DLBCL Subtypes Research Articles

Development and validation of a hierarchical approach for lymphoma classification using immunohistochemical markers.

Accurate lymphoma classification is critical for effective treatment and immunohistochemistry is a cost-effective and time-saving approach. Although several machine learning algorithms showed effective results, they focused on a specific task of classification but not the whole classification workflow, thus impractical to be applied in clinical settings. Thus, we aim to develop an effective and economic machine learning-assisted system that can streamline the lymphoma differential diagnostic workflow using EBER insitu hybridization and immunohistochemical markers. We included pathological reports diagnosed as lymphomas from two cancer centers (Sun Yat-sen University Cancer Center and Peking University Cancer Hospital & Institute). We proposed a hierarchical approach that mimicked the human diagnostic process and employed simplified panels of markers to perform a series of interpretable classification. The diagnostic accuracy for lymphoma pathological subtypes and the markers saving ratio were investigated in both temporal independent population and external medical center. A total of 14,927 patients and corresponding immunohistochemical results from two cancer centers were included. The proposed system had high discriminative ability for differentiating lymphoma pathological subtypes (measured by mean AUC in three validation cohorts, non-Hodgkin and Hodgkin lymphoma: 0.959; non-Hodgkin subtypes: 0.983; B-lymphoma subtypes: 0.868; T-lymphoma subtypes: 0.962; DLBCL subtypes: 0.957). In addition, the system's well selected characteristics can contribute to the development of agreement on panels of markers for differential diagnosis and help minimize cost of immunohistochemical marker techniques (measured by marker saving ratio compared to real clinical settings, internal primary-stage cohort: 16.45% saved, p < 0.001; internal later-stage cohort: 21.73% saved, p < 0.001; external cohort: 3.67% saved, p < 0.001). Machine learning-based hierarchical system using EBER insitu hybridization and IHC markers was developed, which could streamline the workflow by sequentially determining each lymphoma pathological subtype. The proposed system proved to be effective and cost-saving in independent and external validation, thus could be adopted affordably in future clinical practice.

Cystatin SN (CST1) Is a Poor Independent Prognostic Biomarker for Gastrointestinal Diffuse Large B-Cell Lymphoma.

BACKGROUND Gastrointestinal diffuse large B-cell lymphoma (GI-DLBCL) is the most common histological subtype of extra-nodal DLBCL, but the risk factors, prognostic biomarkers, histopathological classifications, and treatment strategies have not had significant progress. Emerging evidence shows that cystatin SN (CST1) is involved in tumor progression in several cancer types, but its role in GI-DLBCL has not been revealed. MATERIAL AND METHODS We established a cohort consisting of 84 patients with GI-DLBCL who underwent surgical resection. The expression of CST1 in the cohort was investigated by immunohistochemistry, which divided the patients into subgroups with low or high expression of CST1. Moreover, the CST1 expression in GI-DLBCL tissues or adjacent GI tissues were compared with RT-qPCR. The correlation between CST1 expression and clinicopathological factors was analyzed with the chi-square test. The prognostic significance of CST1 was estimated by univariate and multivariate analysis, and statistical significance was analyzed with the log-rank test. RESULTS CST1 was aberrantly upregulated in GI-DLBCL tissues compared with in non-tumor GI tissues. High expression of CST1 indicated poor prognosis of GI-DLBCL (P=0.012), and CST1 can be regarded as an independent prognostic biomarker of GI-DLBCL (hazard ratio=3.07). In our study, serum lactate dehydrogenase (P=0.002), performance status (P=0.003), Lugano stage (P=0.002), and International Prognostic Index (P=0.001) were also prognostic factors of GI-DLBCL. CONCLUSIONS CST1 is an independent prognostic biomarker of GI-DLBCL, indicating unfavorable prognosis. Our results suggested that CST1 detection can be a promising method to stratify high-risk patients and guide individual treatment.

Hyper-N-glycosylated SEL1L3 as auto-antigenic B-cell receptor target of primary vitreoretinal lymphomas

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress into primary central nervous system lymphoma (PCNSL). To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed B-cell receptors (BCR) from PVRL patients and tested for binding against human auto-antigens. SEL1L3, a protein with multiple glycosylation sites, was identified as the BCR target in 3/20 PVRL cases. SEL1L3 induces proliferation and BCR pathway activation in aggressive lymphoma cell lines. Moreover, SEL1L3 conjugated to a toxin killed exclusively lymphoma cells with respective BCR-reactivity. Western Blot analysis indicates the occurrence of hyper-N-glycosylation of SEL1L3 at aa 527 in PVRL patients with SEL1L3-reactive BCRs. The BCR of a PVRL patient with serum antibodies against SEL1L3 was cloned from a vitreous body biopsy at diagnosis and of a systemic manifestation at relapse. VH4-04*07 was used in both lymphoma manifestations with highly conserved CDR3 regions. Both BCRs showed binding to SEL1L3, suggesting continued dependence of lymphoma cells on antigen stimulation. These results indicate an important role of antigenic stimulation by post-translationally modified auto-antigens in the genesis of PVRL. They also provide the basis for a new treatment approach targeting unique lymphoma BCRs with ultimate specificity.

Antigen-independent, autonomous B cell receptor signaling drives activated B cell DLBCL.

Diffuse large B cell lymphoma of activated B cell type (ABC-DLBCL), a major cell-of-origin DLBCL subtype, is characterized by chronic active B cell receptor (BCR) signaling and NF-κB activation, which can be explained by activating mutations of the BCR signaling cascade in a minority of cases. We demonstrate that autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia (CLL), can explain chronic active BCR signaling in ABC-DLBCL. 13 of 18 tested DLBCL-derived BCR, including 12 cases selected for expression of IgM, induced spontaneous calcium flux and increased phosphorylation of the BCR signaling cascade in murine triple knockout pre-B cells without antigenic stimulation or external BCR crosslinking. Autonomous BCR signaling was associated with IgM isotype, dependent on somatic BCR mutations and individual HCDR3 sequences, and largely restricted to non-GCB DLBCL. Autonomous BCR signaling represents a novel immunological oncogenic driver mechanism in DLBCL originating from individual BCR sequences and adds a new dimension to currently proposed genetics- and transcriptomics-based DLBCL classifications.

Lymphomas affecting the submandibular glands.

Lymphomas affecting the submandibular glands are very uncommon and few reports are currently available in the literature. Therefore, the aim of the current study is to describe the clinical and microscopic features of an original series of lymphomas affecting the submandibular glands. The pathology files of two institutions were searched for lymphoma cases affecting the submandibular glands. The original hematoxylin and eosin, and immunohistochemical slides were revised by a pathologist for diagnosis confirmation following the revised 4th edition of the World Health Organization classification of tumours of haematopoietic and lymphoid tissues. Clinical data regarding age, sex, clinical manifestation, treatment, follow-up and status at last appointment were retrieved from the patients' medical charts. During the period investigated, 16 cases were included in the study. Females predominated (10:6) with a mean age of 57.8 years-old. Tumors usually presented as asymptomatic swellings. MALT lymphoma represented the most common subtype, followed by diffuse large B cell lymphoma and follicular lymphoma. Three patients died, one of them affected by plasmablastic lymphoma, one by DLBCL and one by MALT lymphoma. Low-grade B cell lymphomas predominate in the submandibular glands, but DLBCL and other subtypes may also be rarely diagnosed in this salivary gland.

