Real World Outcomes Based on Tumor Biology

Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), accounting for 30-40% of NHL cases with an incidence of 7 cases per 100,000 persons per year (Susanibar-Adaniya, 2021). There are two major subtypes of DLBCL based on the cell of origin (COO), germinal center B-cell-like (GCB) and activated B-cell-like (ABC). Around 10-15% of cases are unable to be classified and are termed not otherwise specified (NOS). GCB is generally associated with a more favorable prognosis, showing 3-year progression free survival (PFS) rates of approximately 75% compared to 40-50% for ABC (Sehn, 2021). Chimeric antigen receptor (CAR)-T cell therapy has shown significant responses in relapsed or refractory diffuse large B-cell lymphoma (DLBCL).These treatments have shown objective response rates (ORR) of 52-83% and complete response (CR) rates of 39-58% in relapsed or refractory DLBCL (Abramson, 2020; Locke, 2019; Schuster, 2019; Schuster, 2021). While the CAR-T studies noted the number of patients in the GCB and ABC subtypes, differences in outcomes stratified by subtypes are less well defined. Methods: This is a retrospective, multi-center (Utah, Michigan, North Carolina) study evaluating outcomes of patients with DLBCL treated with CAR-T cell therapies. The primary outcome of this study is PFS at 12 months based on tumor biology (ABC, GCB, NOS). Secondary outcomes include PFS at 6 months, PFS, overall survival (OS) at 6 months, OS at 12 months, OS, and best response to treatment. Kaplan-Meier curves were created for OS and PFS stratified by DLBCL subtype. Patient characteristics and best response to treatment were summarized stratified by DLBCL subtype and compared by Wilcoxon Rank Sum Test or Chi-square test. Results: A total of 99 patients were included in this study. Of these, most were GCB subtype (n=50), followed by NOS (n=39), and ABC (n=10). There was no difference is baseline characteristics between each of these groups (Table 1). The median age was 65 years old, 87% were white, and 62% were male. Patients had received a median of 3 prior lines of treatment prior to CAR-T. The median PFS was not achieved for the ABC subtype, 4.6 months for the GCB subtype, and 3.5 months for those who were NOS (p=0.34) (Figure 1). Median OS was 14.8 months, 13.4 months, and 8 months for ABC, GCB, and NOS, respectively (p=0.083). The complete response rates were: ABC subtype (50%), compared to GCB (32%), and NOS (26%) groups (p=0.34). Progressive disease following CAR-T treatment rates were: NOS (51%), compared to ABC (20%) and GCB (32%) groups (p=0.34). Discussion: The data from this retrospective, multi-center study suggests there may be a trend towards a greater PFS benefit for those with the ABC subtype compared to the GCB and NOS groups. The ABC subtype also showed a numerically higher percentage of complete responses compared to the other groups, but neither were statistically significant differences. The limited number of patients in the ABC group limits the ability to determine a meaningful result currently. There are several limitations of this study. There was a limited sample size in this study, and as a result, statistical significance was unable to be achieved in this analysis. In addition, the percentage of patients in the NOS group in this study (~39%) was much higher than the number reported in the literature (~10-15%). This could be due to the retrospective nature of the study making it difficult to obtain cell of origin information from the time the diagnosis was made and leading to a patient being classified as NOS when they may have had a GCB or ABC subtype. Our study suggests a potential difference seen to treatment with CAR-T therapy based on the tumor biology of the disease. Future studies with a larger cohort to elucidate a difference in CAR-T therapy responses by cell of origin in DLBCL are warranted as this may influence patient treatment selection. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Position of MYC Rearrangements in the Genomic Landscape of Diffuse Large B-Cell Lymphoma

Introduction High grade B-cell lymphoma with MYC and BCL2 and/or BCL6 translocations (HGBL-double hit (DH)/-triple hit (TH) is the most strictly defined molecular class of diffuse large B-cell lymphoma (DLBCL) and is characterized by MYC plus BCL2 rearrangements with- or without BCL6 rearrangement. HGBL DH/TH have an inferior prognosis under standard DLBCL treatment compared to patients without MYC-rearrangements and currently few effective second-line options exist. Still, some HGBL DH/TH respond well to treatment, highlighting the need for further refined markers predictive of treatment resistance and relapse. To distinguish different DLBCL subtypes, two independent DNA based classifications were proposed by the Dana Farber Cancer institute (DFCI) and the National Cancer Institute (NCI, Lymphgen), that distinguish 5- and 7 DLBCL subtypes, respectively. These subtypes are characterized by distinct pathological mechanisms and outcomes. However, in both classifications the number of MYC rearranged cases was insufficient to accurately determine the position of MYC rearrangements in the genomic landscape of DLBCL. We collected a large DLBCL genomic dataset enriched for MYC rearranged cases to evaluate the hypothesis that HGBL DH/TH, or subsets of HGBL DH/TH define a separate molecular class. Methods High quality next generation sequencing (NGS) data for i) mutations, ii) genome-wide chromosomal copy numbers, and iii) MYC, BCL2 and BCL6 rearrangements was assembled for a set of N=706 DLBCL samples from 4 study cohorts (HOVON-84, HOVON-130, PETAL trials and the DFCI dataset) that included N=102 cases with MYC rearrangements including single-hit (SH), HGBL DH and TH. All samples were assigned to genomic subtypes according to published algorithms. Results Clustering all samples following the DFCI approach identified 5 clusters in concordance with published results (Figure 1). We find that MYC SH cases (N=36) are scattered throughout the 5 DFCI subtypes, while the majority of HGBL DH-BCL2/TH cases (N=41/50) fall together in subtype C3, a germinal center B-cell (GCB) rich subtype. Inspection of the genetic features of this HGBL and GCB rich subtype shows that it has many of the genetic characteristics corresponding to transformed follicular lymphomas. DH-BCL6 cases were few (N=16) and predominantly clustered in C1. When classifying samples according to Lymphgen, again we find that the MYC SH cases are scattered throughout the 7 subtypes, while HGBL-DH/TH with BCL2 rearrangement are classified as EZB (N=40/50), substantiating earlier interpretations. DH-BCL6 cases were mostly classified as BN2. Conclusion We recapitulate the 2 DNA-classification methods with a data set enriched for MYC rearranged cases (102/706 samples) and demonstrate that HGBL DH-BCL2/TH form a sub-cluster within DFCIs C3, and Lymphgen's EZB respectively, with genomic characteristics of transformed follicular lymphoma. DH-BCL6 cases were few, but come together in both classifications, DFCI C1 and Lymphgen BN2, consistent with the WHO-HAEM5, in which DH-BCL6 is classified as a separate subtype. MYC SH are not assigned to a specific subtype, also consistent with the WHO-HAEM5. These results provide the currently most robust molecular basis for stratification of DLBCL cases with MYC rearrangement. Figure 1. Cluster heatmap of 706 DLBCLs of which 102 have MYC rearrangements. Clustering was performed by non-negative matrix factorization (NMF) according to DFCI. Top: Classification of samples according to MYC status, Lymphgen assignment, and cell of origin (COO). Right: Genomic alterations (copy number alterations, CNAs; mutations and translocations) enriched in NMF clusters C1 to C5. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Waveline-001: Updated Results from a Phase 1 Dose Escalation and Cohort Expansion Study of Zilovertamab Vedotin (MK-2140) in Non-Hodgkin Lymphoma

