Waldenström Macroglobulinemia Patients Research Articles

Incidence of skin cancer among patients with Waldenström macroglobulinemia: Analysis of the SEER database (2000-2019).

e18893 Background: The association between non-Hodgkin lymphoma, including chronic lymphocytic leukemia, and aggressive skin cancers is well established. We studied the incidence of melanoma and non-melanoma skin cancers (NM) skin malignancies (SM) among patients with Waldenstrom macroglobulinemia (WM). Methods: We used the SEER 17 registry to identify adult [Age > 20] patients(pts) with an index diagnosis (dx) of WM between 2000- 2019. We included ICD-O-3 codes 9671/3 (Lymphoplasmacytic lymphoma) and 9761/ (Waldenstrom macroglobulinemia) to identify index dx. Only first of the two codes was used if both were mentioned for same pt. We excluded patients with diagnosis made from death certificate or autopsy. We used ICD-O-3/WHO 2008 codes assigned to melanoma of skin and other NM SM to identify subsequent diagnosis ( > 2months after index diagnosis) of SM excluding basal cell and squamous cell cancers. SM were categorized using ICD-O-3 codes for histology. Follow up period was till December 2019. Data variables and standardized incidence ratio [SIR] for SM were obtained with multiple primary SIR analyses using SEER Stat v 8.4.0.1 software. Kaplan Meier method was used to estimate overall survival (OS) using R 4.2.2. Results: We identified 8099 patients with WM of which 68 patients (0.84%) had a subsequent dx of SM. WM and SM were diagnosed at median (IQR) ages of 69 (60-77) and 74 (67-81), with a median gap of 52 (30-95) months. Majority of WM survivors with SM were males (51 or 75%), white (100%) and older than 60 years of age (72%) at WM diagnosis. Almost half of the patients who developed SM underwent chemotherapy for WM (n = 35, 45%). The overall risk of developing SM was significantly higher in WM patients as compared to the general population [SIR (95%CI) = 1.34 (1.01-1.74) (p < 0.05) for melanoma and 2.85 (1.52-4.87) (p < 0.05) for NM]. Eleven different types of histology for SM were noted, with malignant melanoma, NOS (n = 30, 44%) and Merkel cell carcinoma (n = 7, 10%) being the most common melanoma and NM subtype (Table). The median survival after SM dx was 68 (55-NA) months. Conclusions: Patient with WM had higher risk of SM (melanoma and NM skin cancers, excluding basal cell and squamous cell cancers) compared to the general population. This study highlights the need for screening and counseling for skin cancers in WM patients in routine clinical practice. [Table: see text]

What differences in outcomes exist in Hispanic patients being treated for lymphoplasmacytic lymphoma and waldenstrom macroglobulinemia?

e19541 Background: Lymphoplasmacytic Lymphoma (LPL) and Waldenstrom Macroglobulinemia (WM) are both rare diseases with little known about their longterm outcomes in ethnic minorities. Overall, the 5- and 10-year survival of this disease has improved over the last 60 years (Blood, PMID: 24948623), but few studies have explored outcomes in Hispanic (HI) patients. We analyzed demographics, treatment patterns and outcomes in patients diagnosed with LPL and WM comparing HI and Non-Hispanics (NH) in the US. Methods: Data was analyzed on LPL and WM patients in the US reported to the Surveillance, Epidemiology, and End Results (SEER) analyzing patient characteristics, age-adjusted incidence rate, and survival rate among HI and NH patients. Results: HI were diagnosed at a younger median age on both LPL and WM cohorts compared to NH (p <0.001). There was a higher percentage of HI patients diagnosed with LPL and WM before the age of 60 compared to that of NH (p <0.001). Regarding race of patients with LPL, most of both HI and NH patients identified as whites (94% vs 86%), followed by blacks (2% vs 6%). Regarding stage, a majority of both HI (50%) and NH (60%) patients diagnosed with LPL were Stage IV. In terms of radiation, 7% of HI patients with LPL received it, compared to 4% of NH patients (p=0.027). On survival analysis for LPL patients, the survival probability at 2, 5 and 10 years (y) of HI vs NH were similar. There was not an OS difference favoring HI/NH (p=0.49). On survival analysis for WM patients, the survival probability at 2, 5 and 10 y of HI vs NH were similar. The median survival time for patients with WM was similar between HI and NH patients and there was not OS difference favoring HI vs NH (p=0.16). On multivariate analysis for LPL and WM, those diagnosed over the age of 80 had worse OS compared to those younger than 60 (95% CI; 4.8-8.1 and 2-3.4, respectively). No OS benefit when adjusting for stage was observed for either LPL or WM. Conclusions: In LPL and WM patients HI patients were diagnosed at younger ages compared to NH for both LPL and WM, and more likely to be diagnosed before the age of 60. The median survival rates were similar between HI and NH patients diagnosed with both LPL and WM. These findings may be confounded by earlier diagnosis in HI patients; however, further studies are needed exploring biological ethnic differences to explain early diagnosis in LPL and WM among HI.