Hysterectomy, oophorectomy and risk of non-Hodgkin's lymphoma.

Hysterectomy is associated with an increased risk for adverse health outcomes. However, its connection to the risk of non-Hodgkin's lymphoma (NHL) remains unclear. The aims of our study were to investigate the associations between hysterectomy, oophorectomy and risk of NHL and its major subtypes (eg, diffuse large B-cell lymphoma [DLBCL]), and whether these associations were modified by exogenous hormone use. Postmenopausal women (n = 141,621) aged 50-79 years at enrollment (1993-1998) from the Women's Health Initiative were followed for an average of 17.2 years. Hysterectomy and oophorectomy were self-reported at baseline. Incident NHL cases were confirmed by central review of medical records and pathology reports. During the follow-up period, a total of 1719 women were diagnosed with NHL. Hysterectomy, regardless of oophorectomy status, was associated with an increased risk of NHL (hazard ratio [HR] = 1.23, 95% confidence interval [CI]: 1.05-1.44). Oophorectomy was not independently associated with NHL risk after adjusting for hysterectomy. When stratified by hormone use, the association between hysterectomy and NHL risk was confined to women who had never used hormone therapy (HR = 1.35, 95% CI: 1.06-1.71), especially for DLBCL subtype (P for interaction = .01), and to those who had undergone hysterectomy before the age of 55. Our large prospective study showed that hysterectomy was a risk factor of NHL. Findings varied by hormone use. Future studies incorporating detailed information on the types and indications of hysterectomy may deepen our understanding of the mechanisms underlying DLBCL development and its potential interactions with hormone use.

An inducible Cd79b mutation confers ibrutinib sensitivity in mouse models of Myd88-driven diffuse large B-cell lymphoma

AbstractDiffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma and constitutes a highly heterogenous disease. Recent comprehensive genomic profiling revealed the identity of numerous molecularly defined DLBCL subtypes, including a cluster which is characterized by recurrent aberrations in MYD88, CD79B, and BCL2, as well as various lesions promoting a block in plasma cell differentiation, including PRDM1, TBL1XR1, and SPIB. Here, we generated a series of autochthonous mouse models to mimic this DLBCL cluster and specifically focused on the impact of Cd79b mutations in this setting. We show that canonical Cd79b immunoreceptor tyrosine-based activation motif (ITAM) mutations do not accelerate Myd88- and BCL2-driven lymphomagenesis. Cd79b-mutant murine DLBCL were enriched for IgM surface expression, reminiscent of their human counterparts. Moreover, Cd79b-mutant lymphomas displayed a robust formation of cytoplasmic signaling complexes involving MYD88, CD79B, MALT1, and BTK. These complexes were disrupted upon pharmacological BTK inhibition. The BTK inhibitor–mediated disruption of these signaling complexes translated into a selective ibrutinib sensitivity of lymphomas harboring combined Cd79b and Myd88 mutations. Altogether, this in-depth cross-species comparison provides a framework for the development of molecularly targeted therapeutic intervention strategies in DLBCL.

Abstract B079: Molecular determinants of sensitivity to Polatuzumab-Vedotin

Abstract Purpose: Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the B cell surface antigen CD79B. When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL overall; however, there is noted heterogeneity in response to Pola-V, with germinal center b-cell (GCB) DLBCL showing no added benefit to the addition of Pola-V compared to standard immunochemotherapy. We aimed to identify molecular determinants of sensitivity or resistance to Pola-V. We hypothesized that these might lead us to innovative strategies to improve on-target tumor killing by Pola-V, or to find novel biomarkers that predict drug resistance. Methods: We employed combined drug-sensitization and CD79B-sorted CRISPR-Cas9 screening to identify molecular determinants of sensitivity to CD79B-directed, tumor killing by Pola-V in 9 cell lines representing different molecular subtypes of DLBCL. Results: Our results reveal the striking impact of epitope glycosylation, specifically a2,6 sialylation, on the binding of Pola-V to CD79B and thereby its ability to kill tumor cells. Specifically, we identify the exact glycosylated residues on CD79A and CD79B which create a sialylated glycan shield around the Pola-V binding site, precluding binding to its target. We show how genetic, pharmacological and enzymatic approaches that remove terminal sialic acid residues from these N-linked glycans lead to enhanced tumor killing by Pola-V. We hypothesize and test multiple methods of targeting this pathway in order to enhance Pola-V killing both in vitro and in vivo. Finally, we reveal a previously unappreciated role for the ubiquitin ligase KLHL6 in regulating CD79B protein abundance and surface expression of the B cell antigen receptor (BCR), including how this pathway is used by physiological germinal center B cells and how it is corrupted to enhance BCR expression in GCB DLBCL. Conclusions: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, we identify how KLHL6 determines expression of the BCR in both physiological and malignant germinal center B cells, and how KLHL6 mutation may modulate sensitivity of GCB DLBCL tumors to Pola-V. Citation Format: Sean R Corcoran, Jaewoo Choi, Rachel E Fenner, Xin Yu, Sebastian Scheich, Galina Schevchenko, Evangelia K Papachristou, Vivian M Morris, Kamal Kishore, Clive S D'Santos, Stefania Pittaluga, George Wright, Jagan Muppidi, Daniel J Hodson, Louis M Staudt. Molecular determinants of sensitivity to Polatuzumab-Vedotin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B079.

Genetic Knockin Approaches to Reconstructing DLBCL-Subtypes from Human Germinal Center B-Cells

