L265P Mutation Research Articles

Polatuzumab Vedotin, Zanubrutinib and Rituximab Achieved Rapid and Deep Response in Previously Untreated Frail and Elderly Diffuse Large B-Cell Lymphoma Patients

Introduction: The incidence of diffuse large B-cell lymphoma (DLBCL) increases with age, while first-line treatment for frail and elderly DLBCL is a debating topic. R-miniCHOP, a reduced intensity regimen of R-CHOP, is currently the standard of care for this population, yet with limitation due to toxicities (Frédéric Peyrade et al., Lancet Oncol, 2011). Chemo-free regimens combining Bruton kinase inhibitors, rituximab and lenalidomide showed fewer toxicities, but compromised on efficacy in elderly DLBCL in multiple previous reports (Pengpeng Xu et al., Lancet Healthy Longev, 2022; Haiyan Yang et al., ASH 2022). First-line treatment balancing efficacy with safety is urgently needed for frail and elderly DLBCL patients. Since polatuzumab vedotin was approved in China for first-line DLBCL this year and reportedly had synergistic effect with rituximab in cell line research (Natsumi Kawasaki et al., Br J Haematol, 2022), we conducted a study of polatuzumab vedotin plus zanubrutinib and rituximab (ZPR) in previously untreated frail and elderly DLBCL to assess the efficacy and safety. Here we present the characteristics of 12 enrolled patients and preliminary results. Methods: Previously untreated DLBCL patients aged over 70 years old, or aged between 60 to 69 with ECOG score 2 to 4 were enrolled in this study. All patients received a triplet regimen of ZPR. Twenty-one days was a cycle. Zanubrutinib was given 160mg twice a day orally from day 1 to day 21. Rituximab was given 375mg/m 2 intravenously on day 1. Polatuzumab vedotin was administered at a dosage of 1.8mg per kilogram intravenously on day 1. Patients would receive 6 cycles of ZPR regimen if assessed partial response (PR) or complete response (CR) after 3 cycles. If assessed CR after 6 cycles of ZPR, patients would receive zanubrutinib 160mg twice a day alone for another 6 cycles. Whole body 18F-FDG PET/CT scan or contrast-enhanced CT scan of cervical, thoracic, abdominal and pelvic parts was scheduled every 3 cycles. Treatment response was assessed by 2014 Lugano response criteria. Adverse events were graded by CTCAE 5.0. The primary endpoint is overall response rate (ORR) after 6 cycles of ZPR. The secondary endpoints are CR rate after 6 cycles of ZPR, and 2-year progression-free survival (PFS). Other endpoint is safety (Figure 1). Results: Twelve patients were enrolled in our study until Jul 24, 2023. Median age was 75 (range: 67-87). Six patients were female and six patients were male. Nine (75%) patients were classified non-GCB type according to cell of origin by Hans algorithm. Nine (75%) patients were assessed stage Ⅲ to Ⅳ by Ann Arbor staging system. Nine (75%) patients had an international prognostic index (IPI) score of 2 or more. One (11%) patient had double-hit risk factor in 9 patients with gene rearrangement results. Six (75%) patients had MYD88 L265P mutation in 8 patients with sequencing results. After a median follow up of 2.1 months, four patients had PET/CT assessment results after 3 cycles of ZPR, and four (100%) of them achieved CR. The ORR after 3 cycles of ZPR regimen was 100% (Figure 2).Grade 3 to 4 adverse events occurred in two patients, that one patient had grade 3 aspartate aminotransferase increase and alanine aminotransferase increase, and the other patient had grade 3 pneumonitis. Grade 1 to 2 adverse events included neutrophil count decrease (16%), dizziness (8%), and epistaxis (8%). All patients recovered from adverse events under best supportive care. There was no discontinuation of ZPR regimen due to severe adverse events. Conclusion: Based on these results in our study, ZPR regimen showed rapid and deep response in previously untreated frail and elderly DLBCL patients with manageable safety profiles. We registered and initiated a phase 2b trial (NCT05940064) to further evaluate the efficacy and safety of ZPR regimen as first-line treatment for this population. Ongoing treatment and follow up of these twelve patients will also be further updated.

Remodeling of the Immune Microenvironment By Oncogenic MYD88 Dictates Immunotherapy Responses across Indolent and Aggressive B-Cell Lymphomas

