Waldenström Macroglobulinemia Patients Research Articles

Impact of the presence and number of chromosomal abnormalities on the clinical outcome in Waldenström Macroglobulinemia: a monocentric experience.

The prognostic and predictive role of specific gene mutations in Waldenström Macroglobulinemia (WM) is well-ascertained whereas the clinical impact of chromosome aberrations is far less known. Recent work has provided initial evidence for an adverse prognostic impact of some aberrations, such as del(6q), while other studies suggest a possible relationship between some clinical features (e.g. advanced age and/or inflammatory status) and specific cytogenetic abnormalities. To add to the still limited knowledge on WM cytogenetics and its clinical implications, we herein report our experience in a cohort of WM patients across 23years. Based on our retrospective study, we found that abnormal karyotype was more represented in older patients and maintained a statistically significant independence from other molecular, clinical, and biological features related to WM. The presence and number of cytogenetic aberrations correlated with inferior overall and progression-free survival outcomes regardless of the type of single chromosome aberration. Our data suggests that the role of the altered karyotype deserves to be further clarified especially in elderly WM patients, in whom cytogenetic abnormalities and disease biology appear to be characterized by a higher degree of complexity.

Serum CXCL13 level is related to treatment response and predicts disease prognosis in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a type of B-cell lymphoma that produces IgM. Our study aimed to investigate the role of CXCL13, a chemokine essential for B lymphocytes, in the evaluation of treatment response and prognosis in WM. We collected serum samples and clinical data from 72 WM patients, with 69 patients receiving systemic therapy and 3 patients opting not to receive treatment. Serum CXCL13 levels at baseline and after six months of treatments were measured by enzyme-linked immunosorbent assay. The median serum level of CXCL13 was 1 539.2pg/ml (range 10.0-21 389.9) at baseline and significantly decreased to 123.1pg/ml (range 0.0-6741.5) after 6months of treatments. At baseline, higher CXCL13 levels were associated with lower hemoglobin levels (p = 0.001), higher β2-microglobulin levels (p = 0.001), lower albumin levels (p = 0.046), and higher IPSS-WM scores (p = 0.013). After 6months of treatment, patients who achieved PR/VGPR had significantly lower CXCL13 levels compared to those with SD (70.2pg/ml vs 798.6pg/ml, p = 0.002). The median follow-up period was 40months (range 4.2-188). Eight patients died during the follow-up period. Overall survival differed based on CXCL13 levels. When grouped by baseline CXCL13 levels, the median OS was 60.0months in patients with serum CXCL13 > 2 000pg/ml, while it was not reached in patients with low CXCL13 levels (p < 0.001). Based on CXCL13 levels after the treatments, the median OS was 74.0months in patients with serum CXCL13 > 200pg/ml, while it was not reached in patients with CXCL13 ≤ 200pg/ml. In a subgroup of 28 patients with a series of serum samples, the increase of serum CXCL13 level was associated with disease progression or the start of next-line therapy (p < 0.001). Our study concludes that serum CXCL13 levels decrease in WM patients treated with various regimens and correlate with treatment response. Detecting serum CXCL13 at baseline or after treatment help in predicting prognosis.

Diagnosis and treatment understanding of Waldenström macroglobulinemia in China: a cross-sectional study

Objective: To conduct a nationwide physician survey to better understand clinicians' disease awareness, treatment patterns, and experience of Waldenström macroglobulinemia (WM) in China. Methods: This cross-sectional study was conducted from February 2022 to July 2022 by recruiting clinicians with WM treatment experience from hematology, hematology-oncology, and oncology departments throughout China. Quantitative surveys were designed based on the qualitative interviews. Results: The study included 415 clinicians from 219 hospitals spread across thirty-three cities and twenty-two provinces. As for diagnosis, the laboratory tests prescribed by physicians for suspected WM patients were relatively consistent (92% -99% recommendation for laboratory, 79% -95% recommendation for pathology, 96% recommendation for gene testing, and 63% -83% recommendation for imaging examination). However, from a physician's perspective, there was 22% misdiagnosis occurred in clinical practice. The rate of misdiagnosis was higher in lower-level hospitals than in tertiary grade A hospitals (29% vs 21%, P<0.001). The main reasons for misdiagnosis were that WM was easily confused with other diseases, and physicians lacked the necessary knowledge to make an accurate diagnosis. In terms of gene testing in clinical practice, 96% of participating physicians believed that WM patients would require gene testing for MYD88 and CXCR4 mutations because the results of gene testing would aid in confirming diagnosis and treatment options. In terms of treatment, 55% of physicians thought that the most important goal was to achieve remission, while 54% and 51% of physicians wanted to improve laboratory and/or examination results and extend overall survival time, respectively. Among patients with treatment indications, physicians estimated that approximately 21% of them refused to receive treatment, mainly owing to a lack of affordable care and disease awareness. When selecting the most appropriate treatment regimens, physicians would consider patient affordability (63% ), comorbidity (61% ), and risk level (54% ). Regimens containing Bruton tyrosine kinase inhibitor (BTKi) were most widely recommended for both treatment-naïve and relapsed/refractory patients (94% for all patients, 95% for treatment-naïve patients, and 75% for relapsed/refractory patients), and most physicians recommended Ibrutinib (84% ). For those patients who received treatment, physicians reported that approximately 23% of patients did not comply with the treatment regimen due to a lack of affordability and disease awareness. Furthermore, 66% of physicians believe that in the future, increasing disease awareness and improving diagnosis rates is critical. Conclusions: This study is the first national physician survey of WM conducted in China. It systematically describes the issues that exist in WM diagnosis and treatment in China, such as a high rate of misdiagnosis, limited access to gene testing and new drugs, and poor patient adherence to treatment. Chinese doctors believe that improving doctors' and patients' understanding of WM is one of the most urgent issues that must be addressed right now.

Biomarker analysis of the ASPEN study comparing zanubrutinib with ibrutinib for patients with Waldenström macroglobulinemia

AbstractThe phase 3 ASPEN trial (NCT03053440) compared Bruton tyrosine kinase inhibitors (BTKis), zanubrutinib and ibrutinib, in patients with Waldenström macroglobulinemia (WM). Post-hoc biomarker analysis was performed using next-generation sequencing on pretreatment bone marrow samples from 98 patients treated with zanubrutinib and 92 patients treated with ibrutinib with mutated (MUT) MYD88 and 20 patients with wild-type (WT) MYD88 treated with zanubrutinib. Of 329 mutations in 52 genes, mutations in CXCR4 (25.7%), TP53 (24.8%), ARID1A (15.7%), and TERT (9.0%) were most common. TP53MUT, ARID1AMUT, and TERTMUT were associated with higher rates of CXCR4MUT (P < .05). Patients with CXCR4MUT (frameshift or nonsense [NS] mutations) had lower very good partial response (VGPR) and complete response rates (CR; 17.0% vs 37.2%, P = .020) and longer time to response (11.1 vs 8.4 months) than patients with CXCR4WT treated with BTKis. CXCR4NS was associated with inferior progression-free survival (PFS; hazard ratio [HR], 3.39; P = .017) in patients treated with ibrutinib but not in those treated with zanubrutinib (HR, 0.67; P = .598), but VGPR + CR rates were similar between treatment groups (14.3% vs 15.4%). Compared with ibrutinib, patients with CXCR4NS treated with zanubrutinib had a favorable major response rate (MRR; 85.7% vs 53.8%; P = .09) and PFS (HR, 0.30; P = .093). In patients with TP53MUT, significantly lower MRRs were observed for patients treated with ibrutinib (63.6% vs 85.7%; P = .04) but not for those treated with zanubrutinib (80.8% vs 81.9%; P = .978). In TP53MUT, compared with ibrutinib, patients treated with zanubrutinib had higher VGPR and CR (34.6% vs 13.6%; P < .05), numerically improved MRR (80.8% vs 63.6%; P = .11), and longer PFS (not reached vs 44.2 months; HR, 0.66; P = .37). Collectively, patients with WM with CXCR4MUT or TP53MUT had worse prognosis compared with patients with WT alleles, and zanubrutinib led to better clinical outcomes.