Background and Significance: The clinical and genetic heterogeneity of DLBCL has led to the recent classification of the disease into five genetically distinct subtypes. The MCD/C5 subtype of DLBCL is defined by genetic alterations of CD79B at the first tyrosine residue of its ITAM motif (CD79B Y196H), crucial for signaling downstream of the BCR, and MYD88 (MYD88 L265P), a signaling adaptor protein essential for Toll Like receptor signaling. CD79B Y196H and MYD88 L265P mutations together define the MCD/C5 subtype and were found to be co-dependent for proliferation and oncogenic BCR-signaling in vitro. However, the mechanism of epistatic interaction between CD79B Y196H and MYD88 L265P is unknown. The MCD/C5 DLBCL subtype is associated with particularly poor clinical outcomes, however only a few human cell lines and a genetic mouse model are available for preclinical testing. Here, we developed a novel genetic approach to engineer the MCD/C5 subtype of DLBCL in primary human tonsillar germinal center B cells. To this end, we introduced the CD79B Y196H and MYD88 L265P mutations as genetic knockin alleles utilizing CRISPR-Cas9 homology directed repair templates (HDRT). In addition, we developed humanized NBSGW mice carrying a mouse Tslp-transgene to enable development of lymph nodes and secondary lymphoid organs to propagate genetically engineered GC B-cells carrying CD79B Y196H and MYD88 L265P knockin mutations for lymphoma development ( Figure 1). Results: Primary human germinal center B-cells were isolated from tonsils and grown on follicular dendritic feeder cells expressing CD40L and IL21. Single strand DNA templates were designed for CD79B, with and without Y196H mutation. Constructs were linked to fluorescent reporters by T2A sites and flanked by homology arms of ~280 bp targeting the guide RNA cut site. The target site was selected to disrupt the endogenous gene, while placing the construct under transcriptional control of endogenous promoter/enhancer elements. We achieved a knock-in efficiency of 5-8%, prior to single-cell cloning and genotyping to confirm the presence of heterozygous CD79B Y196H mutant CD79B Y196 wild-type mutations ( Figure 1). Preliminary biochemical studies show a delayed response and reduced intensity of signaling downstream of the BCR upon stimulation in CD79B Y196H mutant, compared to CD79B Y196 wild-type knockin germinal center B-cells. A similar approach was taken to design temples encoding Exon 5 of MYD88, with and without the L265P mutation, utilizing two guide RNAs to excise and replace Exon 5, linked to a mCherry reporter by a T2A site. Upon successfully introducing the MYD88 HDR templates into our CD79B wild-type and mutant clones to generate four distinct genotypes based on wildtype and CD79B Y196H and MYD88 L265P mutations alone or in combination. In addition, traditional NSG mice lack lymph nodes and secondary lymphoid organs. Lack of lymphoid follicles in NSG mice, owing to defective IL7Rα-signaling, contributed to lack of lymphoma formation observed in the previous model. In our model, we substituted the missing IL7Rα-signal with transgenic expression of murine Tslp, which enables development of embryonic lymphoid tissue inducer (LTi) cells that are required for the development of secondary lymphoid organs and germinal center follicles supporting initiation of human DLBCL development. Conclusion: Previous approaches to model DLBCL starting from germinal center B-cells were based on lentiviral overexpression of mutant oncogenes. Thereby, tonsillar germinal center B-cells with lentiviral BCL2 and BCL6 overexpression did not form lymphomas in NSG mice, however, were permissive to malignant transformation by MYC. Building on this previous work, we propose an improved model for preclinical testing for genetically defined DLBCL subtypes. Our two main improvements involve (i) genetic modeling based on knockin-strategies targeting endogenous loci and transcriptional control elements to phenocopy physiological gene expression levels and transcriptional regulation, and (ii) substitution of defective IL7Rα-signaling by transgenic expression of murine Tslp to rescue development of LTi cells. Transgenic expression of mTslp enables LTi-dependent formation of lymph nodes and secondary lymphoid organs supporting lymphoma development.

Outcomes in Patients with Relapsed Refractory T-Cell/Histiocyte-Rich Large B-Cell Lymphoma Treated with CAR-T Cell Therapies or Salvage Chemotherapy - a Single-Institution Experience

T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL) is a rare and aggressive variant of large B-cell lymphoma. A substantial subset of THRLBCL patients (pts) relapse after frontline anthracycline-based therapy. There is a scarcity of outcome data on therapy options for relapsed/refractory (r/r)THRLBCL, particularly CD19 CAR-T therapy.A recent genetic and spatial analysis provided critical insights into the complex interactions at the tumor-immune interface in THRLBCL. Understanding the mechanisms by which tumor cells evade the immune system is crucial in designing treatment strategies. This retrospective study aims to analyze clinicopathologic data in pts with r/r THRLBCL with the goal of identifying patterns that may influence response rates and long-term survival. METHODS: Pts who were evaluated and treated at MD Anderson Cancer Center (MDACC) with THRLBCL from 1/2005-5/2023 were included in the analysis. We reviewed demographic, clinical and pathological characteristics, treatments, and outcomes through an approved IRB protocol. We excluded cases with ambiguous diagnosis (such as DLBCL with increased infiltrate of T-cells and/or histiocytes), missing treatment, and/or no follow up information. Progression-free survival (PFS) and overall survival (OS) were calculated from start of treatment until progression or death using Kaplan-Meier. Log-rank test was used to evaluate the difference between pt groups. Statistical software SAS 9.4 (SAS, Cary, NC) and S-Plus 8.2 (TIBCO Software Inc., Palo Alto, CA) were used for all analyses. RESULTS: A total of 212 pts were reviewed, and we excluded 86 pts who did not meet selection criteria. Out of 126 pts, 63 with biopsy-confirmed relapse after 1st line chemoimmunotherapy were included in the analysis (Table-1). The median age was 49 (20-79 years); and 49 pts (78%) were males. Twenty-one (34%) pts had documented NLPHL at the time of or predating the THRLBCL diagnosis. At relapse, 93 % had advance stage disease (stage 3 and 4). Splenic involvement was documented in 57% and 80% of pts had one or more sites of extra nodal disease. Most pts who relapsed received front line RCHOP (77%) or R-EPOCH (11%). Twenty-six pts (41%) had primary refractory disease, and 24 pts (38%) relapsed within 12 months. The median time to progression from 1st line treatment was 7.4 months(2.2~145.9 months). The most common 2 nd line therapy was salvage chemotherapy (82%) such as R-ICE (n=41), R-DHAP(n=4), R-GemOx (n=3). Overall response rate (ORR) to 2 nd line salvage chemotherapy was 75% with CR of 57%. For pts who received 2nd line salvage chemotherapy with the intent to undergo HSCT, the PFS at 2 years was 53% (95% CI: 0.4-0.68, p&amp;lt; 0.0001) and OS was 64% (95% CI: 0.5- 0.8, p&amp;lt; 0.372). Among pts who received consolidation with HSCT, (n=30, 57%), 5 pts relapsed at the time of last follow up.With a median follow-up time of 60.1 months (95% CI: 33.6~90.3 months), the median PFS from all 2nd line treatments was 11 months (95% CI: 4.7 ~ NA ) and for pts who received salvage chemotherapy with HSCT intent was 50.26 (95% CI: 7 ~ NA months). The median OS from 2nd line for all pts was 77.3 months (95% CI: 22.2 ~ NA months) and for pts who received salvage chemotherapy with HSCT intent OS was not reached (95% CI: 32 ~ NA months). Among 11 pts who received 3 rd line salvage chemotherapy, the median PFS was 2 months, and only one pt received HSCT. All pts eventually had progressive disease. Eleven pts received CD19 CAR-T (Axi-cel-9, Liso-cel-2),(table-1) 6 pts received as 2 nd line, 4 received as 3rd line and 1 pt received as 4 th line. Best ORR to CAR-T at any lines was 45% with CR of 36.4%. At a median follow-up time of 12.3 months (95% CI: 2.4 ~ NA months), all but one pt had disease progression. Median PFS was 1.7 months (95% CI: 1.1 ~ NA months). 6-month PFS was 21% (Figure 1). One pt who had response experienced prolonged remission of 17 months before relapsing. CONCLUSION: Our study highlights the unique biology and poorer prognosis of TCHRBCL compared to other DLBCL subtypes. Despite the success of CD19 CAR-T therapy in DLBCL, TCHRBCL pts have shown limited response to these therapies, demonstrating the challenges of achieving long-term remission in this group. Our study shows the high unmet need for improved therapies that can target the unique biology in TCHRBCL. We plan to report a comparative spatial analysis evaluating the tumor-immune interface that can contribute to resistance to Immune effector cell therapies at the meeting.