The MYD88 L265P mutation is detected in a variety of lymphoma subtypes, ranging from indolent MALT or lymphoplasmacytic lymphoma (LPL) to aggressive DLBCL with activated B-cell (ABC) phenotype. Whether oncogenic MYD88 L265P constitutes a common founder mutation and how it drives lymphomas with different clinical, histopathological, and immunological characteristics, remains unknown. To address this fundamental question, we screened mice genetically engineered to express an orthologous MYD88 L252P mutation crossed with animals carrying secondary genetic lesions common in MYD88 L265P lymphomas, triggered at two B-cell differentiation stages. Several mouse strains developed indolent or aggressive lymphomas depending on the cell of origin and the secondary genetic lesion. In two of the models, human-like MALT or LPL were progressively driven by mutant MYD88 activated from immature pre-B lymphocytes with either constitutive BCR signaling or BCL2 expression. High-throughput cellular and molecular characterization of the tumor immune microenvironment (TME) by single-cell RNA sequencing coupled with BCR/TCRseq revealed bi-directional B-cell interactions with CD4 + Tfh and Th1 cells from early disease state, promoting survival of B-cell clones through CD40L:CD40 signaling. During progression, IL10-secreting regulatory T (Treg) cells were recruited into the tumor niche through a Ccl5-Cxcr3 axis to suppress CD8 + T-cell cytotoxicity. In patients with MYD88 L265P LPL characterized at progressive disease states, CD40:CD40L interaction between tumor and immune cells took place early while Tregs accumulated at late stage. In the LPL model and in human MYD88 L265P-LPL cells, CD40 signaling decreased effects of standard-of-care drugs including BTK inhibitors. Accordingly, anti-CD40 therapy disrupted B/T-cell interactions and increased ibrutinib activity. In conclusion, oncogenic MYD88 triggered at early B-cell stages promotes indolent lymphomas that are sustained by CD40 signaling, which restricts therapy responses that can be restored upon CD40 blockade. When MYD88 L265P was activated in mature germinal-center B cells together with either Prdm1 or Trp53 deletion, mice developed extranodal ABC-DLBCL (MCD/C5 genomic class). IHC, flow cytometry and single-cell RNAseq revealed that DLBCL with Prdm1 loss showed morphologically heterogeneous B cells with abundant immune and T cells with activated/exhausted phenotypes, which recapitulated the inflammatory/immunosuppressive TME category in DLBCL patients (IN-LME, Kotlov- Cancer-Discovery-2021). In contrast, DLBCL with Tp53 deletion exhibited monotonous B-cell infiltrates with high proliferation index and a TME with depletion of T cells with effector/memory phenotypes and CD4 +CD25 +Foxp3 + Tregs, consistent with the immune depleted TME category (DP-LME). Analysis of 1,039 DLBCL patients demonstrated that majority of MCD cases with MYD88 L265P showed either an IN-LME (43%) or DP-LME (44%). Consistent with the models, ABC-DLBCL patients with MYD88 L265P and TP53 loss/mutation exhibited highly proliferative disease with a DP-LME, while ABC-DLBCLs with MYD88 L265P but without TP53 abnormalities showed an IN-LME. Notably, the number of infiltrating CD4 + T cells correlated with TP53 activity score (PROGeNY) across MCD-DLBCLs. Overall, the presence or absence of TP53 alterations in MYD88 L265P ABC-DLBCL induces TME characterized as DP-LME or IN-LME, respectively. Since DP-LME carries poor response to immune-chemotherapy (Kotlov-Cancer-Discovery-2021), we tested anti-CD19 CAR T-cells in the models. While CAR T-cells were initially effective in the two DLBCL models, progression occurred earlier in DLBCL with DP-LME than in IN-LME, which remained in remission 100 days after cell infusion. Responses correlated with persistence of circulating CD4 + CAR T cells. These data highlight that a TP53-driven DP-LME may limit immunotherapy activity in DLBCL, providing a scientific explanation to the poor outcome of DLBCL patients with TP53 alterations to CD19-directed CAR T cells (Shouval-JCO-2021). Collectively, our study defines the cellular origins and the stepwise genetic alterations of indolent and aggressive lymphomas with MYD88 L265P, dissects distinct immunological paths underlying progression driven by oncogenic MYD88, and builds proof-of-concept for advancing precision immunotherapy in B-cell lymphomas.

Ethnic Diversity in Presentation of Waldenström Macroglobulinaemia and IgM Mgcs in the United Kingdom- a Real-World Data Analysis

Introduction: Waldenström Macroglobulinaemia (WM) is a low-grade B-cell lymphoma characterised by lymphoplasmacytic marrow infiltration with a circulating IgM protein with an incidence of approximately 0.55 cases per 100,000 in the United Kingdom (UK). WM may be asymptomatic, symptomatic and/or associated with IgM-related entities (monoclonal gammopathies) of clinical significance (MGCS). A central aim of the WMUK Charity and Clinical Forum is to systematically upload demographic, clinical data and treatment outcomes from a significant proportion of the UK's WM population. Such data provide important insights on the epidemiology and clinical features of this distinct uncommon disorder. Clinical correlates with ethnicity are important but sparsely characterised. This analysis focusses on baseline demographics and outcomes across different ethnic groups in the UK. Methods: We retrospectively reviewed data from the Rory Morrison WMUK Registry, collating descriptive data of WM, non-IgM lymphoplasmacytic lymphoma and IgM MGCS from 24 centres across the UK, diagnosed between June 1978 and June 2023. Research ethics approval was obtained. Baseline characteristics were compared using χ 2 or Fisher's exact tests (categorical variables) or Wilcoxon Mann-Whitney/Kruskal-Wallis tests (continuous variables). Differences were considered significant at p-values <0.05. Statistical analyses were performed using STATA v17.0 (StataCorp, Texas, USA). Results: 1073 patients with documented self-reported ethnicity and a diagnosis of WM (n=985), non-IgM LPL (n=32) or IgM MGCS (n=88) were included. Those with asymptomatic IgM MGUS were excluded. Of 982 with a diagnosis of WM, 12% (114/985) had concurrent MGCS. Across the entire cohort, IgM MGCS comprised peripheral neuropathy including anti-MAG neuropathy (n=114), cold agglutinin disease/syndrome (n=43), AL amyloidosis (n=20), mixed autoimmune haemolytic anaemia (n=13), cryoglobulinaemia (n=8), Schnitzler syndrome (n=8) and C1 esterase deficiency (n=2). Non-IgM LPL had similar baseline characteristics to WM although a trend towards a lower presenting M-protein (7g/l vs 18g/l, p=0.07). Ethnicity was categorised in accordance with the UK National Census (ethnicity-facts-figures.service.gov.uk). The majority was white (90%; 968/1073: English/Welsh/Scottish/Irish) including 35 patients identified as ‘other’ white. The remaining 105 (10%) were from the following ethnic groups (collectively ‘ethnic minorities’, EM): 54 Asian (22 Indian, 4 Pakistani, 1 Bangladeshi, 7 Chinese, 20 other), 19 Black (5 African, 11 Caribbean, 3 other), 6 Mixed/multiple, 26 Other ethnic group. EM presented at a younger age compared to the white cohort (60 vs 65 years, p<0.001) with a lower presenting M-protein at WM diagnosis (11g/l vs 18g/l, p=0.028). A similar proportion of patients had IgM MGCS in white vs EM cohorts (11% vs 8%, p=0.69). In those with IgM MGCS alone, presenting M-protein was similar across both ethnic categories (5g/l vs 3g/l, p=0.36). MYD88 L265P mutation was assessed in 387 WM patients (36%), of which MYD88 L265P was identified in 88% (322/364) and CXCR4 mutation tested in 91 patients and mutated in 29% (26/91). MYD88-mutated WM was observed less frequently in the EM versus white cohorts (79% vs 90%, p=0.05). A similar proportion presented with symptomatic WM across ethnic groups (56% vs 53%, p=0.56). In those with WM diagnosed since 2010 (n=956), median follow up was 8 years (95% CI 7-8), median overall survival was not reached. International Prognostic Scoring System for WM (IPSSWM) was predictive of overall survival. Overall survival estimates did not differ when comparing ethnic groups. Conclusions: Analysis of the national registry for WM shows that ethnic minorities comprise 10% of WM/IgM MGCS, present with WM at a younger age, a lower M-protein and with a higher proportion of MYD88-wild type cases which may suggest different disease biology. There was no difference in overall survival in our cohort. Limitations include the retrospective analysis of real-world data and incomplete documentation of baseline data. Further analysis of molecular signatures across ethnicities, treatment and outcome is warranted and ongoing.