Incidence and Risk of Secondary Malignancy in Patients with Waldenström Macroglobulinemia: A Population-Based Analysis

Waldenström macroglobulinemia (WM) is a rare lymphoplasmacytic lymphoma which may predispose individuals to development of secondary malignancies (SMs). The Surveillance, Epidemiology, and End Results (SEER) database is a comprehensive registry of cancer patients in the United States reporting on a wide set of demographic variables. Using the SEER-18 dataset, analyzing patients from 2000 to 2018, we aimed to assess the incidence of SMs in WM patients. Patient characteristics such as gender, age, race, and latency were identified, and respective standardized incidence ratios (SIRs) and absolute excess risks (AERs) were calculated to compare to the general population. Of the 4,112 eligible WM patients identified, SMs were reported in 699 (17%) patients. The overall risk of developing SM, second primary malignancy, and secondary hematological malignancy was significantly higher in WM patients compared to the general population. Our findings show that WM patients had a 53% higher risk of SMs relative to the general population, and an AER of 102.69 per 10,000. Although the exact mechanism is unclear, the risk of SM development may be due to genetic predisposition, immune dysregulation, or treatment-induced immune suppression.

Changes in Methylation and Chromatin Accessibility Underlie Subtype Classification and Disease Evolution in Waldenström's Macroglobulinemia

Background: We previously identified three subtypes of Waldenstrom's Macroglobulinemia (WM): B-cell like (BCL), Plasma cell like (PCL), and an intermediate group enriched for early/smoldering WM from which the other two subtypes evolved (Hunter et al, ASH 2022). Diffusion pseudo-time (DPT) analysis suggested all WM samples could be placed on a shared evolutionary path independent of subtype, and when samples were separated into Early and Late DPT values, it mirrored disease progression. The role of epigenetic dysregulation that underlies subtype classification and disease evolution remains poorly understood in WM. Methods: We therefore performed chromatin availability analysis using ATACSeq and bisulfite sequencing of WM patients. ATAC-Seq libraries were constructed from 37 CD19+ BM cells from treatment-naive WM patients along with 6 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood (PB) controls and 3 HD CD138+ plasma cell (PC) samples. Enhanced reduced representational bisulfite sequencing (ERRBS) was also performed on 65 MYD88 mutated and 13 MYD88 wild type (WT) WM patients using CD19-selected BM lymphoplasmacytic cells. For 11 WM MYD88 mutated patients, CD138+ cells were also selected. In addition ERRBS was also performed on paired CD19+ and CD138+ selected samples from 5 IgM multiple myeloma (MM) patients, 6 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood cells; 8 HD CD138+ selected PC; as well as MYD88 mutated BCWM.1, BCWM.2, MWCL-1, RPCI-WM1, OCI-Ly3, TMD-8, HBL-1 and SU-DHL2 cells; MYD88 wild-type OCI-Ly7, OCI-Ly19, Ramos B-cells. To infer 5-hydroxymethylcytosine (5HMC) levels, oxidative bisulfite sequencing was also performed for 73 samples including the HD, MM, WT and WM CD19/138 pairs. All ATAC-Seq and 37 of the WM ERRBS samples were part of our multi-omic study that included 253 treatment naïve patients who underwent whole exome and RNASeq sequencing. Results: A UMAP projection of the CpG ERRBS data clustered WM patients into two distinct groups ( Figure 1). One group clustered directly with HD Memory B-cells (MB) and was greatly enriched for CXCR4 mutated WM patients. The other WM group clustered with IgM MM patients and was largely CXCR4 WT. The findings are consistent with the BCL and PCL WM subtypes, which were confirmed by a perfect assignment correlation with our larger multi-omic data set wherein these subtypes were defined. Notably, BCWM.1, BCWM.2 and MWCL-1 WM cell lines clustered with the WM samples. The 5HMC data displayed no clear clustering of the patient samples but clearly separated all HD samples from those of WM and IgM MM samples, consistent with a role for 5HMC dysregulation in oncogenesis. Differential methylation analysis showed differences by MYD88 and CXCR4 mutation status, deletions in Chr6q, familial history for WM, WM subtype, and stratification by early and late DPT values. Notably, the only comparison that did not yield any differences was WM CD19+ to WM CD138+, suggesting that this may not be a meaningful epigenetic distinction in WM. Similar comparisons were performed on the ATAC peaks. Differential chromatin accessibility was noted near many of the corresponding differentially expressed genes. ATAC peak coverage significantly correlated with the expression of top genes. This was particularly true for WM subtype and DPT comparisons ( Table 1). DNA sequences from differentially accessible peaks between BCL and PCL subtypes were also evaluated for relative TF motif enrichment in open vs. closed chromatin revealing PAX5, FUBP1, and ZNF121 enrichment in BCL subtypes using PCL subtype peaks as a background control. TFs REL, SP1, SPIB, BC11A and JUN were enriched PCL subtype compared with BCL subtype. Correlation between DPT associated genes revealed two clear groups, one associated with stem cell and pre-B-cell genes (Group 1) and the other with inflammatory, myeloid, and T-cell genes (Group 2). Motif analysis of differential peaks correlating with genes in each group demonstrated comparative enrichment for MYC, MXI1, and E2F2 in group 1 while RUNX1, NFKB1, and ATF1 were in enriched in group 2. Conclusions: This is the first independent validation of our previously reported multi-omic driven WM subtype classification. The studies underscore that epigenetic differences underlie the biology of WM subclassification and support a strong role for epigenetic changes driving WM evolution.

Development of a Novel Prognostic Score in Patients with Waldenström Macroglobulinemia Prospectively Enrolled in the Observational NF10 Study of Fondazione Italiana Linfomi

Background. IPSSWM and revised-IPSSWM are the two currently prognostic scores used for survival prediction in symptomatic Waldenström Macroglobulinemia (WM) patients in need of treatment. In addition to age and serum beta-2 microglobulin levels, which are present in both scores, the IPSSWM includes anemia, thrombocytopenia and the concentration of the serum monoclonal component (MC), while revised-IPSSWM includes LDH and serum albumin levels. Both IPSSWM and revised-IPSSWM have been developed in retrospective series and apply only to symptomatic WM patients. Aim. To analyze WM patients prospectively enrolled in the NF10 observational study since 2010 with the aim to define a prognostic score applicable to all WM patients at diagnosis, either symptomatic or asymptomatic. Methods. The progression-free survival (PFS) and overall survival (OS) rate was estimated and plotted using the Kaplan-Meier method, with 95% confidence interval (95% CI), and the effect of covariables on PFS was estimated by means of the Cox proportional hazard (PH) regression. The association with PFS was expressed as hazard ratio (HR) with 95% CI, either in univariable or multivariable regression. All statistical tests were two-sided. The proportionality of risk of the multivariable Cox PH model was checked graphically by means of the scaled Schoenfeld residuals, and the model performance was analyzed using the likelihood displacement method. Starting from a model with 10 variables selected on the basis of clinical and statistical considerations, the final model was selected on considering the Akaike (AIC) and likelihood ratio test. The cut-offs were selected after modelling the covariate with restricted cubic spline in Cox regression. The final model was internally validated using bootstrap techniques to evaluate the discriminant power (C-index), slope shrinkage to check for overfitting, and calibration (comparison between predicted and actual survival). Results. We identified 303 eligible patients from the NF10 study, of whom 185 (60%) needed treatment immediately after diagnosis (42%) or later (18%). These were main patients characteristics: 59% were male, 46% were aged &amp;gt;70 years, 8% had constitutional symptoms, 55% had Hb &amp;lt;12 g/dL, 54% had an absolute lymphocyte count ≥2x10 9/L, 9% had platelets &amp;lt;100x10 9/L, 37% had a serum MC &amp;gt;2 g/dL, 21% had albumin &amp;lt;3.5 g/dL, 23% had LDH &amp;gt; upper limit of normal (ULN), 32% had beta-2 microglobulin &amp;gt;1.5xULN. With a median follow-up of 51 months, 5-year PFS for the entire cohort was 67% (95%CI 60-73%). In univariate analysis, age &amp;gt;70 years (P=0.043), constitutional symptoms (P=0.009), nodal sites (P=0.002), Hb &amp;lt;12 g/dL (P&amp;lt;0.001), absolute lymphocyte count ≥2x10 9/L (P=0.033), serum MC &amp;gt;2 g/dL (P=0.003), high beta-2 microglobulin (P&amp;lt;0.001) and LDH (P=0.001), albumin &amp;lt;3.5 g/dL (P&amp;lt;0.001), C-reactive protein &amp;gt;4 mg/dL (P=0.008) were associated with shorter PFS. In multivariate analysis, age, anemia, hypoalbuminemia and high LDH retained prognostic value and thus entered the final model. Patients were classified into 3 risk groups: low (0-1 factors, 62% of patients), intermediate (2 factors, 24%) or high (3-4 factors, 15%), with a 5-year PFS of 80% (95%CI 70-87%), 49% (95%CI 32-64%) and 25% (95%CI 11-42%), respectively, with C-Harrell=0.744 and robust internal validation and calibration. OS was respectively 96% (95%CI 90-98%), 77% (95%CI 62-87%) and 43% (95%CI 25-60%) for the low, intermediate and high risk group (Figure 1). Conclusions. We developed a prognostic model based on 4 parameters easily attainable in clinical practice, able to predict PFS and OS at diagnosis. This is the first prognostic score for WM developed in a prospective study and applicable to patients presenting either with symptomatic or asymptomatic disease. This prognostic model identifies a subset of high-risk patients with a dismal outcome and a median OS &amp;lt;5 years. External validation in independent cohorts is needed.