Assessment of the Expression of Genes Related to Epigenetic and Chromatin Modifications in Diffuse Large B-Cell Lymphoma Patients Resistant to R-CHOP

Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. The mainstay of frontline therapy remains R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) or R-CHOP-like immunochemotherapy. Patients' responses to R-CHOP therapy vary depending on the stage and/or molecular subtypes of DLBCL. Approximately 40% of advanced-stage DLBCL patients suffer from a refractory or relapsed disease[1]. The outcome for patients with early relapses after rituximab-containing first-line therapy remains poor[2]. DLBCL poses a marked degree of genetic heterogeneity. Here, we compare the expression of genes related to epigenetics and chromatin modifications in DLBCL patients resistant and non-resistant to R-CHOP therapy. Objective The aim of this study is to assess the differences in the expression of genes related to epigenetics and chromatin modification in DLBCL patients that could lead to chemoresistance and failure of R-CHOP treatment. Method The gene expression profile (GEP) was obtained from Gene Expression Omnibus (GEO) database (Accession number: GSE173263) submitted by Dlouhy et al[3]. The GEP was analyzed in DLBCL cases, including 10 early failure cases (defined as refractory to induction treatment or early relapsing (&amp;lt;12 months from diagnosis)) and 29 responding patients, to identify features associated to primary chemoresistance. The comparison was done using the list of genes proposed by Bakhshi, T. et. al[4] , where he selected the genes that were identified to potentially cause epigenetic and chromatin modifications in DLCBL. All the comparisons were done using a T-test. Results There were significant differences in the gene expression related to epigenetics and chromatin modifications between DLBCL patients in the early failure arm versus the remission arm. Patients who had early failure to R-CHOP tended to have higher expression of these genes: SMARCA4 (p=0.010), SETD1B (p=0.018), SETD2 (p=0.024), EP300 (p=0.012), MLL3 (p=0.049), INO80 (p=0.001), CHD8 (p=0.007), NCOR1 (p=0.020), ARID1B (p=0.034), and SETD5 (p=0.007). Compared to the patients who were in remission after R-CHOP therapy, these patients had significantly higher expression of HIST1H1E (p=0.007). Conclusion Gene expression related to epigenetics and chromatin modifications significantly impacts the prognosis of DLBCL patients treated with R-CHOP. Differences in gene expression may lead to chemoresistance, and higher expression levels are associated with a worse prognosis. This study sheds light on the importance of chromatin and epigenetic modifications in DLBCL progression, guiding the development of future therapies.

Favezelimab (anti-LAG-3) Plus Pembrolizumab in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma (DLBCL): Cohort 3 of a Multicohort Open-Label Phase 1/2 Study

Background: Lymphocyte-activation gene 3 (LAG-3) is involved in the regulation of T-cell function and is commonly coexpressed with PD-1 on anergic T cells. Favezelimab (MK-4280), a humanized IgG4 anti-LAG-3 monoclonal antibody, plus pembrolizumab (anti-PD-1) is being investigated in the multicohort phase 1/2 MK-4280-003 efficacy and safety study (NCT03598608) in patients with relapsed or refractory (R/R) hematologic malignancies. Prior analyses of the combination demonstrated antitumor activity and manageable safety in patients with anti-PD-1-naive R/R classical Hodgkin lymphoma (cHL; cohort 1; ORR, 73%; CR, 30%) (Johnson NA et al. Blood. 2022;140(suppl 1):6540-2) and anti-PD-1-refractory cHL (cohort 2; ORR, 29%; CR, 9%) (Timmerman J et al. Blood. 2022;140(suppl 1):768-70). We present results from analysis of patients with R/R DLBCL enrolled in cohort 3. Methods: In this study, a safety lead-in phase (part 1) to determine the recommended phase 2 dose (RP2D) was followed by a dose-expansion phase (part 2). In cohort 3, eligible patients were ≥18 years old, had histologically confirmed R/R DLBCL that had progressed after ≥2 lines of previous therapy, including progression after autologous stem cell transplant (ASCT), had declined ASCT, or were ineligible for ASCT. Patients with Richter transformation were not permitted. In part 1, patients received favezelimab at a starting dose of 200 mg that was escalated to 800 mg plus pembrolizumab at 200 mg IV every 3 weeks (Q3W) per the modified toxicity probability interval method. In the dose-expansion phase, patients received favezelimab at the established RP2D of 800 mg plus pembrolizumab 200 mg Q3W for ≤35 cycles (~2 years). Response assessments were performed at weeks 12 and 24 (PET) and Q12W (CT). Adverse events (AEs) were graded per the NCI CTCAE v4.0. The primary end point was safety. ORR per IWG 2007 criteria by investigator review was a secondary end point. Exploratory end points included duration of response (DOR) and progression-free survival (PFS) per IWG 2007 criteria by investigator review and overall survival (OS). Results: A total of 25 patients with R/R DLBCL were enrolled. Patients had a median age of 73 years (range, 25-87), 10 (40%) had ECOG PS 0, and 15 (60%) had ≥3 prior lines of therapy. Most common subtypes of DLBCL were unspecified DLBCL (n = 12; 48%) and germinal center B-cell DLBCL (n = 6; 24%). At database cutoff (March 2, 2023), 1 patient (4%) was ongoing on treatment and 24 (96%) had discontinued because of progressive disease (n = 14), AEs (n = 2), clinical progression (n = 5), or patient noncompliance/nonstudy anticancer therapy (n = 3); 1 patient (4%) discontinued because of treatment-related AEs. No treatment-related deaths occurred. Sixteen patients (64%) had a treatment-related AE; the most common (≥5%) were cough (16%), increased blood alkaline phosphatase (12%), and hypothyroidism, constipation, infusion related reaction, increased AST, muscle spasms, headache and pruritis (8% each). Grade 3 or 4 treatment-related AEs occurred in 4 patients (16%; 1 grade 3 lymphocytic hypophysitis, 1 grade 3 infectious enterocolitis, 1 grade 3 increased AST, 1 grade 3 increased ALT, 1 grade 3 increased amylase, and 1 grade 4 decreased neutrophil count). AEs of clinical interest occurred in 5 patients (20%); only one grade ≥3 occurred (grade 3 hypophysitis). The median (range) time from first dose to data cutoff was 25.9 (19.7-52.1) months. The objective response rate was 12% (95% CI, 2.5-31.2; [2 CR, 1 PR]). Among 17 patients with a postdose scan, 7 (41%) had a reduction from baseline in target lesion size, and 6 (35%) had ≥50% reduction from baseline. Median DOR was not reached (range, 3.0+ to 16.9+ months); 1 responder had an observed response duration of ≥12 months. Median PFS was 2.1 months (95% CI, 1.1-2.7); 12-month PFS rate was 12%. As of the data cutoff, 20 patients (80%) had died. Median OS was 6.4 months (95% CI, 2.3-15.8); 12-month OS rate was 43%. Conclusion: Favezelimab 800 mg plus pembrolizumab 200 mg had limited antitumor activity in patients with DLBCL in cohort 3. Analyses are underway to identify biomarkers predictive of response to the combination of favezelimab and pembrolizumab. The safety profile was manageable and consistent with that observed in other cohorts in the study.

Response to therapy in Richter syndrome: a systematic review with meta-analysis of early clinical trials.