Background: Zilovertamab vedotin (ZV) is a receptor tyrosine kinase-like orphan receptor 1 (ROR1) targeting antibody-drug conjugate comprising zilovertamab, a proteolytically cleavable linker, and the antimicrotubule agent monomethyl auristatin E. ROR1 is a transmembrane protein absent in mature tissues but highly expressed in certain hematologic malignancies, making it an attractive therapeutic target. Safety and efficacy of ZV are being investigated in the phase 1 WAVELINE-001 study (NCT03833180), which is a dose escalation and cohort expansion analysis in patients (pts) with relapsed and/or refractory (R/R) hematologic malignancies. Initial results showed ZV had a tolerable safety profile and promising antitumor activity in R/R non-Hodgkin lymphoma (NHL; Wang M, et al. NEJM Evid. 2021;1[1]). Updated results, including PFS and OS for mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), and Richter transformation (RT), are presented. Methods: Eligible pts aged ≥18 years of age, with a histological diagnosis of MCL, DLBCL, or RT; had ECOG PS 0-2; and progression during or relapsed after prior systemic therapy, were included. WAVELINE-001 included 3 dosing schedules. Data from Schedule 1 are presented; Schedules 2 and 3 are ongoing. In Schedule 1, pts received ZV IV at a starting dose of 0.5 mg/kg on day 1 Q3W, increasing to 2.5 mg/kg using an accelerated plus 3 + 3 dose escalation design. Primary end point was determination of the maximum tolerated dose and/or recommended dosing regimen (RDR). Secondary end points included safety, ORR, DOR, PFS, and OS. Results: A total of 54 pts were enrolled in Schedule 1, of which 30 pts were treated at the established RDR of 2.5 mg/kg. At data cutoff (May 18, 2022), median follow-up was 15.3 months (range, 0.5-37.8). Median age was 70.0 yrs, 57% of pts were male, 48% had an ECOG PS of 0, 65% had received ≥3 prior therapies, 80% had NHL (FL, n=3; DLBCL, n=15; MCL, n=17; MZL, n=1; RT, n=7), and 20% had other disease (AML, n=3; CLL/SLL, n=7; mixed histology, n=1). MCL pts had received a median (range) of 4 (1-9) prior lines of therapy. All MCL pts received prior BTKi therapy, and none had prior CAR-T or CAR-NK therapy. 59% of MCL pts were Ann Arbor stage IV. DLBCL pts received a median (range) of 4 (1-7) prior lines of therapy, 67% of pts had received prior CAR-T or CAR-NK therapy; 47% of pts were GCB, and 33% were ABC subtype. Five pts (71%) with RT had the DLBCL subtype. No pts with RT received autologous stem cell transplant and received a median (range) of 3 (1-10) prior therapies. Any-cause adverse events (AEs) occurred in 52 (96%) pts. Any-grade treatment-related AEs (TRAEs) occurred in 40 pts (74%); most common (≥20%) were peripheral neuropathy (46%), neutrophil count decreased (35%), fatigue (35%), nausea (28%), and diarrhea (20%). Decreases in hemoglobin and platelet count occurred in 13% and 11% of pts, respectively. Grade 3-4 TRAEs occurred in 28 pts (52%), most commonly neutrophil count decreased (33%), platelet count decreased (11%), and peripheral neuropathy (7%). AEs of special interest regardless of causality occurred in 76% of pts; most common were hematopoietic cytopenia (56%) and peripheral neuropathy (48%). For pts experiencing peripheral neuropathy, most cases were grade 1/2 (41%); 7% were grade 3. Median (range) time to onset of first peripheral neuropathy was 49 (1-162) days. No incidences of tumor lysis syndrome or infusion reactions occurred and no grade 5 TRAEs occurred. AEs led to ZV discontinuation in 12 pts (22%) and interruption or reduction in 24 (44%). ORR was 33% (5/15; 95% CI, 12-62; 3 CR/2 PR) in DLBCL, 53% (9/17; 95% CI, 28-77; 2 CR/7 PR) in MCL, and 57% (4/7; 95% CI, 18-90; 1 CR/3 PR) in RT. No responses were seen among the other indications treated. Median DOR (range) was 4.6 months (2.9-21.5) for DLBCL, 10.0 (0-20.3) for MCL, and 2.8 (0-2.8) for RT. Median PFS (95% CI) was 3.9 months (0.5-7.0) for DLBCL, 11.4 (4.0-NE) for MCL, and 4.7 (0.7-NE) for RT. Median OS (95% CI) was 9.1 months (1.0-NE) for DLBCL, 18.0 (7.1-NE) for MCL, and 24.0 (2.7-NE) for RT. Among 12 pts with NHL (10 DLBCL and 2 RT) who had undergone prior CAR-T/CAR-NK therapy, ORR was 33% (4/12; 95% CI, 10-65). Conclusions: Updated results from WAVELINE-001 support prior analyses showing ZV had a tolerable safety profile and promising response rates, and favorable PFS and OS among heavily pre-treated pts with DLBCL, MCL, and RT, including pts who had received prior CAR-T/CAR-NK therapy.

Mivavotinib, a Syk Inhibitor, in Relapsed/Refractory (R/R) Non-GCB Diffuse Large B-Cell Lymphoma (DLBCL) with or without MYD88 and/or CD79 Mutations: A Phase 2 Study