MINIMAL RESIDUAL DISEASE STATUS IMPROVED THE RESPONSE EVALUATION IN PATIENTS WITH WALDENSTRöM’S MACROGLOBULINEMIA

Minimal residual disease (MRD) has been recognized as an important prognostic factor for survival in hematological malignancies. However, the prognostic value of MRD in Waldenström’s macroglobulinemia (WM) remains largely unexplored. We analyzed 108 newly diagnosed WM patients receiving systematic therapy and assessed MRD by multiparameter flow cytometry (MFC) in the bone marrow. At best response, 34 patients (31.5%) achieved undetectable MRD (uMRD). Hemoglobin > 115 g/L (p = 0.03), serum albumin >35 g/L (p = 0.01), β2-MG ≤ 3 mg/L (p = 0.03), and low-risk International Prognostic Scoring System for WM (IPSSWM) stage (p < 0.01) were associated with a higher rate of uMRD. Improvements in IgM (p < 0.01) and hemoglobin (p = 0.03) were more evident in uMRD patients compared with MRD-positive patients. The 3-year progression-free survival (PFS) was better for uMRD patients compared with those MRD-positive patients (96.2% vs. 52.8%; p = 0.0012). Landmark analysis also showed that uMRD patients had a better PFS than MRD-positive patients after 6 and 12 months. Patients who achieved partial response (PR) and uMRD had a 3-year PFS of 100%, which was significantly higher than that patients with MRD-positive PR (62.6%, p = 0.029). Multivariate analysis showed that MRD positivity was an independent factor for PFS (HR 2.55, p = 0.03). Moreover, the combination of the 6th International Workshop on WM assessment (IWWM-6 Criteria) and MRD had a higher 3-year AUC compared with the IWWM-6 criteria only (0.71 vs. 0.67). MRD based on MFC was an independent prognostic factor for survival in WM, which could improve the precision of response evaluation, especially for patients who had obtained PR. Keywords: diagnostic and prognostic biomarkers, indolent non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.

Minimal residual disease status improved the response evaluation in patients with Waldenström's macroglobulinemia.

Minimal residual disease (MRD) has been recognized as an important prognostic factor of survival in patients with hematological malignancies. However, the prognostic value of MRD in Waldenström macroglobulinemia (WM) remains unexplored. We analyzed 108 newly diagnosed WM patients receiving systematic therapy and assessed for MRD by multiparameter flow cytometry (MFC) using bone marrow samples. Of the total patients, 34 (31.5%) achieved undetectable MRD (uMRD). A hemoglobin level of >115 g/L (P=0.03), a serum albumin level of >35 g/L (P=0.01), a β2-MG level of ≤3 mg/L (P=0.03), and a low-risk International Prognostic Scoring System for WM (IPSSWM) stage (P<0.01) were associated with a higher rate of uMRD. Improvements in monoclonal immunoglobulin (P<0.01) and hemoglobin (P=0.03) levels were more evident in uMRD patients compared with that in MRD-positive patients. The 3-year progression-free survival (PFS) was better in uMRD patients compared with that in MRD-positive patients (96.2% vs. 52.8%; P=0.0012). Landmark analysis also showed that uMRD patients had better PFS compared with MRD-positive patients after 6 and 12 months. Patients who achieved partial response (PR) and uMRD had a 3-year PFS of 100%, which was significantly higher than that of patients with MRD-positive PR (62.6%, P=0.029). Multivariate analysis showed that MRD positivity was an independent factor of PFS (HR: 2.55, P=0.03). Moreover, the combination of the 6th International Workshop on WM assessment (IWWM-6 Criteria) and MRD assessment had a higher 3-year AUC compared with the IWWM-6 criteria alone (0.71 vs. 0.67). MRD status assessed by MFC is an independent prognostic factor for PFS in patients with WM, and its determination could improve the precision of response evaluation, especially in patients who achieved PR.

Mutational Profile in 75 Patients With Anti-Myelin-Associated Glycoprotein Neuropathy: Clinical and Hematologic Therapy Response and Hints on New Therapeutic Targets.