Richter syndrome (RS) represents the clonal evolution of chronic lymphocytic leukemia with histological transformation into a high-grade B cell lymphoma (diffuse large B cell lymphoma - DLBCL) or Hodgkin lymphoma. Considering that RS is an uncommon condition with poor prognosis, few high-quality evidence is available. To overcome this unmet need, this meta-analysis aimed to pool efficacy of early clinical trials in Richter syndrome (DLBCL subtype). MEDLINE, Scopus and Web of Science were searched up to May of 2023 to identify clinical trials decoying efficacy. The pooled complete response, objective response and intension-to-treat failure rates were calculated by pharmacological categories (classical chemotherapy, immunochemotherapy, immunotherapy, Bruton-tyrosine kinase inhibitors, targeted approaches, cell-based therapies and combinatorial regimens) using the Der-Simonian and Laird random-effects model. The Freeman-Tukey double arcsine method was used to estimate variance and confidence intervals. Heterogeneity was assessed using the I2 method. Overall, from 1242 studies identified, 30 were included, pooling data from 509 patients. The higher efficacy rates when, cell-based therapies were excluded, were achieved by immunochemotherapeutic regimens followed by combinatorial regimens, with complete response rates of 21.54% (IC95%14.93-28.87) and 23.77% (IC95% 8.70-42.19), respectively. Bispecific antibodies (alone or coupled with a chemotherapy debulking strategy) overtook Bruton tyrosine kinase inhibitors response rates. The latter, although achieving objective response rates above average, presented scarce complete response rates. Checkpoint inhibitors alone usually do not lead to complete responses, but their effectiveness may improve when combined with other agents, unveiling the importance of immune microenvironmental modulation. This is the first meta-analysis of early clinical trials assessing the impact of different therapeutics in RS. By analyzing the pooled efficacy estimates, our work suggests the role of a tailor-made bridging therapy for young patients with RS eligible for allogeneic hematopoietic stem cell transplantation (alloSCT), formally the only curative strategy.

Deciphering the Clinical Benefit of Pola-R-CHP versus R-CHOP in Different Genetic Subtypes Beyond Cell of Origin in the POLARIX Study

Introduction: In the POLARIX study, polatuzumab vedotin in combination with rituximab plus cyclophosphamide, doxorubicin, and prednisone (Pola-R-CHP) demonstrated prolonged progression-free survival (PFS) vs rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in patients (pts) with previously untreated diffuse large B-cell lymphoma (DLBCL; NCT03274492; Tilly et al. N Engl J Med 2022); the PFS benefit was sustained with longer follow-up (Herrera et al. Blood 2022). Exploratory analyses demonstrated a trend towards greater PFS improvement in activated B cell-like (ABC) DLBCL with Pola-R-CHP vs R-CHOP (Tilly et al. N Engl J Med 2022). Although not yet available for daily practice, more granular molecular subtypes of DLBCL beyond cell of origin (COO) classification have demonstrated poor prognosis with R-CHOP, including EZH2 mutations/ BCL2 translocations (EZB), MYD88/ CD79B-mutated (MCD; as defined by LymphGen; Wright et al. Cancer Cell 2020), and dark zone gene expression signature (DZsig; Alduaij et al. Blood 2023). Here, in a post hoc exploratory analysis, we investigate the prevalence and clinical outcomes of molecularly defined subtypes of DLBCL in pts treated with Pola-R-CHP vs R-CHOP in POLARIX. Methods: All genetic analyses were performed on baseline tumor biopsies. Using mutations derived from whole exome sequencing (WES), pts were allocated to different genetic subtypes, as defined by the LymphGen classifier. DZsig status was determined from global gene expression patterns (GEP) as measured by RNAseq, as previously described (Ennishi et al. J Clin Oncol 2019). COO was determined by NanoString. Hazard ratios (HR) were adjusted for International Prognostic Index score (2 vs 3-5) and age (≤60 vs &amp;gt;60 years). Due to the exploratory nature of the subtype analyses, all statistics are descriptive. Results: WES data were available for 594 pts (Pola-R-CHP, n=292; R-CHOP, n=302), and baseline clinical characteristics were balanced between treatment arms. The observed 2-year PFS rates with EZB and MCD subtypes were numerically higher but not statistically significant in the Pola-R-CHP vs R-CHOP arm ( Table): EZB, 83% (95% confidence interval [CI]: 76-92) vs 75% (95% CI: 65-86), HR 0.61, 95% CI: 0.33-1.13; MCD, 85% (95% CI: 73-98) vs 73% (95% CI: 63-90), HR 0.64, 95% CI: 0.24-1.70. Lower 2-year PFS rates were observed in pts with the BCL6 fusions and NOTCH2 mutations (BN2) subtype treated with Pola-R-CHP vs R-CHOP (HR 1.82, 95% CI: 0.46-7.18; Table). In the subgroup of pts with an undetermined genetic subtype, 2-year PFS estimates were numerically higher with Pola-R-CHP vs R-CHOP (74% vs 62%, respectively; HR 0.70, 95% CI: 0.46-1.07). GEP data were available for 665 pts (Pola-R-CHP, n=331; R-CHOP, n=334); 108 (16.2%) were DZsig+ (Pola-R-CHP, n=52; R-CHOP, n=56). Of those who were DZsig+, 103 (95.4%) had germinal center B cell-like (GCB) DLBCL, 3 (2.8%) had ABC DLBCL, and 2 (1.9%) had an unknown COO. In pts who were DZsig+ and had FISH results, 54/86 (62.8%) had BCL2 translocations, 41/92 (44.6%) had MYC translocations, and 6/40 (15%) had BCL6 translocations (BCL6 FISH was only done in pts with a MYC translocation), resulting in 23/90 (25.6%) pts with double/triple-hit DLBCL. Pts who were DZsig+ were predominantly within the EZB genetic subtype (60.3%). In the R-CHOP arm, pts who were DZsig+ had shorter PFS vs pts who were DZsig− (2-yr PFS: 62% [95% CI: 51-77] vs 73% [95% CI: 68-79], respectively; HR 1.61, 95% CI: 1.02-2.56; Figure). In the Pola-R-CHP arm, no significant difference in PFS was observed between pts who were DZsig+ vs DZsig− (2-yr PFS: 77% [95% CI: 66-89] vs 79% [95% CI: 74-84]; HR 0.97, 95% CI: 0.54-1.75). Improved PFS was demonstrated in pts who were DZsig+ and treated with Pola-R-CHP vs R-CHOP (HR 0.47, 95% CI: 0.24-0.95). Conclusions: In this exploratory biomarker analysis, we recapitulated that pts with molecularly defined DLBCL subtypes, including EZB and MCD by LymphGen and DZsig+ by RNAseq, have poor outcomes with R-CHOP therapy. In pts with the EZB and MCD subtypes, Pola-R-CHP appeared to improve 2-year PFS compared with R-CHOP. Pts with GCB DLBCL who were DZsig+ significantly benefited from Pola-R-CHP vs R-CHOP. In summary, our data indicate that molecular analysis beyond COO leads to identification of distinct molecular subsets of DLBCL that may respond favorably to Pola-R-CHP; future prospective validation is required.