Background: Spleen tyrosine kinase (Syk) is a key signaling protein that activates survival pathways in B-cell lymphomas. The non-germinal center B-cell like (non-GCB) or activated B-cell like (ABC) cell-of-origin subtype of DLBCL has greater reliance on the B-cell receptor pathway and Syk signaling for survival, compared with the GCB subtype. Multiple signaling pathways are activated by Syk in ABC DLBCL (NF-κB and phosphoinositide 3-kinase [PI3K]), whereas only the PI3K pathway is activated by Syk in GCB DLBCL. MYD88 and/or CD79b mutations are strongly enriched within ABC DLBCL and are associated with poor prognosis. Syk hyperactivation has been observed in ABC DLBCL cell lines harboring MYD88/CD79 mutations, with activation of NF-κB via Syk-mediated signaling through both the BCR and the toll-like-receptor (TLR)-MYD88 pathways. Mivavotinib is an orally dosed, potent, and selective Syk inhibitor, which showed potent single agent activity in preclinical studies in ABC and GCB DLBCL cell lines, with the strongest activity against those harboring MYD88 and/or CD79b mutations. In the clinical setting, heavily pre-treated all-comer R/R DLBCL patients receiving single agent mivavotinib at the recommended phase 2 dose (100 mg once daily [QD]) showed an overall response rate (ORR) of 33% (16% complete responses [CR]) with durable responses (median duration of response [DoR] not reached [95% CI: 7 months - NE]) and a favorable safety profile. Subgroups by cell-of-origin showed a substantially higher ORR in patients with the non-GCB subtype (53%; 95% CI: 27, 79) than GCB (22%; 95% CI: 0.11, 0.37), and a median DoR of 15.7 months (95% CI: 2.2, NE) in non-GCB responders. CX-659-401 (NCT05319028) is an ongoing study evaluating two dosing regimens of mivavotinib monotherapy in patients with R/R non-GCB DLBCL, in whom circulating tumor DNA-based next generation sequencing (NGS) will be used to determine MYD88/CD79b mutational status. The goals of this strategy are to confirm the activity of mivavotinib previously observed in non-GCB DLBCL, evaluate its activity in DLBCL according to mutational status of MyD88/CD79b, and optimize the dose and schedule in this biomarker-defined patient population. Methods: This multicenter, open-label, phase 2 study will enroll patients with histologically confirmed de novo or transformed DLBCL that is non-GCB by immunohistochemistry (IHC) per Hans algorithm or ABC by gene expression profiling, including primary mediastinal and primary extranodal DLBCL. Patients must have had at least 2 but no more than 5 prior lines of systemic anticancer therapy. Following standard first-line R-CHOP (or equivalent), patients must have received 2nd-line salvage therapy with or without autologous stem cell transplant (ASCT), and/or CAR-T therapy, unless ineligible for 2nd-line salvage, ASCT, or CAR-T. Additional key eligibility criteria include age >18 years, ECOG performance status 0-2, and FDG-PET-avid measurable disease per 2014 Lugano criteria. DLBCL patients with primary brain or secondary central nervous system involvement with active disease will be excluded. Patients will be randomized to 1 of 2 arms (n=25 planned per arm): (1) 100 mg QD; or (2) 120 mg QD for 2 weeks followed by 80 mg QD thereafter; in 28-day cycles until disease progression or unacceptable toxicity. The primary endpoints are ORR (by independent radiology review according to 2014 Lugano Criteria) and safety/tolerability. Secondary endpoints include DoR, progression-free survival (PFS), overall survival (OS), and CR rate. Exploratory endpoints include pharmacokinetic, biomarker assessments, and investigator-assessed ORR, DoR, PFS, and CR rate. Results of this study will help to define the optimal dose and provide confirmatory efficacy and safety data of mivavotinib in patients with non-GCB DLBCL, including MYD88/CD79b-mutated DLBCL to guide further expansion in these biomarker-defined cohorts.

Biomarker Analysis of Zanubrutinib and Tislelizumab Combination Therapy in Patients with Relapsed/Refractory B-Cell Malignancies

Background/Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma world-wide. Although frontline therapies have yielded positive outcomes in most patients with DLBCL, about one-third of patients are refractory to or relapse after standard therapy (Sehn et al. J Clin Oncol 2005;23(22):5027-33). The antitumor efficacy of zanubrutinib and tislelizumab combination therapy has been demonstrated in patients with B-cell malignancies (Tam et al. Blood 2019;134:1594). Herein, we performed a comprehensive analysis to examine biomarkers associated with response or resistance to zanubrutinib and tislelizumab combination therapy in patients with B-cell malignancies. Methods: Biomarker analysis was performed in 24 patients with published response data determined by the investigator using Lugano 2014 criteria (Cheson et al. J Clin Oncol 2014;32(27):3059-68). Programmed death-ligand 1 (PD-L1) gene amplification was assessed in the baseline tumor of 11 patients with non-germinal center B-cell (GCB) subtype of DLBCL by fluorescence in situ hybridization (Empire Genomics). PD-L1 protein expression was examined in the baseline tumor (8 DLBCL) by immunohistochemistry (IHC) (Ventana PD-L1 [SP263] assay; Roche). DLBCL subtype and gene expression were examined in the baseline tumor (14 DLBCL) by HTG EdgeSeq DLBCL cell of origin assay (HTG Molecular Diagnostics). Gene mutation was examined in the baseline tumor (17 DLBCL) by DNA-seq (CGI NGS 220-Gene Panel assay; Cancer Genetic Inc.). PD-L1 and CD8 protein expression were examined in paired biopsy samples (1 DLBCL, 3 transformed follicular lymphoma [tFL], 1 follicular lymphoma [FL], 1 mantle cell lymphoma) before and 8 days after zanubrutinib treatment by IHC (Ventana SP263, CONFIRM CD8 SP57). Gene expression profiles were examined in paired biopsy samples (3 tFL, 1 FL, 1 chronic lymphocytic leukemia) by RNA-seq (RNA Access assay; Illumina). Results:PD-L1 gene alteration was observed in 2 of 11 patients with non-GCB DLBCL, including 1 with gene amplification and 1 with chromosome 9 polysomy. A higher overall response rate (ORR; 2/2 [100%] vs 3/9 [33.3%]) and complete response (CR) rate (2/2 [100%] vs 2/9 [22.2%]) was observed in patients with PD-L1 gene alteration than in patients without. Two of 8 [25%] evaluable patients with DLBCL harbored PD-L1+ tumor cells (defined using a PD-L1 protein expression cut-off ≥1%). A higher ORR (1/2 [50%] vs 2/6 [33%]) and CR rate (1/2 [50%] vs 1/6 [16.7%]) was observed in patients with PD-L1+ tumor cells than in those without. High mRNA levels of CD3D (P < 0.05), HLA-DRA (P < 0.07), and LAG3 (P < 0.05) were enriched in responders, suggesting an inflamed tumor microenvironment (TME). In contrast, non-responders harbored high mRNA levels of the NF-kB pathway-related gene REL (P < 0.05). High mRNA levels of REL were associated with an inferior clinical outcome to zanubrutinib and tislelizumab combination therapy (P < 0.05). Mutations in the tumor suppressor gene TP53 (5/10 [50%] vs 0/7 [0%]) were enriched in non-responders. Non-responders harbored higher frequency of mutations in genesinvolved in immune evasion (3/10 [30%] vs 1/7 [14.3%]), epigenetic modifications (2/10 [20%] vs 1/7 [14.3%]), and cell survival (2/10 [20%] vs 0/7 [0%]) than responders, suggesting that non-responders may have complex resistance mechanisms. Results from paired tumor biopsies from 5 patients before and after zanubrutinib treatment revealed that zanubrutinib treatment downregulated B-cell and MYC target-related gene sets and upregulated the NK cell-related gene set NK-2. Zanubrutinib treatment seemed to have no effect on the frequency of PD-L1+ cells and CD8+ T cells (using a CD8 expression cutoff of ≥1%) in the TME, although the sample size was too small to draw a definitive conclusion. Conclusion: Patients with PD-L1 gene amplification, PD-L1+ tumor cells, and high mRNA levels of CD3D, HLA-DRA, and LAG3 in baseline tumor tissue may be more responsive to zanubrutinib and tislelizumab combination therapy. A high mRNA level of REL or mutations in TP53 may contribute to resistance of zanubrutinib and tislelizumab combination therapy. Due to the limited number of samples, results must be interpreted with caution.