Anterior segment involvement in vitreoretinal lymphoma: clinical manifestations, molecular findings and in vivo confocal microscopy

BackgroundIntermediate and posterior manifestations of vitreoretinal lymphoma (VRL) are well characterised. However, there is limited information on anterior segment involvement in VRL. This study aimed to describe the anterior manifestations...

Whole Exome Sequencing Analysis of Diffuse Large B-Cell Lymphoma That Progressed to Central Nervous System

Background: Recent sequencing analyses have identified distinct genetic subtypes of diffuse large B-cell lymphoma (DLBCL). The MCD subtype, characterized by MYD88 L265P and CD79B gene mutations, is frequently found in DLBCL that progresses to the central nervous system (CNS), known as secondary CNS lymphoma (sCNSL) (Ollila, Blood 2021). In addition, the MCD subtype is also frequently found in primary CNS lymphoma (PCNSL) (Radke, Nat commun 2022). Therefore, we hypothesized that specific genetic alterations that are present in the initial DLBCL tumors are associated with their progression to the CNS. We aimed to investigate i) the clinical impact of genetic subtypes of DLBCL and ii) genomic mutations that may be associated with sCNSL. To this end, genetic alterations in DLBCL samples at diagnosis that later developed into sCNSL were evaluated using whole-exome sequencing (WES). Methods: A total of 44 patients with DLBCL who developed sCNSL after receiving standard rituximab-containing chemotherapy between 2009 and 2020 were identified from five institutions. sCNSL was diagnosed on the basis of imaging and/or pathological findings. Genomic DNA (gDNA) was extracted from formalin-fixed, paraffin-embedded tumor samples obtained at the initial diagnosis of DLBCL. Finally, 26 of the 44 samples with sufficient gDNA were subjected to WES at BGI Genomic Co. Ltd. Single-nucleotide variants and short indels were detected using Mutect2 and filtered as described in Figure 1. Genetic subtypes were determined using the LymphGen algorithm (https://llmpp.nih.gov/lymphgen/index.php). The association between the clinicopathological features and genetic subtypes was also evaluated. In addition, recurrently mutated genes (>10% in our cohort) were identified and their alteration frequencies were compared with those in a representative general DLBCL cohort (N=574, available in the article by Schmitz, NEJM 2018) and PCNSL cohort (N=30, whole genome sequencing performed, available in the article by Radke, Nat Commun 2022) (Figure 1a). Results: The median age of the 26 patients was 69 years (range, 34-87). At the time of CNS progression, 54, 27, and 19% of the patients had parenchymal CNS disease, extraparenchymal CNS disease, and both, respectively. Immunohistological analysis showed that the non-germinal center B-cell-like (GCB) type, as defined using the Hans algorithm, and dual expression of MYC and BCL2 (DEL) were identified in 65% and 69% of patients, respectively. The median overall survival (OS) from CNS relapse was 16 months (95% confidence interval, 2-30). WES analysis showed that the median mean coverage depth of the 26 samples was 140 (range, 53-299). The median mapping rate was 94% (range, 80-99). As expected, the most frequently mutated genes in our cohort were MYD88 (50%), PIM1 (50%), BTG2 (31%), and CD79B (31%), corresponding to the MCD subtype. The LymphGen algorithm classified the 26 samples into MCD (n=12, 46%), EZB (n=5, 19%, including one with a composite feature of ST2), BN2 (n=1, 4%) and “not classified” (n=8, 31%) (Figure 1b). The CNS-IPI risk, DEL status, and CNS relapse sites did not differ among the genetic subgroups. In addition, univariate analysis showed that non-GCB status, DEL status, and genetic subtype at initial diagnosis did not influence OS. Next, we shortlisted 38 genes whose mutations were identified in three or more samples (>10%) of our cohort as well as in >10% of the PCNSL cohort (by Radke, Nat Commun 2022). We further selected genes that were less frequent (<3%) in the general DLBCL cohort and identified several genes, such as MUC16 and FAT4, that were more enriched in PCNSL and our sCNSL cases (Figure 1a). Metascape analysis (https://metascape.org/gp/index.html#/main/step1) revealed that the top three enriched processes associated with these genes were sensory organ development, cell adhesion via plasma membrane adhesion molecules, and morphogenesis of epithelium. Conclusion: Our study confirmed that MCD was a major genetic subtype of DLBCL that developed sCNSL. We identified recurrently mutated genes that were common in both sCSNL and PCNSL but not in the general DLBCL cohort; these genes may be associated with CNS progression. Validation of our results and investigation of the functions of these genes will help to understand the mechanisms of CNS relapse and develop more accurate markers to predict sCNSL. *Suzuki T and Yokomori R equally contributed to this study.

Orelabrutinib Monotherapy in Patients with Relapsed or Refractory Waldenström's Macroglobulinemia in a Single-Arm, Multicenter, Open-Label, Phase 2 Study: Long Term Follow-up Results