A Randomized, Open-Label, Phase 3 Study of Acalabrutinib in Combination with Rituximab and Reduced Dose CHOP (R-miniCHOP) in Older Adults with Untreated Diffuse Large B-Cell Lymphoma (ARCHED / GLA 2022-1)

Background Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, and its incidence increases with age. Approximately 40% of patients with DLBCL are older than 70 years. Since the addition of the anti-CD20 antibody rituximab to CHOP regime (cyclophosphamide, doxorubicin, vincristine, and prednisone) prognosis in elderly patients has improved but still remains worse compared to their younger counterparts. Adequate dosage of chemotherapy in older adults is limited by comorbidities/frailty, especially in patients &amp;gt;80 years of age. An attenuated chemotherapy regimen, R-miniCHOP, has been evaluated in prospective trials and seems to provide a good balance between safety and efficacy in this vulnerable patient population (Peyrade et al, 2011). However, even with this regime, outcomes remain modest with 2-year overall survival at 50-60%, underlining the need for more effective treatment in this population. Acalabrutinib is a selective, irreversible, second-generation small molecule inhibitor of Bruton Tyrosine Kinase (BTK) with a more favorable safety profile compared to first generation BTK inhibitors. Although, not yet approved for the treatment of DLBCL, acalabrutinib showed promising results both as a single agent (Strati et al, Haematologica 2021) and in combination with full dose R-CHOP (Davies et al, ASH 2022), in patients aged up to 80 years. Furthermore, acalabrutinib carries the potential of improved efficacy in specific subgroups. The activated B-cell like (ABC) DLBCL subtype has been shown to depend on chronic active B-cell receptor signaling involving many downstream kinases including BTK. Additionally, the genetically defined subgroups MCD/C5, N1, and BN2/C1 and also may serve as potential predictive biomarkers for acalabrutinib. Study design and endpoints ARCHED is an investigator initiated, randomized, multicenter, open-label, phase III trial of R-miniCHOP with or without acalabrutinib in older adults with untreated DLBCL. Patients in the standard arm will receive 6 cycles of R-miniCHOP every 21 days followed by 2 cycles of rituximab alone, while patients in the experimental arm will receive the same regimen and additionally acalabrutinib 100 mg p.o. twice daily from day 1 to day 21 for 8 cycles (Figure 1). The primary endpoint of the study is investigator assessed progression-free survival (PFS). Secondary endpoints include overall survival, PFS per blinded independent review, event-free survival, outcomes according to cell of origin and molecular genotype, response rates, duration of response, progression, relapse and central nervous system (CNS) relapse rates as well as toxicity. Key eligibility criteria Patients are eligible for enrolment if they are &amp;gt;80 years old or &amp;gt;60 years old and ineligible for full-dose R-CHOP according to investigator assessment after standardized simplified geriatric evaluation and are diagnosed with untreated CD20+ DLBCL according to the 2017 WHO classification. Primary mediastinal lymphoma, high grade B-cell lymphoma and follicular lymphoma 3B as well as concurrent diagnosis of indolent lymphoma is allowed, but Richter's transformation is an exclusion criterion. Ann Arbor stage I patients can be included only in the presence of concomitant bulky disease (≥ 7.5 cm). Further exclusion criteria are CNS involvement, stroke or intracranial hemorrhage in the past 6 months and relevant organ dysfunction. An ECOG performance status score of 0 - 2 is required for enrolment but an ECOG score of 3 is acceptable if lymphoma associated. Statistical analysis With an expected 5% drop out rate, we aim to recruit 330 patients (165 in each arm) to achieve 80 % power at a 5 % significance level (two-sided) to observe a PFS difference of 15 % in favor of the experimental arm. An interim analysis for efficacy comparing PFS between the two treatment arms will be performed with n=200 patients and 1 year follow-up for the last patient, with approximately 50% of events. Additionally, an interim analysis for safety regarding treatment completion rates and all-cause mortality will take place when 92nd patient in each group has completed the sixth R-mini-CHOP cycle or died. ARCHED is an academic study of the German Lymphoma Alliance and is coordinated by the Saarland University Clinical Trials Unit. This study is funded by AstraZeneca GmbH. The trial is approved in the EU and is open for recruitment in Germany. (EU-CTN: 2022-501187-18-00, NCT05820841).

Identification and Clinical Characterization of CNS Relapse in DLBCL Patients across 19 Prospective Phase 2 and 3 Trials - a GLA/ DSHNHL and LYSA Collaboration

Introduction: Depending on clinical and molecular risk factors, CNS relapse occurs in 1-15% of DLBCL patients (pts) and is associated with dismal outcomes. Despite its important role in further improving DLBCL therapy, a comprehensive and large-scale characterization of secondary CNS relapse remains challenging. Methods: We conducteda retrospective analysis of 7775 pts treated within 19 prospective German and French phase 2/3 trials to identify and characterize DLBCL pts with progression or relapse in the CNS. Pts with histology different from DLBCL (subtypes), not treated with anti-CD20 antibody, or displaying CNS involvement at diagnosis were excluded from the analysis. Results: Among 5289 eligible pts, 159 (3%) experienced progression or relapse in the CNS. Median age at diagnosis was 62 years (range: 19-80 years), 47% of pts were female. In 91% of all cases, reference pathology confirmed the diagnosis of DLBCL. At diagnosis, 28 pts (18%), 62 pts (39%), and 69 pts (43%) had a low (0-1 point), intermediate, and high CNS IPI (4-6 points), respectively. 133 pts received front-line therapy with R/O-CHOP, 19 pts with R-(Mega)CHOEP, and 7 pts with R/O-ACVBP. Following first-line therapy, 54% of pts achieved a CR/CRu. For 131 pts (82%), CNS progress/ relapse represented the first PFS event. Prior to the CNS event, salvage therapy was administered to 30 pts, including 11 pts treated with HDT/ASCT and one pt receiving allogeneic SCT. For 70 out of 158 pts (44%), CNS involvement was classified as intracerebral, 31% showed meningeal, and 3% intraspinal lymphoma involvement. In 22% of pts, CNS involvement was classified as combined including 5 cases with eye involvement. The CNS event occurred as progression without prior CR/ CRu of disease in 61 out of 159 pts (38%). For 155 pts it could be specified that 25% had an isolated progress in the CNS while 12% showed both CNS and concomitant systemic progress. 35% of pts showed an isolated CNS relapse while 28% suffered from a combined relapse. 82% of all CNS events occurred within the first two years after randomization while 29 pts (18%) experienced a late CNS event after 24 months. Notably, 46% of pts with low CNS IPI developed a late CNS event compared to 9% with high CNS IPI at diagnosis (Fisher exact, p&amp;lt;0.001). During the course of disease, 138 pts (87%) showed extranodal lymphoma involvement outside the CNS. The most frequently involved extranodal sites were bone (34%), gastro-intestinal tract (21%), soft-tissue (21%), kidney/ adrenal gland (20%), lung (19%), and liver (18%). In general, pts with CNS event had poor outcomes with a median OS of 3.4 months (95% CI 2.9-4.2) and a 2-year OS of only 15% (10-22%). While sex and initial (CNS) IPI had no impact on OS, we observed significantly worse OS for pts older than 70 years at diagnosis (log rank, p&amp;lt;0.001). OS was poor regardless of the site of CNS manifestation. Pts with isolated CNS disease demonstrated significantly better OS than pts with concomitant systemic involvement (p=0.023). Pts with CNS events occurring later than 24 months from randomization did not show improved OS compared to pts with early relapse/ progression (p=0.19). Treatment categories were reported for 150 pts. 95 pts received immunochemotherapy (IC), 30 pts in combination with radiotherapy (RT). More precisely, 69 pts received salvage therapies containing systemic MTX and 22 pts underwent consolidation with HDT/ASCT (73% of which had thiotepa-based conditioning regimens). Three pts received allogeneic SCT for consolidation. 28 pts were treated with RT alone (+/- intrathecal therapy (IT)). 27 pts received only IT, corticosteroids, or no treatment. Pts consolidated with HDT/ ASCT or allogeneic SCT had superior outcomes with a 3-year OS of 36% (20%-66%) compared to 18% (7.5%-44%) for pts receiving IC and RT, and to 9.7% (3.9%-24%) and 9.5% (2.8-32%) for pts treated with IC or RT alone, respectively ( Fig. 1). Overall, pts achieving a CR after CNS event (38/159, 24%) showed a 3-year OS of 52% (37-73%). Conclusions: This large study including more than 5000 DLBCL pts all treated on prospective clinical trials highlights the unmet medical need to improve the outcome of DLBCL pts suffering from CNS relapse. About one third of pts who underwent transplantation showed long-term survival in contrast to all other pts displaying very dismal outcomes. Novel strategies including targeted therapies and CAR T cells will have to challenge the survival rates reported here.