Efficacy Analysis of Bruton's Tyrosine Kinase Inhibitor Combined Regimen in the Treatment of Elderly Patients with Relapsed/Refractory Primary Central Nervous System Lymphoma

Background Primary central nervous system lymphoma (PCNSL) is a kind of aggressive non-Hodgkin lymphoma that originates in the brain, spinal cord, eyes or pia mater without evidence of systemic involvement, which accounts for about 3% of central nervous system tumors and has a poor prognosis. Most patients will eventually relapse and the 5-year survival rate is 29.6%. Zanubrutinib is a kind of new generation BTK inhibitor, which can penetrate the blood-brain barrier and be used in the treatment of PCNSL. The study aims to investigate the efficacy and safety of a zanubrutinib-based combination regimen in the treatment of elderly relapsed/refractory(R/R) PCNSL. Methods We retrospectively analyzed elderly patients with R/R PCNSL who received zanubrutinib-based combination regimen at the Affiliated Hospital of Nantong University over the past few years. We collected the information of patients diagnosed with PCNSL in the SEER project of the National Cancer Institute (NCI), and analyze the survival status. We used CCK8 and EdU to detect the effect of zanubrutinib on the viability and proliferation of activated B-cell subtype (ABC) diffuse large B-cell lymphoma (DLBCL) cell lines with genotypes similar to PCNSL. Results The 7 elderly R/R PCNSL patients treated with zanubrutinib-based regimen (zanubrutinib, rituximab and temozolomide) showed an overall response rate (ORR) of 85.7% (6/7), a complete response (CR) of 57.1% (4/7) and a partial response (PR) of 28.6% (2/7), and 14.3% (1/7) of patients experienced disease progression (PD). Patients' gender, KPS score, ECOG score, surgical treatment or not, positive cerebrospinal fluid protein (all P>0.05) were not significantly associated with the treatment effect. Pathological type and IESLG score were related to treatment effect (P<0.05). The median progression-free survival (PFS) and median overall survival (OS) of patients in this study were not reached. Through the analysis of clinical data of PCNSL patients in SEER database from 1975 to 2016, it is concluded that elderly patients (age>60 years old) and male PCNSL patients have a worse prognosis, the primary site of PCNSL, treatment methods and the choice of the disease have impact on the prognosis, and there is no significant difference between the disease and other causes in terms of the cause of death. In the survival analysis of elderly patients, gender, surgery and radiotherapy sequence had no significant effect on prognosis, and patients who received chemotherapy had better OS than those who received radiotherapy. The cell viability of ABC subtype DLBCL cell line OCI-Ly10 decreased in a concentration- and time-dependent manner with zanubrutinib and temozolomide (P<0.05). The inhibitory effect of zanubrutinib and temozolomide on the proliferation of OCI-Ly10 cell line was stronger than that of single drug. Conclusion Zanubrutinib-based combination therapy has good efficacy and controllable safety in elderly R/R PCNSL patients. The prognosis of elderly patients is poor, and the OS of chemotherapy patients is better than that of radiotherapy among the treatment options in the pre-BTKi era. Zanubrutinib inhibits cell viability and cell proliferation in ABC subtype DLBCL cell line OCI-Ly10.

ULK1 Blockade Preferentially Targets Germinal Centre B-Cell - Diffuse Large B-Cell Lymphoma Subtype By Attenuating Autophagy, c-MYC and Inflammation

Diffuse Large B-cell Lymphoma (DLBCL) is a heterogeneous lymphoid malignancy. Gene Expression profiling (GEP) has characterized two distinct subsets: Germinal Centre B-cell (GCB) and Activated B-cell (ABC) DLBCL. Oncogenic PI3K-AKT signaling has been identified in primary DLBCL subtypes[1]. PI3K-AKT is activated in response to the loss of PTEN in GCB-DLBCL. In ABC-DLBCL the activation is associated with CD79B mutations and concomitant MYD88 alterations. The Bruton Tyrosine Kinase (BTK) functions as an intermediary kinase. Ibrutinib (BTK inhibitor) has demonstrated promising capability in ABC-DLBCL unlike GCB tumors, warranting further investigations[2]. PI3K-AKT-mTOR signaling is also a known regulator of macro-autophagy (herein referred to as autophagy). ULK1 complex is critical for the initiation of autophagy. Aberrant signaling in this pathway promotes nutritional adaptation to malignant cells and contributes to drug resistance. We hypothesize autophagy facilitates cytoprotection and contributes to drug resistance including R-CHOP and Ibrutinib in GCB-DLBCL. Dual targeting of the BTK with Ibrutinib and autophagy initiating complex(es) would be a strategic method to impair these oncogenic functions. To determine the presence of increased autophagy related genes (ATGs) and its association to R-CHOP treatment outcome, we analyzed the GEP of patients from the REMODL-B trial (R-CHOP: GCB n=240 and ABC n=121)[3]. Gene set enrichment analysis (GSEA) showed no difference between the DLBCL patients indicating mutual autophagy dependency. Genes associated to worst overall survival (OS) in autophagy initiating complex I/ ULK1 complex (ULK1, RB1CC1/FIP200 and ATG13) and complex II (AMBRA1, PIK3C3/VPS34, PIK3R4/VPS15 and USO1/p115) were identified using Kaplan-Meier curves and overlapped according to their respective complexes. Statistical analysis confirmed ULK1 complex was significantly dominate in response to R-CHOP treatment leading to poor OS (p=0.0403). These findings provide a rational to inhibit autophagy via the ULK1 complex in GCB patients. Alike, multiple GCB (n=7) and ABC (n=5) cell lines showed substantial autophagy dependency using LC3B autophagy marker expression. Ibrutinib treatment enhanced autophagic flux in GCB cell lines. Addition of ULK1 complex inhibitor (MRT68921) augmented the anti-tumor activity of Ibrutinib in HT, Oci-Ly1, Oci-Ly18 and SUDHL-6 GCB cell lines and activated caspase dependent apoptosis. Interestingly, ULK1 complex inhibitors (MRT68921/ ULK1-101) induced acute NF-ҡB activation in multiple GCB and ABC lymphoma lines irrespective of their NF-ҡB mutational status. RNA sequencing of GCB and ABC cell lines treated with MRT68921, or vehicle reported diverse ULK1 inhibitory signaling pathways. Oci-Ly1 (GCB, c-MYC amplification) treated cells significantly downregulated genes associated to PI3K-mTOR activity/ signaling, autophagy, metabolism, and IL-6. GSEA reported a substantial decrease in c-MYC transcriptional activation/ pathway and TNF signaling. Immunoblotting data confirmed ULK1 inhibition dephosphorylated c-myc(ser62) in a time dependent manner in Oci-Ly1 and Oci-Ly18. In contrast, Oci-Ly3 (ABC, c-MYC amplification) treated cells upregulated genes involved in JAK-STAT pathway, IL-6, and apoptosis. C-MYC remained unaffected. In summary, GCB-DLBCL patients treated with R-CHOP harbor increased autophagy signatures that potentially dictate treatment outcome. Our in vitro data demonstrates ULK1 inhibition sensitizes GCB cell lines to Ibrutinib due to diverse role in regulating NF-ҡB, c-MYC and inflammation. Unlike, ABC, where ULK1 blockade promotes immunogenic-dependent cell death.