Atypical Stem Cell, Pre-B-Cell, T-Cell and Myeloid Gene Expression Characterizes Early Waldenstrom's Macroglobulinemia Clones Which Diminishes with Advancing Disease and Has Therapeutic Implications

Background: In previous studies using multi-omic data for 253 treatment-naive patients with Waldenstrom's Macroglobulinemia (WM), we identified three subtypes of WM: B-cell like (BCL), Plasma cell like (PCL), and an intermediate clone enriched for early/smoldering WM from which the other two evolved (Hunter et al, ASH 2022). The BCL and PCL subtypes show distinct mutation and transcriptional signatures, as well as clinical characteristics. Diffusion pseudo-time (DPT) analysis suggests all patient tumors, regardless of subtype have a shared evolutionary path and can be divided into Early and Late DPT determined stages that mirror disease progression. Additional analysis of DPT suggests it represents a continuous process akin to clonal evolution. We therefore sought to further characterize the DPT signal in MYD88 mutated WM. Methods: RNASeq was performed on CD19-selected bone marrow (BM) samples from 249 of the 253 treatment-naive WM patients who were MYD88 mutated, as well as 13 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood samples. Whole exome sequencing of CD19-selected BM cells along with CD19-depleted peripheral blood mononuclear controls was also performed for 215 of the WM samples. To assess BM histology, slides from 66 BM paraffin blocks from these WM patients were analyzed. Results: WM patients had median age of 66 (range 31-95 years), BM involvement of 50% (5-95%) and serum IgM level of 3,162 (104-10,321 mg/dL). The median follow-up for the study cohort was 8.8 (range 0.2 - 33.8 years). Patients were stratified into 5 cohorts by DPT values. Analysis of BM involvement by DPT cohorts revealed a stepwise increase with the median involvement rising from 20% in the lowest to 80% in the highest cohort (p&amp;lt;0.0001). Increasing DPT predicted for shorter time from biopsy to first therapy (p=0.0003) but was not predictive of time from diagnosis to first therapy consistent with DPT alignment by stage of WM development. RNASeq analysis revealed high expression of genes atypical of mature B-cells that diminished with increasing DPT ( Figure 1). Among these were the myeloid chemo-attractants CXCL1, CXCL8 and CXCL12 which fell from a median of 4.4, 67.2 and 41.1 to 0.1, 3.6 and 0.8 transcripts per million (TpM) in the lowest and highest DPT cohorts, respectively (p&amp;lt;0.0001). Accordingly, all the top 20 gene ontology (GO) terms associated with DPT were related to activation, degranulation, and migration of myeloid cells and leukocytes. Pathological review of the 66 BM slides ranked by DPT, showed increased early myeloid cells and eosinophils clustering near small lymphoid aggregates, and as DPT increased, increased lymphocytes and mast cells with decreased myeloid cell involvement was observed. By RNASeq, pre-B-cell genes including MYB, the VDJ recombination genes RAG1, RAG2 and DNTT as well as the surrogate light chain genes IGLL1 and VPREB1 fell from 22.5, 10, 13.5, 61.1, 144 and 38.3 to 2.2, 1, 1.1, 9.1, 12.2 and 4.3 TpM, respectively (p&amp;lt;0.0001). T-cell and myeloid genes including CD4, CD8A, FCGR3A (CD16), CD14 and CD33 also decreased with increased DPT from 67.6, 7.1, 80.5, 200, and 23.2 to 2.4, 0.9, 1.7, 3.6 and 0.7 TpM, respectively (p&amp;lt;0.0005). A subclonal stem cell signature was also detected, including CD34 and KIT (CD117), which fell from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively (0&amp;lt;0.0001). CD34, KIT, preBCR, CD4, CD8A, FCGR3A, CEACAM8 (CD66b), and CD33 expression was confirmed by CD19+ light chain restricted flow cytometry in WM but not in HD samples. In addition, the MB and plasma blast genes PRDM1 and XBP1 were elevated in all samples regardless of DPT, consistent with a putative memory B-cell origin. Progression free survival (PFS) in response to proteasome inhibitors revealed strong stratification by subtype (p=0.001), as well as inferior PFS with BTK-inhibitors in low DPT patients (p= 0.044; Figure 2), suggesting less BTK dependence in the early DPT clones. Consistent with this finding, gene set enrichment identified increased BCR signaling with increased DPT. Conclusions: This analysis suggests that stem cell program reactivation and atypical marker expression characterize early WM clones that are ultimately replaced by clones exhibiting more typical B-cell markers with disease progression. The findings may be relevant to treatment selection and provide a framework for investigating subtype and early/late evolutionary staging in the treatment approach to WM.

Establishment of the Bcwm.2 Cell Line As a BTK-Inhibitor Resistant, BCL2 Inhibitor Sensitive in Vitro and In Vivo Study Model for Waldenström's Macroglobulinemia