Plasma Circulating Tumor DNA (ctDNA) as an Alternative to Tissue DNA for Genotyping of DLBCL: Results from the POLARIX Study

Introduction: In the POLARIX study (NCT03274492), polatuzumab vedotin in combination with rituximab plus cyclophosphamide, doxorubicin, and prednisone (Pola-R-CHP) demonstrated prolonged progression-free survival (PFS) versus rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in patients with previously untreated diffuse large B-cell lymphoma (DLBCL; Tilly et al. N Eng J Med 2022); the PFS benefit was maintained with longer follow-up (Herrera et al. Blood 2022). We previously validated the prognostic value of ctDNA at baseline, on treatment, and at the end of treatment in POLARIX (Herrera et al. Blood 2022). Here, we assess and compare the mutation landscape and molecular subtyping of DLBCL by plasma ctDNA versus tissue DNA using the LymphGen classifier. Methods: Baseline plasma ctDNA single nucleotide variants (SNVs) were identified using the AVENIO ctDNA NHL assay (Research Use Only) with an allelic frequency (AF) of 0.5% (Stokowski et al. Blood 2022). Tissue SNVs at baseline were determined by whole exome sequencing (WES) with an AF cutoff of 5%. Plasma-depleted whole blood at baseline was used as a source of germline DNA to filter non-tumor-specific variants for both ctDNA and WES assays. Mutation landscapes in tissue DNA and plasma ctDNA were compared, focusing on genomic regions covered by both assays and variants within the coding regions. LymphGen uses genetic mutations, copy number data, and BCL2/BCL6 rearrangements to classify samples into at least one of seven genetic subtypes (Wright et al. Cancer Cell 2020). Genetic subtypes were defined by the LymphGen classifier using tissue and ctDNA SNVs, per the following: A53 ( TP53 and TP53BP1 mutations), BN2 ( BCL6 translocations and NOTCH2 mutations), EZB ( EZH2 mutations and BCL2 translocations), MCD ( MYD88L265P and CD79B mutations), N1 ( NOTCH1 mutations), and ST2 ( SGK1 and TET2 mutations). Hazard ratios (HR) were adjusted for International Prognostic Index score (2 vs 3-5) and age (≤60 vs &amp;gt;60 years). Results: At baseline, 443 patients had both WES and ctDNA data. When comparing mutation landscapes between tissue DNA and ctDNA, a median 82% (lower quartile: 0.5, upper quartile: 1.0) of tissue SNVs per patient were also found in their matched ctDNA samples. In total, tissue and ctDNA SNVs were found in 163 and 202 genes, respectively. Among these genes, 154 had SNVs in both tissue and ctDNA samples, representing 96% and 78% of mutated genes identified in tissue and ctDNA, respectively. Eleven genes showed statistically significant differences in distribution between tissue and ctDNA (Fisher's exact test, false discovery rate&amp;lt;0.01; Benjamini and Hochberg. J R Stat Soc 1995), including MEF2BNB-MEF2B, MEF2B, BCL10, BNC2, TNFAIP3, RIMS2, CDH19, KIF2B, BCL6, KMT2D and MYC; all but one ( MEF2B) showed higher mutation frequencies in ctDNA than in tissue. The distribution of molecular subtypes is listed in Table 1; due to a lack of information on copy number alterations, the A53 subtype was not included. Based on the tissue SNV data, LymphGen was able to determine the molecular subtypes of 442 patients. Using LymphGen results from tissue SNVs as a reference, 333 (75.3%) patients had the same subtype designation by tissue and ctDNA SNVs, and 109 (24.7%) had different designations ( Table 2). Among the 109 patients, 9 (8.2%) were assigned to a different molecular subtype by ctDNA, 50 (45.9%) patients with tissue SNV-defined molecular subtype were assigned to the undetermined subtype by ctDNA SNVs, and 50 (45.9%) patients with undetermined status by tissue SNVs were classified into distinctive genetic subtypes by ctDNA variants. In a pooled analysis, the 2-year PFS estimates of individual subtypes by tissue versus ctDNA SNVs were consistent ( Table 1). Conclusion: Our analyses demonstrated that the mutation landscape of ctDNA in DLBCL characterized by the AVENIO ctDNA NHL assay resembles that of tumor tissue determined by WES. Patients with molecular subtypes defined by WES or ctDNA had similar PFS outcomes. Overall, these findings support the use of plasma ctDNA as an alternative to tumor tissue for the genotyping of DLBCL.

Molecular Determinants of Sensitivity to Polatuzumab-Vedotin in Diffuse Large B-Cell Lymphoma

Purpose: Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the B cell surface antigen CD79B. When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL overall; however, there is noted heterogeneity in response to Pola-V, with germinal center b-cell (GCB) DLBCL showing no added benefit to the addition of Pola-V compared to standard immunochemotherapy. We aimed to identify molecular determinants of sensitivity or resistance to Pola-V. We hypothesized that these might lead us to innovative strategies to improve on-target tumor killing by Pola-V, or to find novel biomarkers that predict drug resistance. Methods: We employed combined drug-sensitization and CD79B-sorted CRISPR-Cas9 screening to identify molecular determinants of sensitivity to CD79B-directed, tumor killing by Pola-V in 9 cell lines representing different molecular subtypes of DLBCL. Results: Our results reveal the striking impact of epitope glycosylation, specifically a2,6 sialylation, on the binding of Pola-V to CD79B and thereby its ability to kill tumor cells. Specifically, we identify the exact glycosylated residues on CD79A and CD79B which create a glycan shield around the Pola-V binding site, precluding binding to its target. We show how genetic, pharmacological and enzymatic approaches that remove terminal sialic acid residues from these N-linked glycans lead to enhanced tumor killing by Pola-V. We hypothesize and test multiple methods of targeting this pathway in order to enhance Pola-V killing both in vitro and in vivo. Finally, we reveal a previously unappreciated role for the ubiquitin ligase KLHL6 in regulating CD79B protein abundance and surface expression of the B cell antigen receptor (BCR), including how this pathway is used by physiological germinal center B cells and how it is corrupted to enhance BCR expression in GCB DLBCL. Conclusions: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, we identify how KLHL6 determines expression of the BCR in both physiological and malignant germinal center B cells, and how KLHL6 mutation may modulate sensitivity of GCB DLBCL tumors to Pola-V.