Immune Checkpoints Involving the PD-1/PD-L1/L2 and CD73/A2aR Axes and the Immunosuppressive Microenvironment in DLBCL

Background: Immune checkpoint inhibitors that target PD-1/PD-L1 are effective against some solid tumors and Hodgkin's lymphoma (HL). However, the therapeutic benefits of PD-1/PD-L1 inhibitors are limited to a small subset of patients with diffuse large B-cell lymphoma (DLBCL). As immune checkpoint monotherapies rarely elicit effective or long-term responses, targeting the PD-1/PD-L1/L2 pathway combined with other immunosuppressive signaling, such as CD73/A2aR adenosine signaling, has emerged as a promising strategy for cancer treatment. Here, we hypothesized PD-1/PD-L1/L2 and CD73/A2aR contribute to acquiring a CD8+ T cell dysfunctional phenotype. The genetic characteristics and clinical effectiveness of these two immune checkpoints need to be further investigated in DLBCL. Materials and methods: We performed whole-exome sequencing/targeted deep sequencing to investigate the genetic characteristics of PD-1/PD-L1/L2 and CD73/A2aR. The immunosuppressive effect of these two pathways on the tumor microenvironment was evaluated via RNA sequencing. Single-cell RNA sequencing was further applied to investigate the dysfunctional CD8+ T cells. In addition, multiplex immunofluorescence staining was used to quantitatively assess the expression of dysfunctional CD8+ T cells in DLBCL. Results: A frequent copy number gain of 9p24.1, previously described with the upregulation of PD-L1/L2 in HL, was identified in three patients with DLBCL (3/42; 7%), two of which had the non-germinal center B cell-like (GCB) subtype. No CNVs affecting CD73 or A2aR were identified. SP140 was identified as a novel translocation partner for PD-L1 and genomic breakpoint coordinates were mapped to chr9:5467954 and chr2:2311834 00 (NCBI Build 36.1) falling within the 3' untranslated region (UTR) of PD-L1 and the intergenic region downstream of SP140. This translocation partner lead to the upregulation of PD-L1 expression. Additionally, an in-frame PD-L1-PD-L2 inversion caused by the 3' portion of PD-L1 within intron 5 of PD-L1 fused with intron 3 of PD-L2 was also detected in DLBCL. This novel inversion was also ensured to result in the upregulation of PD-L1 expression. However, the expression of PD-L2 was downregulated. Two nonsense mutations and one missense mutation were identified in PD-L1 in three DLBCL patients. The two nonsense variants, p.Arg125* and p.Gln173*, were predicted to generate truncated proteins lacking the transmembrane domain. Another variant, c.302 T>G was located in the coding region of the immunoglobulin subtype with PD-L1 residue isoleucine 101 mutated to serine (p.Ile101Ser). However, PD-L1 expression was not upregulated in these altered samples. Cytogenetic mutations that affected the PD-L1/L2 loci showed no difference in the distribution of subtypes. However, genetic translocations in the PD-L1/L2 loci were primarily associated with the non-GCB subtype of DLBCL. Genetic mutations of CD73 were identified in four cases carrying two missense mutations. One mutation, c.185C>T, encoded the substitution of a highly conserved alanine into valine at position 62 (p.A62V), which is located in the functional calcineurin-like phosphoesterase domain of CD73. CD73 genetic mutations did not increase mRNA and protein expression. However, patients with genetically altered CD73 tended to have a better overall survival than patients with wild-type CD73. Both PD-1/PD-L1 and CD73/A2aR signaling mediated the immunosuppressive microenvironment in DLBCL. The numbers of CD8+ T cells with PD-1 and A2aR expression were positively correlated with the number of dysfunctional CD8+ T cells (R2= 0.974, p = 0.013). According to the grades of dysfunctional CD8+ T cells we defined, grade 1 dysfunctional CD8+ T cells, with either PD-1+ or A2aR+, were significantly associated with poorer survival than grade 0 dysfunctional CD8+ T cells, with both PD-1- and A2aR-; and patients with grade 2 dysfunctional CD8+ T cells showed the worst clinical outcomes. Conclusions: This study describes the additional genetic basis of PD-L1 overexpression and characterizes certain genetic alterations of CD73/A2aR in DLBCL. The degree of T cell dysfunction is correlated with clinical outcomes. Strategies that reverse T cell dysfunction by inhibiting PD-1/PD-L1/L2, particularly in combination with CD73/A2aR, may show potential as effective therapeutic options for DLBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Five-Year Survival Results from the Remodl-B Trial (ISRCTN 51837425) Show Improved Outcomes in Diffuse Large B-Cell Lymphoma Molecular Subgroups from the Addition of Bortezomib to R-CHOP Chemoimmunotherapy

Introduction: The REMoDL-B trial compared R-CHOP versus R-CHOP + bortezomib (RB-CHOP) in patients with diffuse large B-cell lymphoma (DLBCL) stratified by cell of origin (COO) determined by gene expression profile (GEP). The primary analysis of the trial at a median follow-up of 30 months found no overall difference in progression free (PFS) or overall survival (OS) by treatment arm. Retrospective analysis using a gene expression based classifier identified a molecular high grade group (MHG) with distinct molecular features. We present an update of this analysis and the trial final results for patients with lymphomas of activated B-cell (ABC), germinal-centre B-cell (GCB) or MHG type. Methods: REMoDL-B was an open label randomised phase 3 adaptive trial with patients recruited from UK and Switzerland. Eligible patients had untreated, histologically confirmed DLBCL stage I-IV, were >18 year old, ECOG performance status≤2, and had cardiac, lung, renal, and liver function sufficient to tolerate full dose chemotherapy. Patients with sufficient material from initial biopsies for GEP were randomised between R-CHOP or RB-CHOP from cycle 2, stratified by international prognostic index (IPI) and COO. COO classification was performed in the trial with the DLBCL automatic classifier in time for randomisation and reassessed retrospectively adding in the MHG classifier. Time to event analyses were performed using the Kaplan-Meier method and cox regression modelling. REMoDL-B was coordinated by the Southampton Clinical Trials Unit and funded by a grant from Janssen-Cilag and endorsed by Cancer Research UK after independent peer review (C328/A12128). Results: 801 of the 1,077 patients registered were identified as ABC, GCB or MHG type in the final analysis. 128 were unclassified and in 148 the diagnostic biopsy sample was insufficient. Median age was 65 (range 20-86) and 67% were stage III/IV, 48% had abnormal LDH, 29% had bulk, 17% had high IPI and 30% high intermediate IPI, 31% ABC, 59% GCB and 10% MHG. At a median follow up of 64 months in those still alive, there was no overall benefit of bortezomib on PFS or OS (5-year PFS of 64% in R-CHOP and 70% in RB-CHOP HR, 0.81 p=0.085, 5-year OS of 76% vs 79% HR, 0.86; p=0.32). Improved PFS and OS with RB-CHOP was however seen in ABC patients; 5-year PFS of 54% with R-CHOP vs 69% with RB-CHOP (Figure 1); 5-year OS of 67% in the R-CHOP arm vs 80% in the RB-CHOP arm: OS HR 0.58; (95% CI, 0.35 to 0.95) p=0.032. PFS was significantly improved in MHG patients: 5-year PFS 29% with R-CHOP vs 55% with RB-CHOP: HR 0.46 (95% CI 0.26-0.84) p=0.011 (Figure 2). 5-year OS was 48% vs 60% HR, 0.62; 95% (CI, 0.32 to 1.20) p=0.156. Outcomes by LymphGen classification will be presented. Adverse events were similar to those reported previously, with the most common grade 3 or worse being haematological toxicity, reported in 38% R-CHOP patients and 42% RB-CHOP patients. Conclusions: Gene expression profiling can identify subtypes of DLBCL which may benefit from the addition of bortezomib to R-CHOP in initial therapy, specifically, ABC and MHG types. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Special Anatomical Subtypes of Diffuse Large B-Cell Lymphoma Have Distinct Tumor Microenvironments