Background: Waldenström's macroglobulinemia (WM) is an indolent B-cell lymphoproliferative disorder characterized by bone marrow (BM) infiltration with lymphoplasmacytic lymphoma and monoclonal immunoglobulin M (IgM) production. Mutations in MYD88 are present in 95-97% of WM patients. Deletions involving chromosome 6q (del6q) are found in up to 50% of WM patients, and include genes that regulate MYD88, BCL2 and apoptotic signaling. Cell lines can play a pivotal role as disease models, contributing to a comprehensive understanding of WM biology and advancing therapeutic strategies. The availability of cell lines for the study of WM remains limited, and none demonstrate del6q. Patient and Methods: We developed and characterized a novel cell line (BCWM.2) from long-term cultures of CD19 + selected BM lymphoplasmacytic cells from a symptomatic, treatment naive WM patient who then received ibrutinib monotherapy on a clinical trial. The patient attained a major response to treatment, and after 2 years progressed. The patient underwent serial bone marrow biopsies, and extensive genomic and transcriptome analysis of his tumor during the study, and the corresponding cell line established at baseline. An in vivo xenograft mouse model of BCWM.2 was also established in NOD SCID mice, and the origin of the xenografted tumor was confirmed by genomic sequencing and immunohistochemistry. Results: BCWM.2 cells exhibited morphologic and immunophenotypic characteristics resembling lymphoplasmacytic cells and demonstrated robust propagation when co-cultured with HS-5 stromal cells in IMDM medium supplemented with 20% FBS. Flow cytometric analysis revealed that BCWM.2 exhibited an immunophenotype consistent with the source WM patient. Whole genome sequencing demonstrated that both BCWM.2 cells and original patient WM cells carried somatic activating mutation in MYD88 (S243N) and shared trisomy in chromosomes 3 and 12, heterozygous deletion of 6q, and amplification of 6p. Notably, a novel mutation in the SRC family member LYN (I297N; T&amp;gt;A), a component of BCR, was identified in BCWM.2 but not the original patient tumor. The I297N mutation found in the regulatory hinge region is predicted to cause activation of LYN. At time of progression on ibrutinib, the dominant clone that emerged in the patient's tumor also carried the I297N mutation. Additionally, mutated genes in BCWM.2 included histone deacetylase HDAC5, tumor suppressor RUNX3, B-cell transcriptional activator SPI1, and AKAP9 (A-Kinase Anchoring Protein 9). Transcriptome analysis showed shared expression patterns between BCWM.2 and patient tumor samples, including diminished gene expression of chromosome 6q. Drug response testing revealed that BCWM.2 cells did not respond to the BTK-inhibitors ibrutinib, zanubrutinib or pirtobrutinib but were sensitive to the BCL2 inhibitor venetoclax. Furthermore, BCWM.2 cells readily engrafted in NOD-SCID mice by direct subcutaneous injection of 2 x 10^6 cells/mouse. Tumor growth (~500 mm^3) was observed in the mice four weeks post-inoculation. Importantly, follow-up evaluations demonstrated serial increases in human IgM levels in the mouse sera post-engraftment. Moreover, BCWM.2 engraftment was confirmed by immunohistochemistry with tumors exhibiting intermediate lymphoid cell morphology with plasmacytoid differentiation, CD20 and MUM1 expression, clonality for lambda light chain and IgM heavy chain, 60% KI67 staining. Whole exome sequencing of engrafted tumors further confirmed the presence of the MYD88 S243N and LYN I297N mutations detected in the cell line and the patient at the time of ibrutinib progression. Conclusions: BCWM.2 represents a novel, BTK-inhibitor resistant, BCL2 inhibitor sensitive WM cell line that demonstrates MYD88 (S243N) and LYN (I297N) somatic activating mutations, and deletions of 6q. BCWM.2 thereby provides an important new disease appropriate cell model for the in vitro and in vivo study of WM, including the development of targeted therapies for this disease.

CXCR4 S338X Promotes Resistance to Second Generation BTK Inhibitor and BCL-2 Inhibitor in WM Cells

Introduction: Waldenström macroglobulinemia (WM) is an IgM-secreting lymphoplasmacytic lymphoma. CXCR4 somatic mutations are common in WM and are associated with clinical resistance to Bruton's tyrosine kinase (BTK) inhibitors and other therapeutic agents. CXCR4 mutations occur nearly exclusively with MYD88 L265Pmutation, with CXCR4 S338X constituting the most common in WM patients. The aim of this study was to further investigate the effect of CXCR4 S338X on BTK inhibitors (ibrutinib and zanubrutinib) and a BCL-2 inhibitor (venetoclax) resistance. Methods: MWCL-1 cells were transfected with scramble, CXCR4 or CXCR4 S338Xplasmids using Lipofectamine 3000 followed by neomycin selection. CXCR4 expression was confirmed using qPCR and flow cytometry. Transfected cells were cultivated in presence of ibrutinib (5μΜ), zanubrutinib (5μΜ) and venetoclax (1μΜ) for 48 hours. Proliferation was estimated using CCK-8 colorimetric assay while cell cycle was evaluated by propidium iodide staining and flow cytometry. Apoptosis was estimated by Annexin V/7-AAD staining and flow cytometry while the expression of apoptotic genes was determined by qPCR. The statistical analysis was performed using Student's t-test and Graphpad Prism. Results: Drug-induced cytotoxicity was observed only in the case of venetoclax in CXCR4 S338X transfectedcells (p= 0.0007) while increased cytotoxicity was noted in the presence of ibrutinib, zanubrutinib and venetoclax in the case of scramble-transfected cells (p=0.038, p=0.006 and p=0.0006, respectively). Moreover, no significant difference in proliferation rate was noted between CXCR4 S338Xtransfectedcells treated with the therapeutic agents and CXCR4 S338X untreated cells, underlining the S338X role to promote cell proliferation. Interestingly, CXCR4 S338X transfectedcells exhibited apoptosis resistance not only compared to scramble-transfected cells but also in comparison with CXCR4-transfected cells, associated with resistance to all three drug agents (p=0.004, p=0.003 and p=0.034, respectively). In the case of scramble-transfected cells, an increase in apoptosis was demonstrated in parallel with an increase in caspases 8, 9 and 3 in presence of ibrutinib (p=0.006, p=0.0001 and p=0.025, respectively) and zanubrutinib (p=0.027, p=0.007 and p=0.052, respectively), whereas no statistically significant increase in caspase gene expression was observed in the case of CXCR4 S338Xtransfectedcells, highlighting S338X role in the inhibition of intrinsic and extrinsic apoptosis pathways. Lastly, the presence of S338X mutation acted as a mitotic promoting factor when cells treated with the aforementioned drugs showed a high proportion of cells in G2/M phases of cell cycle compared to scramble and CXCR4 controls. Conclusions: The above data demonstrate the role of CXCR4 S338X mutation in drug resistance and suggest the necessity to develop therapeutic strategies to address this issue and overcome this therapeutic barrier in WM. Data on more cell lines and downstream signaling will be presented at the meeting.

Deciphering the Immune Microenvironment in Waldenstrom's Macroglobulinemia

Recent studies have demonstrated the occurrence of somatic mutations of MYD88 and CXCR4 as key players in Waldenstrom's Macroglobulinemia (WM) pathogenesis and disease progression. Despite the significant improvement in the knowledge of the molecular mechanisms supporting WM biology whether immunosuppressive mechanisms could contribute to WM pathogenesis remains unexplored. We interrogated the transcriptome signatures of the bone marrow (BM) microenvironment of WM patients (n:22) as compared to healthy individuals (n:10), we found a significant enrichment for a Treg-signature, and for CD40/CD40L signaling-related genes (FDR &amp;lt;0.001; P &amp;lt;0.01). These findings were confirmed using a transgenic murine lymphoplasmacytic/WM model, where the BM immune microenvironment showed an increased number of infiltrating CD4+ T lymphocytes over CD8+ T cells. Further characterization of the T-cell compartment revealed the presence of abundant CD4+CD25+FoxP3+ Treg cells, with respect to control mice. Murine WM cells recruited a significantly higher number of more abundant Ki67+Treg cells, as compared to B lymphocytes from healthy mice. Collectively, these results suggest that immunosuppressive Treg cells may play in supporting WM disease biology. These findings prompted us to focus on Tregs and their role in supporting WM pathogenesis. We performed bulk RNA sequencing of Tregs isolated from both WM patients (n:14) and healthy donors/HD (n:8); showing a peculiar transcriptome signature characterizing WM-derived Tregs as compared to their normal cellular counterpart. WM patient-derived Tregs presented with transcriptome profiling enriched for FOXP3-target genes and to Treg induction, as compared to healthy donor-derived Tregs; with WM-Tregs showing a significant enrichment for genes related to interferon- and TNFα-related genes via NF-kB. The functional impact of Treg in WM biology was next studied. We observed a significantly higher ability of WM cells to favor both induction and expansion of CD4+CD25+FoxP3+ Tregs, as compared to HD-derived CD19+ B-cells; paralleled by a significantly higher increase of Ki67-positive Treg induced by WM cells as compared to HD-derived CD19+ B-cells. Same results were obtained using WM primary cells. Data were further corroborated by demonstrating significant enrichment of several pro-proliferative- and pro-survival pathways in WM-Tregs, versus HD-counterpart, including MAPK- and PI3K/AKT-related genes. To gain insight into the potential molecular mechanisms responsible for the observed Treg induction in WM, we performed scRNAseq using the whole cell population harvested at the end of the induction assay. We evaluated the B-T-cell interactions at single cell level, adopting a B-&amp;gt;T cross-talk model; and identified a set of four high-priority genes (CD40, TNF, TNFSF14, ICAM2): although all of them were expressed in B cells, only the expression of CD40 was maximal and virtually restricted to the B clusters, with the other three genes showing a more diffuse pattern. Importantly, CD40-ligand was shown to be the interactor expressed within the Treg cell compartment. Given the identified CD40/CD40L axis as a potential regulator of the WM cell/Treg cross-talk, we used the CD40/CD40L inhibitor DRI-C21045; and found a significant decrease in Treg induction and Treg proliferation. Halting CD40/CD40L interaction inhibited Treg induction and growth, also within the context of CXCR4-mutated WM, supported by inhibition of p-AKT and p-ERK in Treg cells. CD40/CD40L blockade led to inhibition of WM cell growth. Overall our studies have demonstrated the existence of a Treg-mediated immunosuppressive phenotype in WM, which can be therapeutically reversed by blocking the CD40L/CD40 axis to inhibit WM cell growth.