Background Orelabrutinib is a novel, small molecule, selective irreversible Bruton tyrosine kinase inhibitor, approved in China for the treatment of patients with relapsed/refractory (R/R) chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), and marginal zone lymphoma (MZL). The previous publication of this study reported that orelabrutinib is highly active in R/R Waldenström's macroglobulinemia (WM), with a well-tolerated safety profile (EClinicalMedicine. 2022;52:101682). We present here the long-term results of this study. Methods This is a prospective, multicenter study of orelabrutinib in patients with WM who had at least one prior line of treatment (ClinicalTrials NO. : NCT04440059). Orelabrutinib was administered orally at a daily dose of 150 mg until disease progression or unacceptable toxicity. Genotyping using MYD88 L265P and CRCR4 S338X mutation were performed with qPCR assay. The primary endpoint was major response rate (MRR) assessed by the Independent Review Committee (IRC) according to IWWM-6 (Br J Haematol. 2013; 160: 171-176). Results Between August 2019 and December 2020, 66 R/R WM patients were assessed for eligibility. Forty-seven eligible patients were evaluated for efficacy at a median follow-up of 31.9 months (interquartile range: 28.0，36.5). As assessed by IRC, the MRR was 80.9%, and the overall response rate was 91.5%. The PFS rates was estimated as 72.1% at 36 months and the median PFS has not been reached. There is no significant difference in PFS across various MYD88 L265P and CRCR4 S338X genotype (P=0.69; Figure 1). The 30-months PFS rate in MYD88 L265P/CXCR4 NEG, MYD88 L265P/CXCR4 S338X, and MYD88 NEG/CXCR4 NEG were 86.2%, 75% and 75%, respectively. Most adverse events were Grades 1 or 2 (63.8%). The common grade 3 or higher adverse events occurred were neutropenia (10.6%), thrombocytopenia (8.5%), and pneumonia (6.4%). Treatment related serious adverse events were reported in 8.5% patients. Adverse events leading to discontinuation occurred in 4 patients (8.5%). One treatment-related death was reported (hepatitis B reactivation). Conclusions Results with longer follow-up continue to show that orelabrutinib has high, deep and durable response in patients with R/R WM. Data support the safety of long-term orelabrutinib treatment in R/R WM, with no new safety signals identified.

Lymphoplasmacytic Lymphoma: An Atypical Presentation with IgG Monoclonal Gammopathy

Introduction Lymphocytic lymphoma (LPL) is a rare type of low-grade mature B-cell non-Hodgkin lymphoma, characterized by a monoclonal population of B lymphocytes, lymphoplasmacytic cells, and plasma cells. In around 95% of LPL cases, the serum immunoglobulin M (IgM) paraprotein is increased, which is correlated to Waldenstrom's Macroglobulinemia (WM), a clinicopathological disease. Only around 5% of LPL cases are associated with the serum paraproteins immunoglobulin G (IgG) or immunoglobulin A (IgA), or light chains alone, or are not linked to a paraprotein. Non-IgM LPLs have a wide range of clinical and pathological features, and MYD88 L265P mutations can occur, though at a lower frequency than WM. Hence, MYD88 testing is advised for prospective therapeutic purposes. Case Presentation A 69-year-old male was referred to a hematologist for uncontrolled epistaxis and periorbital edema. On initial workup, he had high total protein, elevated globulin, hypoalbuminemia, anemia, and proteinuria. Further investigation revealed a high IgG level with low IgM, IgA, and complement levels. Serum Protein Electrophoresis (SPEP): Increased total protein, decreased alpha two and beta globulins with a large monoclonal protein peak migrating in the gamma region, which accounts for 4.77 g/dl of the total 5.38g/dl gamma. Elevated Kappa Quantitative Free Light Chains of 355.39 (3.30 - 19.40mg/L), Decreased Lambda Quantitative Free Light Chains of 1.51 (5.71 - 26.30mg/L), Elevated Kappa Lambda Free Light Chains Ratio of 235.36 (0.26 - 1.65). Serum ImmunoFixation Electrophoresis demonstrated IgG-type kappa monoclonal protein with an additional faint band in IgG kappa. X-Ray skeletal survey showed a possible small lytic area in the inferior aspect of the C2 vertebral body, prompting him to have a cervical spine MRI which revealed no lytic lesions but an 18 mm vertebral body hemangioma inside T2. Molecular testing was positive for MYD88 Mutation c.794T>C (p. L265P) and negative for CXCR4 mutation. Plasma cell myeloma prognostic panel and chromosome analysis were normal. Flow cytometry of bone marrow aspirate showed a kappa light chain restricted plasma cell population (1.6% of total cells) with the following immunophenotype: positive for CD138, CD38, CD45, and negative for CD19, CD20, CD56, CD 117, and lambda light chains. The B-cell population showed kappa light chain restriction with the following immunophenotype: positive for CD19, CD20, and kappa light chains and negative for CD5, CD10, CD11c, CD23, and lambda light chains (Image 2). PET-CT of the whole body was negative for the extension of the disease. Transfusion with the two least incompatible PRBCs had little effect. However, plasmapheresis significantly improved his hemoglobin level by less than 7g/dl. A bone marrow biopsy revealed he had LPL with IgG Monoclonal Gammopathy (Image 1). He received one cycle of CyBorD (Cyclophosphamide, Bortezomib, Dexamethasone), followed by seven cycles of KCD (Carfilzomib, Cyclophosphamide, Dexamethasone) and Rituximab. The patient was diagnosed with low-grade B cell lymphoma with plasmocytic differentiation/lymphoplasmacytic lymphoma, with MYD88 mutation c.794T>C (p. L265P) positivity and CXCR4 mutation negativity. After plasmapheresis, the IgG level dropped to 1958. Follow-up SPEP revealed decreased alpha2 and beta globulins, decreased free lambda light chains, and increased K/L ratio. The patient was treated with Acalabrutinib, and his anemia, epistaxis, and coagulopathy resolved. Repeat PET-CT scan revealed no evidence of illness progression. Discussion LPL is not always linked with IgM monoclonal gammopathy; it can also be associated with IgG or IgA monoclonal gammopathy, light chains alone, or no monoclonal paraprotein. Non-IgM LPL has a high prevalence of extramedullary involvement, a lower rate of MYD88 mutation, and few hyper viscosity symptoms. The MYD88 mutation does not allow WM and Non-IgM LPL to be distinguished from other lymphoproliferative disorders with similar clinical and immunophenotypic features. The case described is unique as the patient has high levels of IgG (11,200) at presentation. In contrast to our patient, who has LPL with IgG Monoclonal gammopathy, approximately 95 percent of LPL patients have IgM Monoclonal gammopathy, which corresponds with WM. As a result, clinicians should be on the lookout for LPL that lacks an IgM Monoclonal Paraprotein.

Targeting MyD88: Therapeutic mechanisms and potential applications of the specific inhibitor ST2825.