Background The heterogeneity of diffuse large B-cell lymphoma (DLBCL) and its diverse clinical course encouraged the World Health Organization (WHO) to recognize specific subtypes based on anatomical location. These anatomical subtypes are accompanied by specific mutational profiles and gene-expression patterns. For example, the poor-prognostic primary DLBCL of immune-privileged sites (IP-DLBCL) originating in the central nervous system or testis have frequent NF-κB-activating mutations and a predominantly activated B-cell (ABC) phenotype. In contrast, we recently showed that good-prognostic bone DLBCL (B-DLBCL) have frequent mutations in immune-modulating and epigenetic regulating genes and a germinal center B-cell (GCB) phenotype (PMID: 34478526). We hypothesize that these remarkable anatomical site-specific enrichments for tumors with a distinct molecular signature are associated with different local anti-tumor immune responses. Here, IP-DLBCL and B-DLBCL could be considered opposing ends of a spectrum and we compared the composition of the tumor microenvironment (TME) of these entities to each other and to DLBCL with nodal localization only (nodal-DLBCL). Method This retrospective study used formalin fixed and paraffin embedded biopsy specimens that were selected from clinically, histologically and molecularly well-annotated cases of IP-DLBCL (n=29), B-DLBCL (n=21) and nodal-DLBCL (n=18). All cases were diagnosed according to the revised 2016 WHO classification. High grade B-cell lymphomas (double/triple hit) were excluded. Imaging mass cytometry (Hyperion) was used to define the TME at single-cell resolution and with intact spatial relationships. The panel comprised 41 antibody-markers for deep phenotyping of four major cellular compartments: tumor cells, tumor-infiltrating lymphocytes, myeloid- and stromal cells. Image output was processed to create single-cell data, as described before (PMID: 34196108). Cell phenotyping was performed by supervised clustering after dimension reduction using opt-SNE algorithm (OMIQ software). Cluster abundances were depicted as median (±IQR) per thousand of total cells per patient(‰). Kruskal-Wallis testing was used to determine statistical differences between abundances. Results In total 1,7M single cells (range 7-49k) were obtained and 90 unique cellular phenotypes were defined. Differences in composition of the four cellular compartments were compared between IP-DLBCL and B-DLBCL (Figure 1). The lymphocyte compartment of IP-DLBCL was significantly enriched with granzyme B expressing lymphocytes, mainly cytotoxic T cells (IP 9,4‰ ± 8,8 versus B-DLBCL 2,7‰ ± 3,8; p<0,01), including activation related markers like CCR4 and Ki-67. In contrast, B-DLBCL was characterized by a substantial influx of T cells, more specifically T-helper cells (B-DLBCL 49,9‰ ± 32,5 versus IP 35,4‰ ± 24,6; p<0,01), including T-regulatory cells (B-DLBCL 2,7‰ ± 4,4 versus IP 0,9‰ ± 1,5; p<0,01) and cells expressing ICOS. T-helper 1 cells, expressing TBET, were less abundant (B-DLBCL 0,3‰ ± 0,5 versus IP 1,0‰ ± 0,6; p<0,01). The myeloid compartment was generally increased in B-DLBCL and comprised of HLADR and/or CD11c expressing dendritic cells (B-DLBCL 84,3‰ ± 39,8 vs IP 32,5‰ ± 31,9; p<0,01). In the stromal compartment, a significantly higher expression of activation markers related to the "stromal1" gene signature (PMID: 34597589) in B-DLBCL was identified (B-DLBCL 15,4‰ ± 12,4 vs IP 10,98‰ ± 9,0; p=0,04). Immune checkpoint markers like PD1, LAG3, TIM3, TIGIT and VISTA were generally low in expression within the entire cohort. In nodal-DLBCL both IP- as well as B-DLBCL-like phenotypes were identified. Conclusion This study shows significant differences in the tumor microenvironment of DLBCL subtypes based on anatomical site. In IP-DLBCL, substantial presence of cytotoxic T cells-with activated phenotypes-was identified. In contrast, in bone-DLBCL the TME was generally non-cytotoxic and exhibited immune regulatory elements. Altogether, knowledge on these distinct tumor microenvironments may help understanding heterogeneity between these subtypes and advocates correlation with efficacy of emerging treatments like bispecific antibodies (for the cytotoxic TME) and CAR T-cell therapy (for the non-cytotoxic/immune regulatory TME). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MTOR inhibition amplifies the anti-lymphoma effect of PI3Kβ/δ blockage in diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease that exhibits constitutive activation of phosphoinositide 3-kinase (PI3K) driven by chronic B-cell receptor signaling or PTEN deficiency. Since pan-PI3K inhibitors cause severe side effects, we investigated the anti-lymphoma efficacy of the specific PI3Kβ/δ inhibitor AZD8186. We identified a subset of DLBCL models within activated B-cell–like (ABC) and germinal center B-cell–like (GCB) DLBCL that were sensitive to AZD8186 treatment. On the molecular level, PI3Kβ/δ inhibition decreased the pro-survival NF-κB and AP-1 activity or led to downregulation of the oncogenic transcription factor MYC. In AZD8186-resistant models, we detected a feedback activation of the PI3K/AKT/mTOR pathway following PI3Kβ/δ inhibition, which limited AZD8186 efficacy. The combined treatment with AZD8186 and the mTOR inhibitor AZD2014 overcame resistance to PI3Kβ/δ inhibition and completely prevented outgrowth of lymphoma cells in vivo in cell line- and patient-derived xenograft mouse models. Collectively, our study reveals that subsets of DLBCLs are addicted to PI3Kβ/δ signaling and thus identifies a previously unappreciated role of the PI3Kβ isoform in DLBCL survival. Furthermore, our data demonstrate that combined targeting of PI3Kβ/δ and mTOR is effective in all major DLBCL subtypes supporting the evaluation of this strategy in a clinical trial setting.

Mutational profiling of circulating tumor DNA and clinical characteristics in lymphoma: Based on next generation sequencing.