Quantitative MYD88 L265P Analysis Represents a Powerful Tool for Assessing Disease Response and Evaluating Clinical Trial Performance in Waldenstrom's Macroglobulinemia

Serum IgM measurements represent the current standard for assessing changes in disease burden in Waldenström's Macroglobulinemia (WM). Many agents used to treat WM can significantly affect serum IgM levels without impacting the underlying burden of clonal lymphoplasmacytic cells thereby producing discordant findings. Such agents can raise (CD20-directed monoclonal antibodies, IMiDs), or lower (BTK-, proteasome-, MTOR- inhibitors) serum IgM levels, and impact categorical response assessment. Somatic mutations in MYD88 are found in 95-97% of WM patients, and support tumor growth through activation of multiple pro-survival pathways that include HCK/BTK, SYK and ERK. Nearly all MYD88 mutations in WM are of the L265P variant. Previous studies by us and others have shown that serial quantitative measurements of both bone marrow (BM) and peripheral blood (PB) MYD88 L265P (c.978 T&amp;gt;C) by allele-specific PCR can be used to assess changes in underlying BM disease burden (Blood 121:2051-2058; Leukemia 28:1698-1704). However, the utility of using quantitative allele-specific MYD88 L265P (qMYD88 L265P) analysis has not been studied in prospective clinical trials. As such, we performed a comprehensive study of qMYD88 L265P response assessment utilizing BM and PB CD19-selected tissue across 5 prospective clinical studies in WM: Ixazomib, Dexamethasone and Rituximab (IDR; NCT02400437) Ibrutinib monotherapy (IBR; NCT02604511); Venetoclax monotherapy (VEN; NCT02677324); Ibrutinib plus Ulocuplomab (IBR/ULO; NCT03225716); and Ibrutinib plus Venetoclax (IBR/VEN; NCT04273139). Changes in MYD88 L265P ΔCt were assessed as reported above with ΔCt = Mutant Ct - Wild-type Ct, and higher ΔCt values indicating a lower mutant allele burden, with each ΔCt unit reduction representing a 50% decrease in mutant MYD88 L265P burden. Changes in MYD88 L265P ΔCt were compared to changes in underlying BM disease burden and categorical response assessment using the current (IWWM-11) response criteria (Semin Hematol. 60:97-106). Across all five trials, BM (r=0.52; p&amp;lt;0.001) and PB (r=0.43; p&amp;lt;0.001) MYD88 L265P ΔCt changes from baseline were highly correlated at best response with corresponding changes in underlying BM disease burden determined by repeat BM biopsies. As shown in Fig. 1, comparing changes from baseline in BM MYD88 L265P ΔCt assessments across studies showed marked differences, with greatest reductions observed in patients treated with IBR/VEN, IDR, and VEN alone, than for those treated with IBR alone or IBR/ULO. Similar findings were also observed for corresponding PB MYD88 L265P ΔCt assessments from baseline, with changes in PB MYD88 L265P ΔCt strongly correlating with those of BM MYD88 L265P ΔCt findings across all 5 clinical trials (r=0.67; p&amp;lt;0.001), thereby signifying the ability to use PB MYD88 L265P ΔCt assessments to evaluate treatment responses. We next compared findings from BM and PB MYD88 L265P ΔCt to changes from baseline in serum IgM levels across all 5 trials. At best response, major categorical responses denoted by &amp;gt;50% reduction in serum IgM using IWWM-11 criteria showed commensurate decreases in BM and PB MYD88 L265P ΔCt from baseline in patients receiving IBR/VEN, IDR and VEN alone ( Fig. 2). In contrast, minimal changes in BM and PB MYD88 L265P ΔCt from baseline were observed at best response for most major responders on IBR or IBR/ULO, including individuals who achieved very good partial responses denoted by &amp;gt;90% decrease in IgM by IWWM-11 criteria. In this first prospective evaluation of BM and PB qMYD88 L265P response assessment, we show that both BM and PB L265P qMYD88 analysis can provide more accurate assessment of treatment related changes in disease burden over the current standard of IgM response assessment alone, and can be used to more robustly evaluate clinical trial performance byidentifying treatments or regimens that produce more meaningful tumor reductions in WM patients.

Novel Isoforms Identified By Isoseq Analysis Drive Expression Differences in Key Genes That Delineate the Subtypes of Waldenstrom's Macroglobulinemia

Background: Alternative isoforms can alter regulatory motifs, binding partners, localization, and function of gene or even inhibit its function. While isoform regulation in humans is generally well described, there are still many tissue specific isoforms that remain to be fully documented. Moreover, mutations and broad dysregulation of transcriptional and RNA processing machinery is common in cancer resulting in additional novel isoforms. Analysis of 249 MYD88 mutated patients from our multi-omic data set of 253 treatment-naive patients with Waldenstrom's Macroglobulinemia (WM) identified three subtypes of WM: B-cell like (BCL), Plasma cell like (PCL), and an intermediate clone enriched for early/smoldering WM from which the other two evolved. (Hunter et al, ASH 2022) The BCL and PCL subtypes have distinct clinical as well as mutational and transcriptional characteristics. To investigate isoform usage, including novel disease related isoforms, we utilized PacBio single molecule real-time (SMRT) sequencing which can produce reads up to 10Kb allowing for end-to-end single transcript sequences for most genes. Methods: PacBio Isoseq analysis was performed on RNA isolated from CD19+ bone marrow (BM) samples from 11 WM patients; CD19+CD27+ memory B-cells (MB) from two healthy donors; and CD138+ plasma cell (PC) from two healthy donors. SMRT analysis data was aligned and processed using minimap2 with Cupcake. Observed isoforms were filtered, categorized, and annotated using Squanti. High confidence novel isoforms were then added into hg38 Gencode annotation which was used to realign our existing high depth Illumina 150bp paired end RNASeq data set of 249 untreated MYD88 mutated WM patients using STAR and Salmon. DTU analysis and functional annotation was performed using the isoformSwitchAnalyzeR package from Bioconductor in R. Results: The 11 patients in PacBio novel isoform discovery cohort had a median age of 63 (range 48-78 years) at time of sample acquisition; serum IgM of 2,150 (598-5,830 mg/dL); and BM involvement of 75% (range 20-95%). Six (55%) were female; 4 (36%) had a familial history of WM; 4 (36%) had a familial history of other B-cell malignancies; and 3 (27%) were sporadic. MYD88 and CXCR4 mutations were present in 10/11 (91%) and 6/11 (55%) patients, respectively. In the MYD88 mutated RNASeq cohort, 102/249 (41%) were female, the median age at biopsy was 66 (range: 31-95 years), BM involvement was 50% (5-95%), and the median serum IgM was 3,162 (104-10,321 mg/dL). PacBio analysis added 194,997 novel isoforms to the gencode annotation which ranged in function from minor alterations in untranslated regions (UTRs) to the introduction of novel coding sequences. Of the 3,134 genes differentially expressed between BCL and PCL, 1,175 (37.5%) and 1,149 (36.6%) had greater that 50% of their expression assigned to novel isoforms for BCL and PCL, respectively (p=NS), which highlights the impact of the improved annotation. The top result from the DTU analysis was DUSP22, a negative regulator of MAPK and ERK1/2 signaling ( Figure 1). DUSP22 was overexpressed in early/smoldering WM and PCL, with overexpression driven by a novel internal start with a partial intron inclusion. These novel isoforms express only half of DUSP22's functional domain but also contain novel 5' signal peptide sequences targeting the transcript to the endoplasmic reticulum for translation. Another top result was FCRLB, an FC receptor that can promote survival in B-lymphocytes. In HD and BCL, the minimal expression corresponded to a canonical non-coding isoform, while in PCL and early/smoldering WM most of the expression resulted from novel coding isoforms that contained the core protein coding regions of the gene. Other genes with significant DTU included HDAC4, HDAC9, and ARID5B all of which are critical modulators of chromatin accessibility. Functional validation of novel isoforms and DTU analysis of additional genomic, and clinical factors is ongoing. Conclusions: Novel transcripts identified by PacBio IsoSeq analysis drive expression of key genes including the important MAPK/ERK regulator DUSP22, and FC receptor FCRLB that promotes survival of B-lymphocytes. Other genes of interest included modulators of chromatic accessibility. Our findings demonstrate important new insights into isoform usage associated with WM subtype in WM and provide novel insights into the underlying biology of the disease.

