CXCR4 Mutations Research Articles

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.

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 <0.001; P <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->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.

Outcomes of Patients with Relapsed/Refractory Lymphoplasmacytic Lymphoma Treated with Venetoclax: A Multicenter Retrospective Analysis

Outcomes of Patients with Relapsed/Refractory Lymphoplasmacytic Lymphoma Treated with Venetoclax: A Multicenter Retrospective Analysis

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.

Treatment Selection and Clinical Outcomes in Lymphoplasmacytic Lymphoma/Waldenstrom Macroglobulinemia (LPL/WM) - a Single Center Analysis

Introduction: Lymphoplasmacytic lymphoma (LPL) is as an indolent neoplasm of B-lymphocytes with plasmacytic differentiation causing bone marrow infiltration along with variable involvement of lymph nodes/or spleen. Most common subtype of LPL, Waldenstrom Macroglobulinemia (WM) involves IgM monoclonal gammopathy of any concentration. Due to widespread availability of targeted therapy drugs (BTKi), the treatment landscape of LPL is changing. The aim of this study is to analyze disease characteristics, patterns of real-world treatment selection and its impact on clinical outcomes (Objective response rate (ORR), Progression free survival (PFS) and Overall survival (OS). Methods: A retrospective analysis of patients (age ≥18 years) with diagnosis of LPL/WM between January 1, 2003, to March 31, 2023, seen in consultation at Roswell Park Comprehensive Cancer Center was performed. ORR (CR, VGPR, PR and MR) was calculated according to response criteria at 6 th International Workshop for WM. Statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC). OS and PFS were reported using standard Kaplan-Meier methods. Multivariate cox regression modeling was used to assess associations between first line regimen and survival outcomes (OS / PFS). Predictor variables, namely age at diagnosis, gender, Charlson Comorbidity Index (CCI) score, and rIPSS (revised International Prognostic Score System) were included in multivariable models. Results: A total of 127 patients were included in this study. The median age at diagnosis was 68 years (range 41 to 88), of which 75 (59.1%) were men. Majority of the population was White (n=112, 88.2%) and had ECOG of 0 (n=70, 67.3%). Monoclonal gammopathy of IgM subtype was present in 114 (94.2%), IgG in 3 (2.5%) and IgA in 1 (0.8%) patient. MYD88 mutation testing was positive in 69.8%, negative in 14% and unknown in 16.3% patients. Two out of 10 patients tested for CXCR4 mutation were positive. Sixteen (28.1%) patients had hyperviscosity symptoms and underwent plasmapheresis with first line treatment. These patients had a mean IgM level of 6249.5 mg/dL and mean M-spike of 4.53 g/dL. Of 121 patients with data on treatment strategy, 20 (16.5%) were monitored with observation alone. The most common treatment modality used in first line was chemo-immunotherapy (C-I) in 61 (50.4%) patients followed by Rituximab immunotherapy (R-I) in 20 (16.5%) patients and BTK inhibitors in 12 (9.9%) patients. ORR was highest 90.7% (39/43) in chemoimmunotherapy group, 87.5% (7/8) in BTKi group and 71.4% (10/14) in Immunotherapy group. Bendamustine-Rituximab was the most common used treatment regimen (n=42), with resultant CR in 20% and VGPR in 23.3% patients. At median follow-up duration of 36.9 months, median OS was 113.9 months in R-I, 159.8 months in C-I and not reached (NR) in BTKi group. The median PFS was 50.2 months in R-I, 61.2 in C-I and 23 months in BTKi group. Although Multivariate Cox Regression analysis failed to show statistically significant difference in PFS, there is tendency towards improved OS in C-I group (p=0.08) among all the groups when adjusted for age, sex, CCI. Conclusion: In our single center experience, chemoimmunotherapy combination with Bendamustine-Rituximab is the most used treatment choice in first line settings followed by single agent anti-CD20 therapy and BTKi. Although Multivariate Cox Regression analysis failed to show statistically significant difference in PFS, there is a tendency towards improved OS in the chemoimmunotherapy subgroup (p=0.08). The shorter PFS in BTKi group is likely due to toxicity of targeted therapy leading to treatment interruption and change of therapy.

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.

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.

Clinical, biological, electrophysiological and therapeutic profile of patients with anti-MAG neuropathy according to MYD88L265P and CXCR4 mutations and underlying haemopathy.