Liquid biopsy has been experimented with to identify the mutation of lymphoma based on next-generation sequencing (NGS). We applied NGS analysis to circulating tumor DNA (ctDNA) in 20 lymphoma patients. Then, we compared treatment outcomes, and clinical characteristics among these patients, then investigated mutational profiling. Two independent cohorts of 241 patients with mature B cell lymphoma in Mature B-cell malignancies data set (MBN) data set and 50 diffuse large B-cell lymphoma (DLBCL) patients in DLBCL data set, were used to examine the association between gene mutations and prognosis. We found ctDNA positive group had significantly more relapsed/PD (7/12, 58.3%) and less CR/PR patients (1/12, 8.3%) compared to negative group (0, 0%) (5/8, 62.5%) (p < 0.001). Somatic alterations were identified in 12 of 20 patients and the total 11 mutations were: Ataxia telangiectasia mutated (ATM), TP53, BCL2, BTG2, CD28, EP300, IDH2, IRF8, JAK3, NOTCH1, and NRAS. ATM (S2168L) was found in SLL and TLBL for the first time. BTG2 (c.292_293del), CD28 (P119T), IRF8 (E74D) and NOTCH1 (c.4348 G > A) were newly detected in DLBCL, angioimmunoblastic T-cell lymphoma, primary central nervous system lymphoma, and BCL for the first time respectively. We also disclosed an unreported mutation EP300 (c.1058_1059insC) in DLBCL. Our cases implied ctDNA detection consistent with the FISH of tissue samples to some extent, speculating new molecular subtypes of DLBCL, finding some potential drug-resistant mutations, and suggesting disease recurrence. Moreover, in MBN and DLBCL datasets, patients with TP53 mutation had a significantly shorter OS (all p < 0.05) in both circulating free DNA and tumor tissue. The mutations (no SNP) of NOTCH1 (all p < 0.05) significantly contributed to worse OS in the two cohorts.

The path towards consensus genome classification of diffuse large B-cell lymphoma for use in clinical practice.

Diffuse large B-cell lymphoma (DLBCL) is a widely heterogeneous disease in presentation, treatment response and outcome that results from a broad biological heterogeneity. Various stratification approaches have been proposed over time but failed to sufficiently capture the heterogeneous biology and behavior of the disease in a clinically relevant manner. The most recent DNA-based genomic subtyping studies are a major step forward by offering a level of refinement that could serve as a basis for exploration of personalized and targeted treatment for the years to come. To enable consistent trial designs and allow meaningful comparisons between studies, harmonization of the currently available knowledge into a single genomic classification widely applicable in daily practice is pivotal. In this review, we investigate potential avenues for harmonization of the presently available genomic subtypes of DLBCL inspired by consensus molecular classifications achieved for other malignancies. Finally, suggestions for laboratory techniques and infrastructure required for successful clinical implementation are described.

A bone-based 3D scaffold as an in-vitro model of microenvironment-DLBCL lymphoma cell interaction.

About 30% of patients with diffuse large B-cell lymphoma (DLBCL) relapse or exhibit refractory disease (r/r DLBCL) after first-line immunochemotherapy. Bone marrow (BM) involvement confers a dismal prognosis at diagnosis, likely due to the interaction between neoplastic cells and a complex tumor microenvironment (TME). Therefore, we developed a 3D in-vitro model from human decellularized femoral bone fragments aiming to study the role of mesenchymal stromal cells (MSC) and the extracellular matrix (ECM) in the adaptation, growth, and drug resistance of DLBCL lymphoma cells. The 3D spatial configuration of the model was studied by histological analysis and confocal and multiphoton microscopy which allowed the 3D digital reproduction of the structure. We proved that MSC adapt and expand in the 3D scaffold generating niches in which also other cell types may grow. DLBCL cell lines adhered and grew in the 3D scaffold, both in the presence and absence of MSC, suggesting an active ECM-lymphocyte interaction. We found that the germinal center B-cell (GCB)-derived OCI-LY18 cells were more resistant to doxorubicin-induced apoptosis when growing in the decellularized 3D bone scaffold compared to 2D cultures (49.9% +/- 7.7% Annexin V+ cells in 2D condition compared to 30.7% + 9.2% Annexin V+ 3D adherent cells in the ECM model), thus suggesting a protective role of ECM. The coexistence of MSC in the 3D scaffold did not significantly affect doxorubicin-induced apoptosis of adherent OCI-LY18 cells (27.6% +/- 7.3% Annexin V+ 3D adherent cells in the ECM/MSC model after doxorubicin treatment). On the contrary, ECM did not protect the activated B-cell (ABC)-derived NU-DUL-1 lymphoma cell line from doxorubicin-induced apoptosis but protection was observed when MSC were growing in the bone scaffold (40.6% +/- 5.7% vs. 62.1% +/- 5.3% Annexin V+ 3D adherent cells vs. 2D condition). These data suggest that the interaction of lymphoma cells with the microenvironment may differ according to the DLBCL subtype and that 2D systems may fail to uncover this behavior. The 3D model we proposed may be improved with other cell types or translated to the study of other pathologies with the final goal to provide a tool for patient-specific treatment development.

Hypoxia-related tumor environment correlated with immune infiltration and therapeutic sensitivity in diffuse large B-cell lymphoma.

Background: Due to the high heterogeneity of diffuse large B-cell lymphoma (DLBCL), traditional chemotherapy treatment ultimately failed in one-third of the patients. Big challenges existed in finding how to accurately predict prognosis and provide individualized treatment. Hypoxia, although being a key factor in the development and progression of DLBCL, plays its role in DLBCL prognosis, which has yet to be fully explored. Methods: Data used in the current study were sourced from the Gene Expression Omnibus (GEO) database. DLBCL patients were divided according to different hypoxia-related subtypes based on the expressions of hypoxia-related genes (HRGs) relevant to survival. Differentially expressed genes (DEGs) between subtypes were identified using the limma package. Using univariate Cox regression and least absolute shrinkage and selection operator (LASSO) analyses, the prognostic signature was established to calculate risk scores. The tumor microenvironment (TME) in low- and high-risk groups was evaluated by single-sample gene set enrichment analysis (ssGSEA) and ESTIMATE. The chemotherapeutic sensitivity in two groups was assessed by IC50 values. Results: DLBCL patients were clustered into two hypoxia-related subtype groups according to different gene survival and expressions associated with increasing oxygen delivery and reducing oxygen consumption, and these two subtype groups were compared. Based on the differential expression, a risk model was established using univariate cox and LASSO regression analyses, FNDC1, ANTXR1, RARRES2, S100A9, and MT1M. The performance of the risk signature in predicting the prognosis of DLBCL patients was validated in the internal and external datasets, as evidenced by receiver operating characteristic (ROC) curves. In addition, we observed significant differences in the tumor microenvironment and chemotherapeutic response between low- and high-risk groups. Conclusion: Our study developed novel hypoxia-related subtypes in DLBCL and identified five prognostic signatures for DLBCL patients. These findings may enrich our understanding of the role of hypoxia in DLBCL and help improve the treatment of DLBCL patients.

EZH2 inhibitor DZNep blocks cell proliferation of GCB-DLBCL cells by upregulating p16

Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Two main subgroups of DLBCL include germinal center B-cell-like (GCB) and activated B-cell-like (ABC). Molecular profiling can further classify DLBCL into four subtypes: MCD (both CD79B and MYD88 L265P), BN2 (NOTCH2 mutation or BCL6 fusion), N1 (NOTCH1 mutation), or EZB (EZH2 mutation or BCL2 fusion). EZH2 inhibitors were recommended for patients with the EZB subtype of DLBCLs; however, little is known about the therapeutic mechanisms. Our results showed that DZNep arrested G1/S phase of GCB-DLBCL cells and inhibited the cell proliferation in vitro through upregulation of p16 by demethylating its promoter. These results suggest that DZNep may have potential as a novel therapeutic agent for DFLBL therapy. This agent may serve as a novel molecular agent to be applied to GCB DLBCL.