Multi-Center Clinical Validation of a Mass Spectrometry Immunoassay for the Diagnosis and Monitoring of Multiple Myeloma and Associated Disorders

Introduction: Mass spectrometry (MS) technology holds great promise for the investigation of monoclonal proteins (M proteins) in peripheral blood. We present results from a multi-center clinical validation study using quantitative immunoprecipitation MS (QIP-MS). QIP-MS combines isotype-specific immunopurification with matrix-assisted laser-desorption ionization MS, and offers automated, sensitive detection, isotyping and quantification of M proteins. It reports the mass/charge ratio (m/z) of the involved light chain, which serves as molecular fingerprint for monitoring the M protein. Methods: The study included 460 diagnosed monoclonal gammopathy (MG) patients (160 multiple myeloma (MM), 112 smoldering MM (SMM), 120 monoclonal gammopathy of undetermined significance (MGUS), 47 Waldenström's Macroglobulinemia (WM), and 21 AL amyloidosis), and 170 disease controls for assessing diagnostic sensitivity and specificity. Sixty-four MM patients with 439 follow-up samples and 10 WM patients with 91 follow-up samples were included to evaluate the ability of QIP-MS to detect M protein changes related to treatment. Median follow up was 19 and 15 months, respectively. Serum samples were retrospectively analyzed at three sites. QIP-MS was carried out using the automated EXENT® solution (in development, The Binding Site, part of Thermo Fisher Scientific). The assay's diagnostic sensitivity and specificity were calculated based on categorizing results as positive or negative. A positive result was defined in baseline samples as the presence of an M protein which was either an intact immunoglobulin ≥0.200 g/L or a light chain only. Results by QIP-MS were also compared to serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). QIP-MS response categories were defined based on M protein changes per international guidelines criteria, and compared to response categories assigned by the treating physician. Complete response (CR) was defined as absence of the M-peak that was observed at baseline, using isotype and m/z value of ±4 as criterion for identity. Results: The overall diagnostic sensitivity of QIP-MS in this study was 95.0%: 93.3% for MGUS; 100.0% for SMM; 94.4% for MM; 100.0% for WM; and 71.4% for AL amyloidosis. The diagnostic specificity of the assay was 68.2%. QIP-MS identified an M protein in more MG patients compared to SPE: 437 (95.0%) vs 398 (86.5%). The positivity rate by QIP-MS vs SPE was 93.3% vs 84.2% in MGUS; 100% vs 92.9% in SMM; 94.3% vs 85.0% in MM; 100% vs 100% in WM and 71.4% vs 47.6% in AL amyloidosis. Method comparison demonstrated a Passing-Bablok slope of 0.8 to 1.2 between QIP-MS and SPE for the quantification of M proteins for each disease group and for each isotype, except for monoclonal IgM and in WM (slope of 1.58). In SPE-positive MG patients, the overall concordance between QIP-MS and IFE for M protein isotype was 97%. The overall concordance rate between QIP-MS response categories and standard response assignment was 55% for MM and 56% for WM: 48% for progressive disease (PD); 63% for stable disease (SD); 46% for minimal response (MR); 71% for partial response (PR); 66% for very good partial response (VGPR); and 25% for CR in MM patients. Among 73 responses categorized as CR, QIP-MS produced a positive result for the original clone in 55 (75.3%) cases. Concordance rates in WM patients were 71% for PD%; 30% for SD; 38% for MR; and 74% for PR; no VGPR or CR were reported by either method. Conclusions: In this study, QIP-MS demonstrated the potential for same or superior diagnostic sensitivity compared to SPE, high concordance with IFE for the M protein isotype and good quantitative agreement with SPE measurements of the M proteins. It reported higher IgM values compared to SPE, likely due to reliance on turbidimetric immunoglobulin measurement for quantitations. Diagnostic specificity was impacted by the identification of minor M proteins, not detectable by SPE, and whose clinical significance requires further investigation. QIP-MS demonstrated “moderate” to “fair” agreement for response assignment in MM and WM, respectively, mostly due to the detection of residual M proteins in a significant proportion of patients in CR, in line with its enhanced analytical sensitivity. These data support the use of QIP-MS as an aid in the diagnosis and monitoring of MGs.

Mutational Landscape of Bone Marrow CD19 and CD138 Cells in Waldenström Macroglobulinemia (WM) and IgM Monoclonal Gammopathy of Undetermined Significance (IgM MGUS)

Background Waldenström's Macroglobulinemia (WM) is an incurable B-cell neoplasm characterized by serum monoclonal immunoglobulin M (IgM) and clonal lymphoplasmacytic cells infiltrating the bone marrow (BM). Smoldering WM (sWM) is the asymptomatic/indolent form with a high risk of progressing to symptomatic WM requiring treatment whereas IgM monoclonal gammopathy of undetermined significance (IgM MGUS) is an early precursor stage of WM. Despite recurrent and activating mutations, including MYD88, CXCR4, ARID1A, KMT2D, and CD79B have been identified, the genetic basis for WM and the risk of progression of IgM MGUS to WM remain to be fully elucidated. Methods Based on the mutational profiling of WM and our previous wide transcriptome analyses on BM CD19+ and CD138+ cells of patients with WM and IgM MGUS, we decided to investigate the mutation status of these patients as follows: WM (n=10), sWM (n=7), and IgM MGUS (n=5), by performing high throughput targeted AmpliSeq next generation sequencing on 117 target genes potentially involved in B-cell lymphomagenesis and in the risk of progression of IgM MGUS to WM (Trojani A. et al. Cancers, 2021). Results We identified genetic aberrations in multiple genes, including previously described classic mutations in MYD88 (L260P alias L265P) found in 82.3% (14/17) of WM/sWM patients and in 60% (3/5) of IgM MGUS subjects. The CXCR4 mutation (S338Ter) was detected in 23.5% of CD19+ cells of WM patients, whereas it was absent in IgM MGUS subjects. Interestingly, we also identified new mutated genes, including WNK2 somatic mutations affecting 47% of WM patients, for which a recurrent allelic variant (V1635Ter) was observed, and BCL9 with a recurrent frameshift variant (P516Lfs) identified in 29.4% of WM patients exclusively in the CD19 cell fraction. The BCL9 (P516Lfs) variant was also detected in the B cells of one IgM MGUS patient. Moreover, sequencing evaluation revealed recurrently frameshift or missense mutations involving NFKB2 (L473Afs), PTPN13 (P1546Tfs), CARD11 (S622del), KMT2C (I823T), and ATM in WM and IgM MGUS patients (Figure 1). Of interest, KMT2C (also known as MLL3) exhibited a somatic hypermutated phenotype in both CD19+ and CD138+ cells of WM and IgM MGUS samples. The KMT2C mutations were not mutually exclusive with those involving KMT2D in our cohort of patients. Conclusion The present genomic analysis revealed new insights into the mutational landscape of Waldenström Macroglobulinemia, providing evidence of the recurrence of some variants in subjects with IgM MGUS as well. Notably, we uncovered new genetic mutations with somatic variants recurrently observed (n≥3) in KMT2C, WNK2, BCL9, PTPN13, CARD11 and NFKB2 genes. These recurring somatic events provide a genomic basis for future research and a better understanding of the WM pathogenesis as well as the risk of transformation of IgM MGUS to WM.