Myeloid differentiation factor-88 (MyD88) is a crucial adapter protein that coordinates the innate immune response and establishes an adaptive immune response. The interaction of the Toll/Interleukin-1 receptor (IL-1R) superfamily with MyD88 triggers the activation of various signalling pathways such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), promoting the production of a variety of immune and inflammatory mediators and potentially driving the development of a variety of diseases. This article will explore the therapeutic potential and mechanism of the MyD88-specific inhibitor ST2825 and describe its use in the treatment of several diseases. We envision future research and clinical applications of ST2825 to provide new ideas for the development of anti-inflammatory drugs and disease-specific drugs to open new horizons for the prevention and treatment of related inflammatory diseases. This review analysed relevant literature in PubMed and other databases. All relevant studies on MyD88 inhibitors and ST2825 that were published in the last 20 years were used as screening criteria. These studies looked at the development and improvement of MyD88 inhibitors and ST2825. Recent evidence using the small-molecule inhibitor of ST2825 has suggested that blocking MyD88 activity can be used to treat diseases such as neuroinflammation, inflammatory diseases such as acute liver/kidney injury, or autoimmune diseases such as systemic lupus erythematosus and can affect transplantation immunity. In addition, ST2825 has potential therapeutic value in B-cell lymphoma with the MyD88 L265P mutation. Targeting MyD88 is a novel therapeutic strategy, and scientific research is presently focused on the development of MyD88 inhibitors. The peptidomimetic compound ST2825 is a widely studied small-molecule inhibitor of MyD88. Thus, ST2825 may be a potential therapeutic small-molecule agent for modulating host immune regulation in inflammatory diseases and inflammatory therapy.

The oncogenic human B-cell lymphoma MYD88 L265P mutation genocopies activation by phosphorylation at the Toll/interleukin-1 receptor (TIR) domain

MYD88 is the key signaling adaptor-protein for Toll-like and interleukin-1 receptors. A somatic L265P mutation within the Toll/interleukin-1 receptor (TIR) domain of MYD88 is found in 90% of Waldenström macroglobulinemia cases and in a significant subset of diffuse large B-cell lymphomas. MYD88-L265P strongly promotes NF-κB pathway activation, JAK-STAT signaling and lymphoma cell survival. Previous studies have identified other residues of the TIR-domain crucially involved in NF-κB activation, including serine 257 (S257), indicating a potentially important physiological role in the regulation of MYD88 activation. Here, we demonstrate that MYD88 S257 is phosphorylated in B-cell lymphoma cells and that this phosphorylation is required for optimal TLR-induced NF-κB activation. Furthermore, we demonstrate that a phosphomimetic MYD88-S257D mutant promotes MYD88 aggregation, IRAK1 phosphorylation, NF-κB activation and cell growth to a similar extent as the oncogenic L265P mutant. Lastly, we show that expression of MYD88-S257D can rescue cell growth upon silencing of endogenous MYD88-L265P expression in lymphoma cells addicted to oncogenic MYD88 signaling. Our data suggest that the L265P mutation promotes TIR domain homodimerization and NF-κB activation by copying the effect of MY88 phosphorylation at S257, thus providing novel insights into the molecular mechanism underlying the oncogenic activity of MYD88-L265P in B-cell malignancies.

The Prevalence of MYD88 L265P and TNFAIP3 Mutations and Their Correlations with Clinico-Hematological Profile in Egyptian Patients with Diffuse Large B Cell Lymphoma.

Activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic active B-cell receptor signaling and a constitutive activation of the NF-KB pathway. MYD88 L265P mutation occurs as a driving force of NF-KB overactivity in ABC-DLBCL. Nonetheless, in cases of DLBCL, the MYD88 L265P mutation has not yet been investigated in association with the tumour necrosis factor alpha induced protein3 (TNFAIP3) mutation. To investigate the frequency of MYD88 and TNFAIP3 mutations in DLBCL and their association to the clinico-hematological profile. We used real-time polymerase chain reaction in order to search for MYD88 L265P and TNFAIP3 mutations in 100 DLBCL patients. MYD88 L265P In 20% of cases, the CT heterozygous genotype was discovered. CT heterozygous genotype was more common in ABC type, stage IV, greater IPI groups, extra-nodal infiltration, and BM infiltration. It was also linked to a shorter OS. TNFAIP3 mutation GA heterozygous genotype was detected in 18% of the patients, with ABC-DLBCL subtype accounting for 77.8%. The GA heterozygous genotype was usually related with stage IV, extranodal infiltration, and a reduced life expectancy. MYD88 L265P and to lesser extent TNFAIP3 mutations are major mutations in ABC- DLBCL and may be predictive factors for poor OS in ABC- DLBCL patients.

PRELIMINARY FINDINGS OF A PHASE II STUDY OF CHEMO‐FREE COMBINATION OF POMALIDOMIDE, ORELABRUTINIB, RITUXIMAB WITH SEQUENTIAL HIGH‐DOSE METHOREXATE IN NEWLY DIAGNOSED PRIMARY CNS LYMPHOMA