The multilobated morphology is still a better prognosis factor of diffuse large B-cell lymphoma in the R-CHOP era.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of B-cell lymphoma. Although the multilobated subtype of DLBCL has been observed since the 1970s, little is known about the clinical significance of this unique variant in the era of rituximab, cyclophosphamide, hydroxydaunorubicin, oncovin, prednisone/prednisolone (R-CHOP) therapy. In this study, the retrospective clinicopathological analysis of 312 patients diagnosed with DLBCL showed that the multilobated DLBCL group comprised 11% of the cases and was predominantly male (p = 0.027), achieved complete remission in the first therapy (p = 0.023), and exhibited germinal center B-cell phenotypes in the Hans algorithm (p = 0.025). The multilobated DLBCL groups had a better prognosis in overall survival (OS) and progression-free survival (PFS) than the non-multilobated DLBCL group (OS, p = 0.006; PFS, p = 0.010). In the multivariate Cox regression analyses for OS, independent prognosis factors were high soluble IL-2 receptor (p = 0.025), high risk of International Prognostic Index, and multilobated morphology (p = 0.031). The most characteristic copy number gains found in more than 50% of the cases were located at 1q, 3p, 10q, 12q, and 14q. Overall, the multilobated morphology in DLBCL exhibits a good outcome in the R-CHOP era.

Establishment and characterization of a new activated B-cell-like DLBCL cell line, TMD12

We report the establishment of a novel activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) cell line, designated as TMD12, from a patient with highly refractory DLBCL. ABC-DLBCL is a subtype with a relatively unfavorable prognosis that was originally categorized using gene expression profiling according to its cell of origin. TMD12 cells were isolated from the pleural effusion of the patient at relapse and passaged continuously in vitro for >4 years. The cells displayed cluster of differentiation (CD)19, CD20, CD22, CD38, human leukocyte antigen-DR isotype, and κ positivity and CD5, CD10, CD23, and λ negativity, as detected using flow cytometric analysis. The chromosomal karyotypic analysis, including the spectral karyotyping method, confirmed t(1;19)(q21:q13.1), del(6q23), gain of chromosome 18, and other abnormalities. Mutation analyses, including whole-exome sequencing, revealed that TMD12 cells harbored mutations in MYD88 and CD79B, indicating an ABC subtype. TMD12 cells exhibited chronic active B-cell receptor signaling and constitutive activation of the nuclear factor κB pathway, which is typically associated with sensitivity to a specific Bruton tyrosine kinase inhibitor, ibrutinib. Intriguingly, TMD12 cells displayed moderate resistance to ibrutinib and lacked activation of Janus kinase/signal transducers and activators of transcription 3 signaling, another hallmark of this DLBCL subtype. Treatment with an inhibitor against tumor progression locus 2 (TPL2), a multifunctional intracellular kinase that is activated particularly downstream of Toll-like receptors or MYD88 and IκB kinase α/β (IKKα/β), suppressed the proliferation of TMD12 cells, implying the possible involvement of the TPL2-p105 pathway in the tumorigenesis of ABC-DLBCL. Because only a limited number of ABC-DLBCL cell lines are currently available, TMD12 cells might provide a useful tool in the search for novel druggable targets for this intractable lymphoma.

Abstract A15: Clinico-pathologic and molecular characteristics of primary extranodal germinal-center B-cell like diffuse large B-cell lymphoma (EN GCB-DLBCL)

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease and in 30-40% cases can present or relapse at extranodal sites. Primary extranodal DLBCL (EN-DLBCL) is defined as disease presenting as clinically dominant extranodal masses with no or only minor nodal disease. EN-DLBCL is predominantly of the activated B-cell (ABC) and less commonly of germinal center B-cell (GCB) like DLBCL subtype. We aimed to identify genetic characteristics of EN GCB-DLBCL to gain insights into extranodal tropism and aggressive clinical behavior. Methods: Patient data was collected via retrospective chart review. Inclusion criteria consisted of pathologically confirmed diagnosis of GCB-DLBCL by the Hans algorithm, extra-nodal involvement at diagnosis or relapse, and availability of next-generation sequencing (NGS). Overall (OS) and progression-free survival (PFS) were analyzed using the Kaplan-Meier method. Results: Fifteen patients were identified. The median age at diagnosis was 64 years (range 33-89) and majority were men (62.5%). Two patients had purely nodal disease at presentation that relapsed at EN sites, while the remainder (13/15) had predominantly EN disease. EN sites included central nervous system [CNS] (n = 5), bone marrow (n = 4), epidural (n=4), bone (2), paranasal sinus (1), testes (1), stomach (n = 2), lung/pleura (n=2), and liver (n=3). Nine patients had disease in multiple EN sites. All patients had advanced stage disease and median IPI score was 3 (range 3-4). On FISH/cytogenetic analysis, two patients had MYC and four patients had BCL2 rearrangements. Copy gains in BCL6, BCL2 and MYC were seen in 2, 1 and 4 patients respectively. No patients had evidence of MYC and BCL2 and/or BCL6 rearrangements (double hit). TP53 deletion was seen in 1 patient. On NGS, prominent gene mutations identified were KMTD2 (8/15, 53%), TP53 (5/15, 33%), TNFRSF14 (4/15, 27%), and evidence of aneuploidy with abnormalities in chromosome 12 (5/15, 33%), chromosome 6 (4/15, 27%), and chromosome 8 (4/15, 27%), including both losses and gains of function. Notably, MYD88 (3/15, 20%), CARD11 (2/15, 13%), and IRF4 (2/15, 15%) mutations, representative of BCR/TLR4 pathway activation and NF-kB signaling were seen at initial presentation. All patients were treated with chemo-immunotherapies incorporating CNS prophylaxis and 2 patients received salvage CAR T-cell therapy. Median OS was 1.3 years and median PFS was 11.3 months. Nine patients (63%) progressed, with CNS relapses in 5 and of these, 7 patients died. Conclusions: This study shows that EN GCB-DLBCL is molecularly similar to EN ABC-DLBCL; specifically, activation of NF-kB pathway signaling through gene mutations involving the MCD (MyD88/CD79B) cluster is prominent, and this potentially enables extranodal tropism. The complex karyotype and TP53 mutations may contribute to chemoresistance and aggressive disease biology. Our results provide valuable molecular information and can aid in personalizing treatment strategies for this uncommon, aggressive DLBCL subset. Citation Format: Akshat Patel, Brian Lue, Kiran A Kumar, Farrukh T Awan, Jeffrey Gagan, Weina Chen, Praveen Ramakrishnan Geethakumari. Clinico-pathologic and molecular characteristics of primary extranodal germinal-center B-cell like diffuse large B-cell lymphoma (EN GCB-DLBCL) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A15.