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.

Ibrutinib and Venetoclax in Symptomatic, Treatment-Naive Patients with Waldenström Macroglobulinemia

Background: BTK inhibitors and BCL2 antagonists as monotherapy are highly active and well tolerated in Waldenström macroglobulinemia (WM). We initiated a prospective study evaluating ibrutinib and venetoclax in treatment-naive WM. Study therapy was stopped on March 31, 2022, after four ventricular arrhythmia events, including two grade 5 events. Herein, we present follow-up data after stopping therapy to assess ongoing safety and response durability of this combination in WM. Methods: This was an investigator-initiated, multicenter, prospective phase II study in symptomatic treatment-naïve WM patients. Study therapy was given in 4-week cycles. Intended therapy consisted of ibrutinib at 420 mg/day on cycle 1. Venetoclax was added on cycle 2 at 100 mg/day for one week, 200 mg/day for one week, and 400 mg/day for two weeks. Ibrutinib 420 mg/day and venetoclax 400 mg/day were given together for cycles 3-24. All patients underwent baseline laboratory studies, a bone marrow biopsy with MYD88 and CXCR4 genotyping, and CT scans to evaluate extramedullary disease. Responses were assessed using modified IWWM6 criteria. The study was registered under clinicaltrials.gov (NCT04273139). Results: Between July 2020 and January 2022, 45 (30 males; 15 females) patients were enrolled. Baseline characteristics were: median age 67 (range 40-82 yrs); serum IgM 4297 (range 572-9211 mg/dL); hemoglobin 10.2 (range 7.8-15.3 g/dL); bone marrow involvement 60% (5-90%); presence of lymphadenopathy (&amp;gt;1.5 cm) 25 (56%); and splenomegaly (&amp;gt;15 cm) 13 (29%). MYD88 L265P was detected in all patients, and CXCR4 mutations in 17 (38%; 10 nonsense and 7 frameshift). Median time on treatment was 10.2 months (range 1.9-20.8) before treatment was halted. Overall response rate was 100%, and major response 96%. Best responses included very good partial response (VGPR; n=19, 42%); partial response (n=24, 53%); and minor response (n=2, 4%). CXCR4 mutations were associated with lower VGPR rates (29% v 50%; p=0.18). At a data cutoff date of May 31, 2023, the median study follow-up is 24.4 months (range 3-33.6). There were 12 progression events, including two treatment-emergent deaths and one transformation to DLBCL. Five patients started a new therapy. At 24 months, progression-free survival (PFS) rate was 76% (95% CI 59-86; Figure A), next treatment-free rate was 89% (95% CI 72-96), and overall survival (OS) rate was 96% (95% CI 84-99). CXCR4 mutations did not impact PFS (p=0.89; Figure B), time to next treatment (TTNT) (p=0.43), or OS (p=0.27). Median time after end of therapy (EOT) was 13.3 months (95% CI 12.9-13.5). There were 11 progression events after EOT, and the 12-month PFS rate after EOT was 79% (95% CI 63-88; Figure C). CXCR4 mutations (p=0.35) or time on therapy (&amp;lt;12 vs. ≥12 months; p=0.76) did not impact PFS after EOT. There was a trend towards longer PFS after EOT in patients who had attained VGPR (p=0.12; Figure D). No treatment-emergent events, especially arrhythmia, were observed after treatment stopped. Thirty-one patients underwent a bone marrow biopsy one year after EOT. No emergent mutations, especially in BTK and TP53, were detected. Conclusion: In symptomatic, treatment-naive patients with WM, the combination of ibrutinib and venetoclax was shown to be highly effective with a VGPR rate of 42% and a 24-month PFS rate of 76%, despite stopping therapy early due to unexplained ventricular arrhythmia. VGPR attainment might predict longer PFS after EOT, and PFS was similar in patients who received &amp;lt;12 vs. ≥12 months of therapy. No treatment-emergent toxicities, including arrhythmia, occurred after EOT. The combination of ibrutinib and venetoclax holds significant promise. However, given that the cause of the unexpected ventricular arrhythmia remains unclear, it is challenging to recommend the combination at the current dose and schedule. The potential for shorter treatment durations or safer combinations offer hope for maintaining efficacy while mitigating adverse effects.

Clinical Characteristics, Treatment Approach and Long-Term Outcomes of 678 Patients with Symptomatic Waldenstrom's Macroglobulinemia: Comprehensive Insights from a Spanish Registry of IgM Gammapathies

Introduction The pathogenesis of Waldenstrom's macroglobulinemia (WM) involves the clonal expansion of lymphoplasmacytic B cells in bone marrow, producing monoclonal immunoglobulin M (IgM). The heterogeneity of clinical manifestations in this uncommon disease represents the treatment approach more challenging. Multicenter registries may help in improving health care and scientific research. PRAME is a Spanish national registry of WM and IgM-related disorders with comprehensive data collection, providing insights for a better understanding of WM. Methods This is a retrospective, multicenter study that included newly diagnosed symptomatic WM (SWM) patients, evaluated at (24) Spanish hospitals between 1990 and 2023. All patients had confirmed diagnoses of SWM according to the International Workshop Consensus. Data collection on patients' demographics and disease epidemiology, clinical characteristics, diagnosis, treatment approaches and outcomes were performed from patients' local medical files. Results From 1796 total patients included in the Spanish registry for IgM gammopathies, a total of 678 newly SWM patients were identified. The median age was 71(range 35-94) years, 37% of patients had aged ≥ 75 years and 63% were male. The most common symptom at diagnosis was anemia (43%), followed by B symptoms (30%). Fifteen percent had peripheral neuropathy (PN) as the first WM symptom (39% with sensitive motor PN). Hyperviscosity was only observed in 15%. The free light chain isotypes were mostly kappa (75.4%). Less than 5% of the total cohort, had an ECOG ≥3, at diagnosis. Among 403 patients, who had samples available for assessing MYD88L265P by quantitative allele-specific PCR, 75% were positive for the mutation. However, out of the 199 patients assessed for CXCR4(S388X) mutation, only 12% were positive. The first line of therapy was started in 98% of patients. Plasmapheresis was indicated in 10%, with a median number of 2 sessions. The most frequent indications for initiating the treatment were anemia (48%), B symptoms (13%), and neuropathy (9%). Symptoms related to IgM monoclonal and extramedullary involvement were seen in 15% (56% associated with hyperviscosity) and 11.3%, respectively. The main first-line treatment regimens received were: chlorambucil (37%), dexamethasone, rituximab and cyclophosphamide (DRC)(21%), rituximab monotherapy (13%), bendamustine/rituximab (BR) (9.1%), Bruton tyrosine kinase (BTK) inhibitors (7%), and bortezomib, dexamethasone and rituximab (BDR) (5.5%). The overall and major response rates (ORR and MRR) were 67% and 58%, respectively. (Table 1). The MRR was higher among patients who received BR as the first line of therapy (88%), with a 26% of complete response (CR) and 21% of very good partial responses (VGPR). The CR+VGPR rates with other common regimens were 17% for DRC, 9% for BDR and 21% for BTK inhibitors, respectively. With a median follow-up of 96 months (IQR, 43 to 192), the median overall survival (OS) was 8.7 years (95% confidence interval [CI]:7.3-10.1) and the median progression-free survival (PFS) was 6.8 years (95% CI:6.2-7.5) with no relevant improvement over the last years. (Figure 1) Conclusion In the current registry, we observed a high proportion of younger patients with good performance status at diagnosis. Among newly diagnosed SWM patients, BR demonstrated ongoing superiority as a front line of therapy, leading to deeper responses compared to other therapeutic schemes. There were no significant differences in OS between different times of diagnosis. This Spanish registry for SWM contributes to advancing knowledge and the continual enhancement of treatment strategies for WM.