Background: High-dose methotrexate (HD-MTX) is widely used as front-line treatment in patients with primary central nervous system lymphoma (PCNSL). Both preclinical models and clinical data suggested pomalidmide and orelabrutinib were effective in PCNSL and the combination of them may have synergy in DLBCL. Aims: We aimed to evaluate the tolerability and efficacy of pomalidomide, orelabrutinib and rituximab (POR) with the sequential addition of HD-MTX chemotherapy in newly diagnosed PCNSL. Methods: This is an investigator-initiated, single-arm phase II study, and immunocompetent patients with untreated PCNSL were enrolled. Patients were treated with pomalidomide 4 mg once per day on days 1-14, orelabrutinib 150 mg once daily continuously, and rituximab 375 mg/m2 intravenous once on day 1 of each 21-day cycle. After four cycles, HD-MTX chemotherapy was sequentially added for two additional cycles. The primary endpoint was overall response rate (ORR) at the end of four cycles of POR. Circulating tumor DNA detection of cerebrospinal fluid was performed. This trial was registered at www.clinicaltrials.gov (#NCT 05390749). Findings: The data cut-off date was 15 February 2023, and thirteen patients were enrolled in this study. The median age was 58 years (range 34-79 years). Four patients had eye involvement and one patient had leptomeninges involvement. Eight patients performed ctDNA analysis and all had MYD88 l265p mutation. One patient discontinued the experimental treatment due to treatment-related adverse events (TRAE) and eleven patients finished four cycles of POR and were evaluable. The ORR after four cycles of POR was 90.1% and the complete response rate was 36.4%. Twelve patients who finished at least 1 cycle were evaluated for safety analysis. The most common grade 3 or 4 AEs were neutropenia (G3, n = 1; G4, n = 2), thrombocytopenia (G3, n = 2), rash (G3 n = 1, G4 n = 1), ALT increased (G3, n = 1) and anemia (G3, n = 1). With a median follow-up of 4 months, one patient relapsed and died of PCNSL. Conclusion: This is the first study to treat newly diagnosed PCNSL with a targeted therapy combination before chemotherapy. POR produced a high ORR with good tolerance. This suggested the potential of noncytotoxic first-line therapies for PCNSL. Encore Abstract—previously submitted to EHA 2023 The research was funded by: CAMS Innovation Fund for Medical Sciences (CIFMS) 2021-I2M-C&T-B-005. Keywords: aggressive B-cell non-Hodgkin lymphoma, immunotherapy, molecular targeted therapies No conflicts of interests pertinent to the abstract.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Diffuse large B-cell lymphoma (DLBCL) is an aggressive but potentially curable lymphoma. Rituximab, cyclophosphamide, vincristine, doxorubicin and prednisone (R-CHOP) is the standard first-line regimen with cure rates nearing 60%. Over the last years many efforts have been made aiming at improving outcomes of first line therapy but none has resulted in improved overall survival over R-CHOP. Recently, genetic subtypes of DLBCL that go beyond the cell-of-origin and have distinct biology, clinical characteristics and different likelihood of response to specific therapies have been identified. One of these entities is represented by the MCD subtype harboring either the MYD88 L265P mutation, the CD79A/B mutation, or both. MCD has a dismal outcome when treated with R-CHOP but is sensitive to BTK inhibition. In addition to the genetic definition of specific subtypes, the possibility to use circulating tumour DNA (ctDNA) response assessment together with PET-response may establishe a new strategy for treatment decisions in patients with DLBCL. Methods: SAKK 38/19 (NCT04604067) is an ongoing exploratory multicohort phase II trial in patients (pts) with previously untreated DLBCL not otherwise specified (NOS). Adult pts with CD20-positive DLBCL NOS, ECOG performance status 0–2, candidates for 6 cycles of R-CHOP are eligible. At baseline, a liquid biopsy is perfomed and pts with MCD subtype are treated with the combination of R-CHOP plus acalabrutinib for 6 cycles (cohort A). Non-MCD pts receive 2 cycles of R-CHOP and based on the combined PET and ctDNA response assessment after cycle 2 they are allocated to one of three different cohorts: R-CHOP for 4 additional cycles in combination with acalabrutinib followed by 2 months of acalabrutinib single-agent (cohort B); 2 additional cycles of R-CHOP and 2 administrations of single-agent rituximab (cohort C); 4 additional cycles of R-CHOP (cohort D). Co-primary endpoints of the trial are: Progression free survival (PFS) (in Cohorts A, C and D) and complete remission (CR) rate (in Cohort B) Secondary endpoints: Adverse events in the cohorts treated with acalabrutinib-R-CHOP; overall survival; PFS in cohort B; CR rate in cohorts A, C and D; overall response rate; duration of response. The research was funded by: -Astra Zeneca -Hubacher Fund Keywords: aggressive B-cell non-Hodgkin lymphoma, liquid biopsy, ongoing trials Conflicts of interests pertinent to the abstract A. Stathis Honoraria: Eli Lilly, Bayer, Roche, Novartis, Janssen Oncology, AstraZeneca Research funding: ADC Therapeutics, Pfizer, Roche, Novartis, Bayer, Eli Lilly, MEI Pharma, Cellestia, Debiopharm Group, Merck/MSD, Abbvie, Amgen, AstraZeneca, Incyte, Loxo, and Philogen F. Hitz Consultant or advisory role Abbvie, Takeda and Roche G. Gritti Consultant or advisory role Roche, Takeda, Kite-Gilead, Ideogen, Genmab Educational grants: Janssen, Beigene, Sandoz Other remuneration: Clinigen, Ideogen, Beigene, Incyte, Novartis T. Zenz Honoraria: Roche, Abbvie, AZD, Beigene, Janssen, Gilead, Novartis

Ibrutinib response in a patient with refractory mixed essential cryoglobulinemia.