Phase Ib/II Study of Multi-Targeted Therapy with Venetoclax, Ibrutinib, Prednisone, Obinutuzumab, and Lenalidomide (ViPOR) in Relapsed/Refractory (R/R) Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Foundational studies have revealed essential oncogenic pathways in DLBCL, triggering the development of drugs targeting distinct survival pathways in this malignancy. While many of these agents are active in DLBCL as monotherapy, they rarely induce deep responses or cure. Based on our identification of drug synergy in DLBCL models, we hypothesized that targeting multiple survival pathways concurrently could be curative in DLBCL. We developed a 5-drug combination regimen (ViPOR) that targets DLBCL survival sustained by constitutive B-cell receptor (BCR) signaling (ibrutinib, lenalidomide, prednisone) and by BCL2 (venetoclax), and also enlists the innate immune system using obinutuzumab. To maximize drug exposure and minimize toxicity, we administered all agents in non-continuous cycles for fixed duration in R/R DLBCL. Methods: R/R DLBCL pts with adequate organ function were eligible. In Ph I, pts were treated at 4 doses of venetoclax (200-800 mg) PO D2-14 to identify the MTD. An initial 12d venetoclax ramp-up was given in combination with fixed-dose ibrutinib 560 mg PO D1-14, prednisone 100 mg PO D1-7, obinutuzumab 1000 mg IV D1-2, and lenalidomide 15 mg PO D1-14. Ph II expansion cohorts of R/R GCB and non-GCB DLBCL were included at the MTD. Max 6C of ViPOR q21d were given without maintenance. TLS, G-CSF, and PCP prophylaxis were given to all pts. Baseline CT, PET, BM, and tumor biopsies were performed with CT after C1, 2, 4, and 6 and PET after C6. CT was then performed q3m x 1y, q4m x 1y, q6m x 1y, then q12m x 2y. Tumor genomics and ctDNA (clonoSEQ) were studied. Results: 50 DLBCL pts were enrolled (25 DLBCL NOS, 17 HGBCL-DH-BCL2, 3 HGBCL-DH-BCL6, and 5 THRLBCL). 52% and 48% were GCB and non-GCB subtype by IHC, respectively, with transformed lymphoma in 34%. Median age was 61y (range 29-77), with stage 3-4 disease in 92%, elevated LDH in 86%, &amp;gt;2 extranodal sites in 56%, and IPI &amp;gt;3 in 68% of pts. Median prior txs were 3 (range 1-9), with 40% post-CAR-T pts and 58% refractory. A single DLT of G3 intracranial hemorrhage occurred, and venetoclax 800 mg was identified as the MTD. Heme AEs were most common, with G3-4 neutropenia in 24%, thrombocytopenia in 23%, and anemia in 7% of cycles. Febrile neutropenia occurred in 3 (1%) cycles. The only G3-4 non-heme AE in &amp;gt;10% pts was hypokalemia (28%). G3 A.fib occurred in 3 pts, and G4 TLS occurred in 1 pt, which resolved. Other common any grade non-heme AEs (% pts) included diarrhea (68%), hypokalemia (67%), nausea (45%), rash (35%), and fatigue (33%). Dose reductions occurred in 17% of pts, and 8% discontinued tx due to AE. Of 48 evaluable pts (2 came off tx prior to restaging), ORR was 54% (26/48), and CR was 38% (18/48). Responses were observed across all molecular DLBCL subtypes, including a CR rate of 62% (8/13) in non-GCB DLBCL, 53% (8/15) in HGBCL-DH-BCL2, 25% (2/8) in other DLBCL, and a PR rate of 33% (4/12) in GCB DLBCL (non-DH) (Fig. 1A). CR rate was 20% (4/20) and 19% (5/27) in post-CAR-T and refractory pts, respectively. With a median FU of 40m, 72% of CRs are ongoing, with a 2-year PFS and OS of 34% and 36%, respectively. By histology, 2-year PFS was 47%, 38%, 38%, and 8% in HGBCL-DH-BCL2, non-GCB DLBCL, other DLBCL, and GCB DLBCL (non-DH), respectively (Fig. 1B). 2-year PFS was 30% and 21% in post-CAR-T and refractory pts, respectively. MRD was undetectable in 38% (16/42) of pts at end of therapy (EoT), and in 93% (14/15) of pts in PET CR at EoT. Elevated baseline ctDNA or detectable ctDNA during or at EoT were associated with significantly inferior PFS and OS, as were quantitative PET parameters (elevated baseline TMTV and TLG). Two genetic subtypes known to rely on BCR-dependent NF-kB signaling - MCD and N1 - had a significantly higher CR rate (5/6, 83%) than all other genetic subtypes (4/22, 18%; p=0.0066). Conclusions: This is the first study to show the feasibility and curative potential of multi-targeted therapy in R/R DLBCL. ViPOR was well tolerated across all ages in R/R DLBCL with rare febrile neutropenia. ViPOR was most effective in non-GCB DLBCL, as expected from its reliance on BCR signaling and BCL2. ViPOR was also highly active in HGBCL-DH-BCL2, possibly due to inhibition of MYC-driven apoptosis by BCL2 in this subtype. Durable remissions and likely cure were observed in non-GCB DLBCL (38%) and HGBCL-DH-BCL2 (47%), including pts relapsed after or refractory to CAR-T (30%). Multicenter Ph II testing is in development to confirm the activity of ViPOR in R/R non-GCB DLBCL and HGBCL-DH-BCL2.

Relationship Between Clinical Factors of Lymphoma DLBCL GCB and Non-GCB Subtype with Ki-67 Proliferation Index in Siloam Karawaci Hospital 2014 - 2018

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt; Non-Hodgkin large cell B-lymphoma (DLBCL: Diffuse Large B-cell Lymphoma) were classified into two subtypes of Germinal Center B-Cell Like (GCB) and non-Germinal Center B-Cell Like (non-GCB), which GCB has a better prognosis. The Ki-67 antigen is one of the most reliable markers of cell proliferation. This research aims to establish the relationship between proliferation index Ki-67 with DLBCL subtypes and to evaluate the utility of Ki-67 proliferative index as a predictive marker for predicting stages of lymphoma.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Methods: &lt;/strong&gt;We obtained 60 cases of patient samples DLBCL in Siloam Hospital Lippo Karawaci in 2014-2018. Clinical and pathological data were obtained from medical records. Chi-square methods were used to analyze data.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Result:&lt;/strong&gt; There were more ≤60 years compared to &amp;gt;60 years. In this study, there were more male patients than female patients. lymphoma provided in extranodal were higher than lymphoma in nodal form.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; There is a relationship between Ki-67 proliferation index with lymphoma GCB and non-GCB subtypes and stage of lymphoma, which can be used as a predictive factor in predicting the stage of lymphoma.&lt;/p&gt;