Anti-MAG neuropathies are associated with an IgM monoclonal gammopathy of undetermined significance (MGUS) or with a malignant haemopathy. Our objective was to determine whether the presence of a haemopathy or somatic mutations of MYD88 and CXCR4 genes influences disease presentation and response to rituximab (RTX). We included 79 patients (mean age 74years, disease duration 9.68years) who had a bone marrow aspiration with morphologic and immunophenotypic analysis. MYD88L265P and CXCR4 mutations were analysed in peripheral B cells. Information collected included: inflammatory neuropathy cause and treatment sensory sum score (ISS), MRC testing, overall neuropathy limitation scale (ONLS), Rash-built Overall Disability Score (RODS), ataxia score, anti-MAG titres, peak IgM dosage, neurofilament light chain levels, motor and sensory amplitudes, motor unit index (MUNIX) and motor unit size index (MUSIX) sum scores. Efficacy of RTX was evaluated at 12months in 26 patients. Malignant haematological disorders were discovered in 17 patients (22%): 13 Waldenstrom macroglobulinemia, 3 marginal zone lymphoma and one mantle cell lymphoma. MYD88L265P mutation was detected in 29/60 (48%) patients and CXCR4 in 1 single patient. Disease severity, biological and electrophysiological data and response to RTX were comparable in patients with MGUS/lymphoma and patients with/without MYD88L265P mutation. ISS was lower and MUSIX higher in patients improved by RTX. MYD88L265P mutation and underlying haemopathies are not predictive of a more severe disease. However, in cases of resistant and progressive neuropathy, they provide an opportunity to prescribe newly available drugs such as Bruton tyrosine kinase inhibitors.

Multicenter Prospective Phase II Study of Rituximab Combined, Bortezomib, Lenalidomide, Dexamethasone Followed By Lenalidomide Maintenance (R-VRD) in Patients with Waldenstrom's Macroglobulinemia (KMM1803)

Backgroud: Waldenström macroglobulinemia (WM) is an indolent B-cell malignancy characterized by the presence of immunoglobulin M (IgM) monoclonal gammopathy and lymphoplasmacytic bone marrow infiltration. Recently, the superior efficacy of BTK inhibitors and other novel agents has been established. However, BTK inhibitors has been shown limited efficacy in patients with CXCR4 mutation and MYD88 wild type. Therefore, the importance of chemotherapy which do not contain BTK inhibitors still remain. This study was conducted to estimate the efficacy and safety of the combination of rituximab, bortezomib, lenalidomide and dexamethasone in patients with WM in South Korea. Methods : Between Aug. 2018 and Aug. 2022, a total of 39 patients were included in this study at 12 academic institutes in South Korea. The primary end-point of the study was the median progression-free survival (PFS), and the secondary end-points were the overall survival (OS), overall response rate (ORR), minimal residual disease (MRD) achievement and toxicities. Eligible patients were confirmed clinicopathological diagnosis of WM who has Measurable and symptomatic disease meeting at least 1 of the recommendations from the Second International Workshop on Waldenström Macroglobulinemia (IMWWM) for requiring treatment. Pateints who received rituximab treatment within the last 12 months before the first dose of study drug were excluded. Patients received the 28-day cycle of Rituximab (375 mg/m 2 IV on day 1), Bortezomib (1.3 mg/m 2 SC on day 1, 8, 15), Lenalidomide (15 mg per oral day 1-21) and dexamethasone (20 mg iv or oral day 1-4). Enrolled patients were received induction therapy with rituximab and bortezomib, lenalidomide and dexamethasone every 4 weeks for 6 cycles and then lenalidomide (10 mg on day 1-21) maintenance for 2 years. All patients were checked MYD88 and CXCR4 mutation at the baseline work up of this study and MRD achievement was checked by MYD88 PCR every 6 months for 3 years. Results : The median (range) age was 65.2 years. Lymphadenopathy were noted around 50%. In this study, MYD88 mutation was noted 74.3% and CXCR4 mutation was noted 20%, respectively. The response rates were following: CR in 4 (13.3%), VGPR in 3 (10.0 %) PR in14 (46.7 %), MR in 3 (10.0%) and < MR in 6 (20.0 %). Overall response rates (≥PR (partial response)) rate was 70.0 % and more than MR were 80%. 2yrs PFS and OS were 56% and 93%, respectively. We are now checking MRD by MYD88 PCR for ongoing patients. The detailed response rates and survival outcomes will be reported soon according to MYD88 and CXCR4 mutation status. And comparison of survival difference according to MRD negativity will be also announced after finishing data collection of MRD. Conclusions: R-VRD regiment could be helpful for MYD88 mutation negative or CXCR4 mutation positive patients. BTK inhibitors show superior response and survival outcomes for patients with MYD88 mutation positive and CXCR4 mutation negative. So, we are supposed that R-VRD could be optional treatment for patients who are not suitable for BTK inhibitors.