A 74-year-old male presented with fatigue, parasthesiae, Raynaud's phenomena, visual disturbances, and recurrent lower extremity rash (left). Investigations revealed an elevated serum cryoglobulin, rheumatoid factor >1320 IU/ml (ref. <15 IU/mL), undetectable C4, and C3 of 25 mg/dL (ref. 87–200 mg/dL). A small amount of IgM kappa monoclonal protein (M spike of 2.5%) and polyclonal IgG were identified by immunofixation. A diagnosis of mixed (Type II) cryoglobulinemia was made. Testing for Hepatitis B and C was negative. Positron emission/computed tomography (PET-CT) showed no evidence of lymphoma. Although the bone marrow appearance raised suspicion for Waldenstrom macroglobulinemia (WM), testing for MYD88 L265P mutation, which is commonly associated with this disorder, was negative. Of note, scattered CD20-positive B-cells comprised only ∼5% of bone marrow cellularity. The patient's rash gradually evolved into large ulcers over bilateral medial malleoli. Despite treatment with immunosuppression, rituximab, and plasmapheresis, they expanded to 9.3 × 8.9 × 0.3 cm on the left and 8.6 × 5.6 × 0.4 cm on the right (middle), requiring extensive use of opioids and significantly interfering with patient's mobility. A second bone marrow biopsy was again suggestive of WM. However, the frequency of abnormal B-cells was even lower (1.7%), likely reflecting prior therapy. Therefore, B-cells were purified by flow sorting using antibodies against CD19, CD20, and CD38 prior to molecular testing. Critically, this approach revealed the presence of MYD88 L265P mutation, supporting the diagnosis of WM and justifying therapy with Ibrutinib, a Bruton kinase inhibitor which is highly effective in this disease. Starting Ibrutinib 420 mg daily was a clear turning point in the patient's clinical course. After just a few days, he reported a drastic improvement in fatigue and leg ulcer pain. Within the following 2 months, he became pain-free and no longer required narcotics. By the end of the 9 months of therapy, the ulcers were completely healed (right) and quantitative IgM went down from 1058 to 325 mg/dL after the first 3 months. Nearly 2 years since starting the treatment, the patient is asymptomatic except for mild parasthesiae. He continues on Ibrutinib at a lower dose (280 mg daily). Cryoglobulinemia (CG) is a rare multisystem disorder which is caused by immunoglobulins with a capacity to aggregate at lower temperatures (cryoglobulins). Notably, even a small amount of cryoglobulin in the serum is sufficient to cause disease. Hence, a low-burden lymphoproliferative disorder that serves as the source of monoclonal cryoglobulin may be difficult to diagnose, precluding the use of targeted therapies. In agreement with a single prior report [1], our case shows that Ibrutinib can be remarkably effective in refractory CG due to WM. Unexpectedly, we found that despite a near-complete resolution of symptoms with Ibrutinib, the patient's cryoglobulins/IgM paraprotein persisted. In addition, the rheumatoid factor was still elevated, and C4 complement remained undetectable. This suggests that the therapeutic benefit of Ibrutinib may be explained not only by anti-tumor effect but also anti-inflammatory properties, as demonstrated by its efficacy in COVID [2], graft-versus-host disease [3], and autoimmune conditions [4]. Finally, our case shows that enriching for B-cells prior to molecular analysis may aid the diagnosis of occult lymphoproliferative disorder in other patients, for example those with IgM monoclonal gammopathy of clinical significance, who may also benefit from a trial of targeted therapy. H. Jamali wrote the manuscript. D. Wu performed cell sorting and molecular analysis. L. Soma performed morphological assessment of the bone marrow. M. Linenberger contributed to clinical management of the patient (plasma exchange). M. Wener provided rheumatology expertise and co-managed the patient with LS. L. Silberstein provided haematology expertise, co-managed the patient with MW, and wrote the manuscript. The authors have nothing to report. The authors received no specific funding for this work. None of the authors received financial compensation from an external source in return for writing or publishing this paper. The patient consented to the use of their clinical data and photographs for this correspondence.

Treatment of Patients with Waldenström Macroglobulinaemia: Clinical practice update from the Myeloma Foundation of Australia Medical and Scientific Advisory Group.

Waldenström Macroglobulinaemia (WM) is an indolent B-cell malignancy characterised by the presence of IgM paraprotein, bone marrow infiltration by clonal small B lymphocytes with plasmacytic differentiation, and the MYD88 L265P mutation in >90% of cases. Traditionally, WM has been treated with chemoimmunotherapy. Recent trials have demonstrated the efficacy and safety of Bruton's Tyrosine Kinase inhibitors (BTKi) in WM, both as monotherapy and in combination with other drugs. There is emerging evidence on use of other agents including BCL2 inhibitors and on treatment of rare presentations of WM. In this update, the Medical and Scientific Advisory Group ofMyeloma Australia review available evidence on treatment of WM since the last publication in 2017 and provide specific recommendations to assist Australian clinicians in the management of this disease. This article is protected by copyright. All rights reserved.

Combining MYD88 L265P mutation detection and clonality determination on CSF cellular and cell-free DNA improves diagnosis of primary CNS lymphoma.

Diagnosis of primary central nervous system lymphoma (PCNSL) is challenging, and although brain biopsy remains the gold standard, cerebrospinal fluid (CSF) constitutes a less invasive source of lymphomatous biomarkers. In a retrospective cohort of 54 PCNSL cases tested at diagnosis or relapse, we evaluated the contribution of immunoglobulin heavy chain (IGH) gene clonality and MYD88 L265P detection on both CSF cell pellets and supernatants, in comparison with cytology, flow cytometry, interleukin (IL)-10 and IL-6 quantification. Clonality assessment included a new assay to detect partial IGH-DJ rearrangements. Clonal IGH rearrangements and/or MYD88 L265P mutation were detected in 27 (50%) cell pellets and 24 (44%) supernatant cell-free (cf) DNA. Combining analyses on both compartments, 36 (66%) cases had at least one detectable molecular marker, present only in cfDNA for 9 (16%) of them. While cytology and flow cytometry were positive in only 7 (13.0%) and 9 (17.3%) cases respectively, high IL-10 levels were observed in 36 (66.7%) cases. Overall, taking into account molecular and cytokine results, 46/54 (85%) cases had at least one lymphomatous biomarker detectable in the CSF. These results show that this combination of biomarkers evaluated on both cell pellet and supernatant CSF fractions improves significantly the biological diagnosis of PCNSL.

Transcriptome Analysis of Diffuse Large B-Cell Lymphoma Cells Inducibly Expressing MyD88 L265P Mutation Identifies Upregulated CD44, LGALS3, NFKBIZ, and BATF as Downstream Targets of Oncogenic NF-κB Signaling.

During innate immune responses, myeloid differentiation primary response 88 (MyD88) functions as a critical signaling adaptor protein integrating stimuli from toll-like receptors (TLR) and the interleukin-1 receptor (IL-1R) family and translates them into specific cellular outcomes. In B cells, somatic mutations in MyD88 trigger oncogenic NF-κB signaling independent of receptor stimulation, which leads to the development of B-cell malignancies. However, the exact molecular mechanisms and downstream signaling targets remain unresolved. We established an inducible system to introduce MyD88 to lymphoma cell lines and performed transcriptomic analysis (RNA-seq) to identify genes differentially expressed by MyD88 bearing the L265P oncogenic mutation. We show that MyD88L265P activates NF-κB signaling and upregulates genes that might contribute to lymphomagenesis, including CD44, LGALS3 (coding Galectin-3), NFKBIZ (coding IkBƺ), and BATF. Moreover, we demonstrate that CD44 can serve as a marker of the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) and that CD44 expression is correlated with overall survival in DLBCL patients. Our results shed new light on the downstream outcomes of MyD88L265P oncogenic signaling that might be involved in cellular transformation and provide novel therapeutical targets.