Hepatitis B Virus Infection Status and Reactivation Analysis in Multiple Myeloma in China

Background: China is a highly endemic area for Hepatitis B virus (HBV) infection and HBV reactivation is an event of concern in treating patients with hematologic malignancy. However, there were very few reports regarding HBV reactivation in patients with multiple myeloma (MM) in China. Since the approval of novel agents, especially immunotherapy such as monoclonal antibody and chimeric antigen T cell immunotherapy, the risk of HBV reactivation has increased. Therefore, the study aimed to evaluate the HBV infection status in MM and explore the incidence and risk factors of HBV reactivation in MM patients in China. Methods: Newly diagnosed MM patients with available serologic assays for HBV infection were retrospectively enrolled between June 30, 2008 and December 30, 2022 in West China Hospital, Sichuan University. Demographic and clinical data were obtained from the “HemaTank” Chinese Multiple Myeloma Database (HCMMD). Meanwhile, patients diagnosed with Waldenstrom Macroglobulinemia (WM) at the same time were enrolled as the control group. HBV reactivation was defined as the occurrence of one of the following: ≥ 2 log increase in HBV-DNA levels from baseline level, detection of HBV-DNA with level &amp;gt; 100 IU/ml in a person with undetectable HBV DNA at baseline or HBsAg-negative becoming positive. The penalized maximum likelihood logistic regression for rare events was applied to identify independent risk factors related to HBV reactivation. Results: The study recruited 3040 MM patients with a median age of 62 (range 14~98) and 240 WM patients with a median age of 63 (range 25~92). The HBsAg-positive rates were similar between MM and WM (9.4% vs 10.8%, P=0.49), but both the HBcAb-positive rates and HBeAb-positive rates in WM were significantly higher than MM (76.3% vs 60.7%, P&amp;lt;0.001; 37.5% vs 25.1%, P&amp;lt;0.001). The analysis of co-expression of HBV markers shows that the prevalence of HBsAg-/Anti-HBs+/anti-HBC- in MM was significantly higher than WM (16.7% vs 8.3%, P&amp;lt;0.001) and HBsAg-/Anti-HBs-/anti-HBC+ in MM was significantly lower than WM (18.3% vs 27.1%, P&amp;lt;0.001). However, he prevalence of HBsAg+/ HBeAg+/anti-HBC+ and HBsAg+/ anti-HBeAb+/anti-HBC+ were similar between MM and WM (0.5% vs 1.3%, P=0.158; 8.6% vs 8.8%, P=0.503). The baseline characteristics of HBsAg-positive MM and HBsAg-negative MM were summarized in Table 1. The rates of HBsAg-positive MM were significantly increased in young male patients (P&amp;lt;0.05). The level of ALT and AST, and the incidence of HBV reactivation, liver cirrhosis and pulmonary disease were significantly higher in HBsAg-positive MM than in HBsAg-negative MM (P&amp;lt;0.05). Of the 2147 MM patients who have received at least one line of chemotherapy (Table 1), 924 (43%) MM patients received bortezomib-containing or lenalidomide-containing regimens as the frontline therapy, while only 21 (1.0%) received a daratumumab-containing regimen as the frontline therapy. During hospital-documented follow-up visits, 26 (1.21%) MM patients have experienced HBV reactivation. According to the multivariate analysis of risk factors for HBV reactivation in MM, liver cirrhosis (OR 8.63, 95%CI 2.95-25.24, P&amp;lt;0.001), autologous stem cell transplantation (OR 6.03, 95%CI 2.47-14.72, P&amp;lt;0.001) and HBsAg-positive (OR 14.45, 95%CI 6.50-32.14, P&amp;lt;0.001) were independent risk factors associated with high prevalence of HBV reactivation. The ROC curve of the model integrating the three factors is shown in Figure 1, and the AUC was 0.8584. Conclusions: Among Chinese MM patients with multiple myeloma, a high proportion of MM patients presented with resolved hepatitis B virus infection, whereas HBV reactivation was pretty rare. However, long-term HBV DNA levels monitoring is still needed for MM patients at high risk for HBV reactivation.

Real-Life Multicentre Study on 547 Patients Affected By Waldenstrom Macroglobulinemia Treated with Chemo-Immunotherapy: Which Is the Best and Most Used First-Line Treatment?

Waldenström Macroglobulinaemia (WM) is an indolent lymphoma still primarily managed with chemo-immunotherapy, which remains a key first-line treatment option, although recently BTKIs have emerged as potential therapies for untreated patients. The aim of our study was to evaluate and compare efficacy and safety of different chemo-immunotherapeutic regimens commonly used in Italy for frontline WM treatment. A retrospective analysis was conducted on 547WM patients enrolled between 2008-2022 from 14 Italian haematological centres. Among them, 245 received the BR scheme (bendamustine-rituximab), 116 were treated with DRC (dexamethasone-rituximab-cyclophosphamide), and 129 were treated with various other regimens including monotherapy with chlorambucil or cyclophosphamide as well as more aggressive combinations, such as Chl-R (chlorambucil-rituximab), FCR (fludarabine-cyclophosphamide-rituximab) or R-CHOP (rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone), 48 patients received rituximab monotherapy for neurological symptoms. The main focus of our analysis was on the two major treatment groups: BR and DRC. There were notable differences between these two groups in terms of age at treatment (69 vs 70 years old, respectively), CIRS (more patients with CIRS&amp;gt;6 in DRC) and revised IPSSWD (higher risk rates in BR). Patients treated with BR tended to be younger, more fit but also had a more aggressive disease. Overall survival (OS) curves did not show significant differences between the two schemes (5-year OS 87.5% for BR and 93.0% for DRC, p=0.42). Similarly, the analysis of progression free survival (PFS) curves demonstrated a better PFS at 5 years for BR (79.2%) compared to DRC (54.5%) (p&amp;lt;0.001; figure 1A). The median PFS was 63.6 months for DRC, while it was not reached for BR. When analysing BR-treated patients separately (standard dose and reduced initial dose) both groups showed better PFS curves than DRC (p&amp;lt;0.001; figure 1B). Multivariate analysis identified the choice of the treatment (BR vs DRC) as the sole significant variable impacting PFS (RR 1.76). Notably, approximately 33% of the patients treated with BR received an initial bendamustine dose lower than the standard dose of 90 mg/m 2 for first-line therapy with 91% of these cases receiving 70 mg/m 2. The two schemes BR and DRC were well tolerated with no significant reduction in administered cycles (16.3% for BR vs 20.7% for DRC). However, dose reduction was more common for BR (14.3%) compared to DRC (6.0%). In conclusion, this large Italian retrospective real-life study showed excellent outcomes for unselected WM patients treated with chemo-immunotherapy. Despite the emergence of new treatment options, chemo-immunotherapy remains a highly effective treatment modality for these patients. The long-term follow-up confirmed better PFS for BR-treated patients, while OS, remained comparable between BR and DRC. Moreover, the BR scheme exhibited excellent PFS even with a reduced initial bendamustine dose.