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 (>1.5 cm) 25 (56%); and splenomegaly (>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 (<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 <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.

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.

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.

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.

Indolent Lymphoma: High CR and VGPR Rate with Fixed Duration Bendamustine, Rituximab and Acalabrutinib in Waldenstroms Macroglobulinaemia (BRAWM)

Background: Waldenström's macroglobulinaemia (WM) is an uncommon lymphoproliferative disorder. Although many options are available, an optimal first-line therapy for WM has not been defined. We postulated that combining bendamustine and rituximab (BR) with a next generation BTK inhibitor would result in deeper responses as measured by complete response (CR) and very good partial response (VGPR) rates, and provide a longer duration of response. Objectives: The primary objective of this trial is to document the CR and VGPR rates Methods: The BRAWM clinical trial combines BR with acalabrutinib in a one-year, fixed duration treatment course including six cycles of BR and 12 months of acalabrutinib (6 months of monotherapy). This ongoing trial is taking place at 9 clinical sites across Canada and 44 patients have been enrolled as of July 1, 2023, with a recruitment goal of 59. Results: Median age of participants was 68; 34 were male; 3 participants were low risk, 21 intermediate and 20 high risk. 27 participants completed combination therapy, 17 completed monotherapy and 7 were followed-up at 18 months, and 3 at 24 months after first dose. Table 1 shows the observed Clinical Responses using 6 th IWWM; 2 participants improved from a VGPR to a CR between cycles 7 and 12, both of whom have not reached month 18 for assessment. The interim primary endpoint of combined CR and VGPR rate is 13/17 (77%), with no participants having progressed at this interim analysis. 3 participants discontinued treatment early: 1 at cycle 7 (with a VGPR) who experienced an adverse event requiring treatment; 1 at cycle 3 with inter-current illness, and 1 at cycle 2 for personal reasons not related to treatment. The total observed Treatment Related Adverse Events (TRAEs) in combination therapy was 188 amongst 35 participants, and n= 24 in 10 participants during monotherapy. There were 24 Grade 3/4 TRAEs (combination n= 20, monotherapy n= 4). Total observed Serious Adverse Events was 18, and 7 TRSAEs 7; 6 during combination therapy (3 febrile neutropenia and 1 for each fever, allergic reaction and bowel obstruction) and 1 pneumonia during monotherapy. Grade 3/4 TRAEs resulted in 29 dose interruptions in 13 participants, and 2 dose reductions. Mutational analysis of participants with adequate sample for assessment (n=35), showed 33 with MYD88 mutation, 10 with a CXCR4 mutation, and 1 with a TP53 mutation. Minimal residual disease (MRD) analysis of Peripheral Blood (PB) and Bone Marrow, using next generation sequencing, is underway. Initial review shows that most evaluable patients (those with samples at two or more time-points) have become MRD undetectable in PB. Uni- and multi-variate analyses of variables that may be associated with outcomes is underway. Conclusions: Bendamustine, rituximab and acalabrutinib front-line therapy for WM is safe and well tolerated and initial clinical results show that this treatment induces a high percentage of CR + VGPRs. Table 1: Clinical Responses using 6 th IWWM. *The 2 participants observed to have a CR at month 12 have not reached month 18 for progression or survival follow-up.

Clonal Relationship and Mutation Analysis in Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia Associated With Diffuse Large B-cell Lymphoma

Patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) occasionally develop diffuse large B-cell lymphoma (DLBCL). This mostly results from LPL/WM transformation, although clonally unrelated DLBCL can also arise. LPL/WM is characterized by activating MYD88 L265P (>95%) and CXCR4 mutations (~30%), but the genetic drivers of transformation remain to be identified. Here, in thirteen LPL/WM patients who developed DLBCL, the clonal relationship of LPL and DLBCL together with mutations contributing to transformation were investigated. In 2 LPL/WM patients (15%), high-throughput sequencing of immunoglobulin gene rearrangements showed evidence of >1 clonal B-cell population in LPL tissue biopsies. In the majority of LPL/WM patients, DLBCL presentations were clonally related to the dominant clone in LPL, providing evidence of transformation. However, in 3 patients (23%), DLBCL was clonally unrelated to the major malignant B-cell clone in LPL, of which 2 patients developed de novo DLBCL. In this study cohort, LPL displayed MYD88 L265P mutation in 8 out of eleven patients analyzed (73%), while CXCR4 mutations were observed in 6 cases (55%). MYD88 WT LPL biopsies present in 3 patients (27%) were characterized by CD79B and TNFAIP3 mutations. Upon transformation, DLBCL acquired novel mutations targeting BTG1, BTG2, CD79B, CARD11, TP53, and PIM1. Together, we demonstrate variable clonal B-cell dynamics in LPL/WM patients developing DLBCL, and the occurrence of clonally unrelated DLBCL in about one-quarter of LPL/WM patients. Moreover, we identified commonly mutated genes upon DLBCL transformation, which together with preserved mutations already present in LPL characterize the mutational landscape of DLBCL occurrences in LPL/WM patients.

Ibrutinib and venetoclax as primary therapy in symptomatic, treatment-naïve Waldenström macroglobulinemia

AbstractConcurrent Bruton tyrosine kinase and BCL2 inhibition has not yet been investigated in Waldenström macroglobulinemia (WM). We performed an investigator-initiated trial of ibrutinib and venetoclax in symptomatic treatment-naïve patients with MYD88-mutated WM. Patients received ibrutinib 420 mg once daily (cycle 1), followed by a ramp-up of venetoclax to 400 mg daily (cycle 2). The combination was then administered for 22 additional 4-week cycles. The attainment of very good partial response (VGPR) was the primary end point. Forty-five patients were enrolled in this study. The median baseline characteristics were as follows: age 67 years, serum IgM 43 g/L, and hemoglobin 102 g/L. Seventeen patients (38%) carried CXCR4 mutations. Nineteen patients (42%) achieved VGPR. Grade 3 or higher adverse events included neutropenia (38%), mucositis (9%), and tumor lysis syndrome (7%). Atrial fibrillation occurred in 3 (9%), and ventricular arrhythmia in 4 (9%) patients that included 2 grade 5 events. With a median follow-up of 24.4 months, the 24-month progression-free survival (PFS) and overall survival (OS) rates were 76% and 96%, respectively, and were not impacted by CXCR4 mutations. The median time on therapy was 10.2 months, and the median time after the end of therapy (EOT) was 13.3 months. Eleven of the 12 progression events occurred after EOT, and the 12-month PFS rates after EOT were 79%; 93% if VGPR was attained, and 69% for other patients (P = .12). Ibrutinib and venetoclax induced high VGPR rates and durable responses after EOT, although they were associated with a higher-than-expected rate of ventricular arrhythmia in patients with WM, leading to early study treatment termination. This trial was registered at www.clinicaltrials.gov as #NCT04273139.

Advances in Treatment of Waldenström Macroglobulinemia.

The discovery of recurring somatic mutations, in particular MYD88 and CXCR4 mutations, in Waldenström macroglobulinemia (WM), a rare B-cell lymphoproliferative disorder, led in the last decade to the development of several therapeutic agents with high efficacy. This review aims to provide an overview of available treatments in WM and novel agents, focusing on studies published over recent years. There is no international consensus on the best first-line option in treatment-naïve patients. Randomized clinical trials are rare in WM and there has been no prospective comparison of chemoimmunotherapy and BTK inhibitors in the frontline setting. Chemoimmunotherapy and BTK inhibitors, the two feasible and most widely used treatments in first-line treatment, represent very different options in terms of duration of therapy, route of administration, cost, and adverse effect. In addition to tumor genotype and patient comorbidities, choice of therapy in WM should take into account these parameters. Results of ongoing and future clinical trials evaluating fixed-duration combinations with BTK inhibitors and novel agents are awaited.

P-422 Impact of early response and CXCR4 mutation status on progression-free survival with ibrutinib-based therapy in Waldenström macroglobulinemia: a post hoc analysis of the iNNOVATE trial

P-422 Impact of early response and CXCR4 mutation status on progression-free survival with ibrutinib-based therapy in Waldenström macroglobulinemia: a post hoc analysis of the iNNOVATE trial

Waldenström macroglobulinemia: a review of pathogenesis, current treatment, and future prospects.