Rapid detection of the MYD88 L265P mutation for pre- and intra-operative diagnosis of primary central nervous system lymphoma.

The myeloid differentiation primary response gene 88 (MYD88) L265P mutation is a disease-specific mutation of primary central nervous system lymphoma (PCNSL) among the central nervous system tumors. Accordingly, this mutation is considered a reliable diagnostic molecular marker of PCNSL. As the intra-operative diagnosis of PCNSL is sometimes difficult to achieve using histological examinations alone, intra-operative detection of the MYD88 L265P mutation could be effective for the accurate diagnosis of PCNSL. Herein, we aimed to develop a novel rapid genotyping system (GeneSoC) using real-time polymerase chain reaction (PCR) based on microfluidic thermal cycling technology. This real-time PCR system shortened the analysis time, which enabled the detection of the MYD88 L265P mutation within 15 min. Rapid detection of the MYD88 L265P mutation was performed intra-operatively using GeneSoC in 24 consecutive cases with suspected malignant brain tumors, including 10 cases with suspected PCNSL before surgery. The MYD88 L265P mutation was detected in eight cases in which tumors were pathologically diagnosed as PCNSL after the operation, while wild-type MYD88 was detected in 16 cases. Although two of the 16 cases with wild-type MYD88 were pathologically diagnosed as PCNSL after the operation, MYD88 L265P could be detected in all eight PCNSL cases harboring MYD88 L265P. The MYD88 L265P mutation could also be detected using cell-free DNA derived from the cerebrospinal fluid of two PCNSL cases. Detection of the MYD88 L265P mutation using GeneSoC might not only improve the accuracy of intra-operative diagnosis of PCNSL but also help the future pre-operative diagnosis through liquid biopsy of cerebrospinal fluid.

Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors.

Inhibitors of phosphatidylinositol 3-kinase (PI3K) and Bruton tyrosine kinase (BTK) represent a recognized option for the treatment of patients affected by indolent B cell lymphomas. However, small molecules as single agents show limited success in their ability in inducing complete responses, with only partial remission achieved in most patients, suggesting the need for combination therapies. IRAK4 is a protein kinase downstream of the Toll-like receptor signaling (TLR), a driver pathway of secondary tumor° resistance in both hematological and solid tumor malignancies. Activation of IRAK4 upon TLRs and IL-1 receptor (IL-1R) stimulation and through the adaptor protein MYD88 initiates a signaling cascade that induces cytokine and survival factor expression mediated by the transcription factor NF-κB. MYD88-L265P encoding mutations occur in diffuse large B-cell lymphomas, in lymphoplasmacytic lymphomas and in few marginal zone lymphomas (MZL). The IRAK4 inhibitor emavusertib (CA-4948) has shown early safety and clinical activity in lymphoma and leukemia patients. In this preclinical study, we assessed emavusertib effectiveness in MZL, both as single agent and in combination with targeted agents, with a particular focus on its capability to overcome resistance to BTK and PI3K inhibitors. We showed that the presence of MYD88 L265P mutation in bona fide MZL cell lines confers sensitivity to the IRAK4 inhibitor emavusertib as single agent. Emavusertib-based combinations improved the sensitivity of MZL cells to BTK and PI3K inhibitors, including cells with a secondary resistance to these agents. Emavusertib exerted its activity via inhibition of NF-κB signaling and induction of apoptosis. Considering the early safety data from clinical trials, our study identifies the IRAK4 inhibitor emavusertib as a novel compound to be explored in trials for patients with MYD88-mutated indolent B cell lymphomas as single agent and as combination partner with BTK or PI3K inhibitors in unselected populations of patients.

Waldenström macroglobulinemia: 2023 update on diagnosis, risk stratification, and management.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, lymphadenopathy, and rarely hyperviscosity. Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. The L265P mutation in MYD88 is detectable in more than 90% of patients and is found in most IgM MGUS patients. MYD88 is not required for the diagnosis. Age, hemoglobin level, platelet count, β2 microglobulin, LDH, and monoclonal IgM concentrations are characteristics that are predictive of outcomes. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-monotherapy is inferior to regimens that combine it with bendamustine, an alkylating agent, a proteosome inhibitor, or a BTK inhibitor. The preferred Mayo Clinic induction is either rituximab and bendamustine (without rituximab maintenance) or zanubrutinib. Bortezomib, cyclophosphamide, fludarabine, thalidomide, everolimus, Bruton Tyrosine Kinase inhibitors, carfilzomib, lenalidomide, bendamustine, and venetoclax have all been shown to have activity in relapsed WM. Given WM's natural history, the reduction of therapy toxicity is an important part of treatment selection.

Waldenström Macroglobulinemia and Non-IgM-Type Lymphoplasmacytic Lymphoma Are Genetically Similar

Introduction: Waldenström macroglobulinemia (WM) represents a subset of lymphoplasmacytic lymphoma (LPL) with the immunoglobulin (Ig)M paraprotein. MYD88 L265P and CXCR4 mutations are common mutations in WM patients, and mutations in ARID1A and KMT2D (MLL2) have also been reported. However, little information has been accumulated on genetic changes in LPL with other paraproteins like IgG. Methods: We therefore aimed to evaluate genetic differences between WM and LPL with non-IgM paraprotein (non-IgM-type LPL) using targeted next-generation sequencing (NGS) in 20 Japanese patients (10 with WM, 10 with non-IgM-type LPL). Results: Mutations were detected in ARID1A (10%), CXCR4 (20%), MYD88 (90%), and KMT2D (0%) for WM patients and in ARID1A (10%), CXCR4 (20%), MYD88 (70%), and KMT2D (10%) for non-IgM-type LPL patients. No significant differences were identified. No mutations were detected in NOTCH2, PRDM1, CD274 (PD-L1), PDCD1LG2 (PD-L2), RAG2, MYBBP1A, TP53, or CD79B. Discussion: Mutant allele frequency in MYD88 L265P did not differ significantly between WM and non-IgM-type LPL. Most mutations detected by NGS were subclonal following MYD88 L265P, although one non-IgM-type LPL patient harbored only CXCR4 S338X mutation. Our NGS analyses reveal genetic characteristics in LPL patients and suggest genetic similarities between these two subsets of LPL, WM and non-IgM-type.