Waldenström macroglobulinemia (WM) is a chronic B-cell lymphoproliferative disorder characterized by lymphoplasmacytic cell overgrowth in the bone marrow and increased secretion of IgM immunoglobulins into the serum. Patients with WM have a variety of clinical outcomes, including long-term survival but inevitable recurrence. Recent advances in disease knowledge, including molecular and genetic principles with the discovery of MYD88 and CXCR4 mutations, have rapidly increased patient-tolerable treatment options. WM patients may benefit from chemotherapy regimens that include rituximab-based regimens, alkylating drugs, proteasome inhibitors, monoclonal antibodies, and drugs targeting Bruton tyrosine kinase inhibitors. In light of these advancements, patients can now receive treatment customized to their specific clinical characteristics, focusing on enhancing the depth and durability of their response while limiting the adverse effects. Despite the rapidly developing therapeutic armament against WM, a lack of high-quality evidence from extensive phase 3 trials remains a significant challenge in the research. We believe clinical outcomes will keep improving when new medicines are introduced while preserving efficacy and minimizing toxicity.

THE INCIDENCE, CLINICAL APPLICATION AND PROGNOSTIC SIGNIFICANCE OF MYD88 AND CXCR4 MUTATION IN CHINESE PATIENTS WITH LYMPHOPLASMACYTIC LYMPHOMA/WALDENSTRöM MACROGLOBULINEMIA

Introduction: Waldenström macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) is a rare, B-cell lymphoma with highly prevalent somatic mutations including MYD88, and CXCR4. The mutation rates of LPL/WM in China and the direct comparison among different deletion means are still not clear. Methods: 387 patients with LPL/WM were included in this study. Tumor cells were collected from 357 un-sorted BM 28 PB and 2 lymph nodes (FFPE). MYD88L265P mutation were detected by Sanger sequencing (FGS), NGS, allele-specific quantitative polymerase chain reaction (ASPCR), and droplet digital polymerase chain reaction (ddPCR). CXCR4 mutation were detected by Sanger sequencing, NGS, and ASPCR. Results: 386 patients were assessed for MYD88L265P mutation, of whom 263 were tested by FGS, 235 by NGS, 311 for AS-PCR, 118 for ddPCR. The MYD88L265P mutation was found in 88.6% of the patients. ASPCR and ddPCR had the highest sensitivity of 97.1% and 96.8%. FGS and NGS failed to detected the mutation in low tumor load. A high false-negative rate (FNR) for MYD88 by FGS and NGS was observed in patients with tumor load less than 10% (FNR: 35.5% and 20%). There was no significant difference in the MYD88 mutation rate detected by ddPCR and ASPCR among patients with different tumor load groups (p = 0.794). MYD88 mutation was detected in a high incidence of 88.5% in less than 1% infiltrated WM tumor cells specimens by ddPCR and ASPCR. CXCR4 mutation testing was performed in 358 patients of LPL/WM, comprising 301 with FGS, 236 with NGS, and 311 with ASPCR. Overall, 31.2% patients were identified as having the CXCR4 mutation. NGS exhibited the highest sensitivity among the three detection means. MYD88 wild-type patients had a significantly lower Hb levels (median 79 g/L vs. 90 g/L, p = 0.015), lower proportion of males (54.5% vs. 51.8%, p = 0.012) and a significantly higher proportion elevated β2-MG (84.2% vs. 65.6%, p = 0.021), and LDH (30.2% vs. 11.9%, p = 0.001). However, there were no significant differences in either PFS or OS between MYD88L265P wild-type and MYD88L265P mutation group (p = 0.35, p = 0.42). Patients with CXCR4 mutation tended to be older (median 64 vs. 60, p < 0.001) and displayed lower hemoglobin level (median 81 g/L vs. 93 g/L, p = 0.008), higher serum IgM level (median 36.1 g/L vs. 31.6 g/L, p < 0.001), and higher baseline BM involvement (by FCM, median 15.1% vs. 7.1%, p < 0.001) compared with CXCR4 wild-type patients. Notably, CXCR4 mutation group had significantly worse survival compared with the wild-type group (p = 0.03, p = 0.02). The research was funded by: National Nature Science Foundation of China (81970187, 82170193, 81920108006, and 81900203) and the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2022-I2M-1-022, 2021-I2M-C&T-B-081) Keywords: diagnostic and prognostic biomarkers, indolent non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.