Neuropathy with antibodies to myelin-associated glycoprotein (MAG) is the most common paraproteinemic IgM neuropathy. Recently, the mutational profile of the MYD88 and CXCR4 genes has been included in the diagnostic workup of IgM monoclonal gammopathies. The objective of our study was to assess the prevalence of MYD88 L265P and CXCR4 S338X gene variants in patients with anti-MAG antibody neuropathy. Secondary aims were to evaluate possible correlations between the mutational profile and neuropathy severity, antibody titers, and treatment response. Seventy-five patients (47 men, mean age at molecular analysis 70.8 ± 10.2 years; mean disease duration 5.1 ± 4.9 years) with anti-MAG antibody neuropathy were recruited. Among them, 38 (50.7%) had IgM monoclonal gammopathy of undetermined significance, 29 (38.7%) Waldenstrom macroglobulinemia (WM), and 8 (10.6%) chronic lymphocytic leukemia/marginal zone lymphoma/hairy cell leukemia variant. Molecular analysis was performed on DNA from the bone marrow mononuclear cells in 55 of 75 patients and from peripheral mononuclear cells in 18 of 75 patients. Forty-five patients were treated with rituximab, 6 with ibrutinib, 2 with obinutuzumab-chlorambucil, and 3 with venetoclax-based therapy. All the patients were assessed with the Inflammatory Neuropathy Cause and Treatment (INCAT) Disability Scale, INCAT Sensory Sum Score, and MRC Sum Score at baseline and follow-up. We considered as responders, patients who improved by at least 1 point in 2 clinical scales. Fifty patients (66.7%) carried the MYD88L265P variant, with a higher frequency in WM and naive patients (77.2% vs 33.3%, p = 0.0012). No patients harbored the CXCR4S338X variant. There were no significant differences in hematologic data (IgM levels, M protein, and anti-MAG antibody titers), neuropathy severity, or response to rituximab in MYD88-altered and MYD88 wild-type patients. Nine of 11 (81.8%) patients treated with novel targeted drug, according to the MYD88 status, responded to treatments. MYD88L265P variant has a high prevalence (66.7%) in anti-MAG antibody neuropathy representing a potential effective mutational target for Bruton tyrosine kinase inhibitors. MYD88L265P variant, however, does not seem to be a prognostic factor of neuropathy severity or response to rituximab. In patients not responding or becoming refractory to rituximab, a tailored therapy with new effective target therapies should be considered.

Systematic literature review of quality-of-life questionnaires in Waldenström macroglobulinaemia-need for a disease-specific tool.

Systematic literature review of quality-of-life questionnaires in Waldenström macroglobulinaemia-need for a disease-specific tool.

Clonotypic analysis of immunoglobulin heavy chain sequences among 44 patients with Waldenström macroglobulinemia

To analyze the correlation between the mutational status of immunoglobulin heavy chain variable (IGHV) gene with the prognosis of patients with Waldenström macroglobulinemia (WM). Immunoglobulin heavy chain gene (IGH) clonotypic sequence analysis was carried out to assess the mutational status of IGHV in the blood and/or bone marrow samples from 44 WM patients. The usage characteristics of IGHV-IGHD-IGHJ gene was explored. The most common IGHV subgroup was IGHV3, which was similar to the data from the Institute of Hematology of Chinese Academy of Medical Science. IGHV3-23 (20.45% vs. 15.44%) and IGHV3-74 (11.36% vs. 7.35%) were the main fragments used, which was followed by IGHV4 gene family (15.91% vs. 24.26%). However, no significant correlation was found between the IGHV4 usage and the prognosis of the patients. Should 98% be taken as the cut-off value for the IGHV mutation status, only 5 patients had no IGHV variant, and there was no correlation with the prognosis. Based on the X-tile analysis, 92.6% was re-selected as the cut-off value for the IGHV variant status in such patients. LDH was increased in 26 patients (59.1%) without IGHV variant (P < 0.05), whilst progression-free survival (P < 0.05) and overall survival (P < 0.05) were significantly shorter compared with those with IGHV variants. The usage characteristics of IGHV-IGHD-IGHJ in our patients was similar to reported by the Institute of Hematology of Chinese Academy of Medical Science, albeit that no correlation was found between the IGHV4 usage and the prognosis of the patients. Furthermore, 98% may not be appropriate for distinguishing the IGHV variant status in WM patients.

Report of consensus panel 5 from the 11th international workshop on Waldenstrom's macroglobulinemia on COVID-19 prophylaxis and management

Consensus Panel 5 (CP5) of the 11th International Workshop on Waldenstrom's Macroglobulinemia (IWWM-11; held in October 2022) was tasked with reviewing the current data on the coronavirus disease-2019 (COVID-19) prophylaxis and management in patients with Waldenstrom's Macroglobulinemia (WM). The key recommendations from IWWM-11 CP5 included the following: Booster vaccines for SARS-CoV-2 should be recommended to all patients with WM. Variant-specific booster vaccines, such as the bivalent vaccine for the ancestral Wuhan strain and the Omicron BA.4.5 strain, are important as novel mutants emerge and become dominant in the community. A temporary interruption in Bruton's Tyrosine Kinase-inhibitor (BTKi) or chemoimmunotherapy before vaccination might be considered. Patients under treatment with rituximab or BTK-inhibitors have lower antibody responses against SARS-CoV-2; thus, they should continue to follow preventive measures, including mask wearing and avoiding crowded places. Patients with WM are candidates for preexposure prophylaxis, if available and relevant to the dominant SARS-CoV-2 strains in a specific area. Oral antivirals should be offered to all symptomatic WM patients with mild to moderate COVID-19 regardless of vaccination, disease status or treatment, as soon as possible after the positive test and within 5 days of COVID-19-related symptom onset. Coadministration of ibrutinib or venetoclax with ritonavir should be avoided. In these patients, remdesivir offers an effective alternative. Patients with asymptomatic or oligosymptomatic COVID-19 should not interrupt treatment with a BTK inhibitor. Infection prophylaxis is essential in patients with WM and include general preventive measures, prophylaxis with antivirals and vaccination against common pathogens including SARS-CoV-2, influenza, and S. pneumoniae.

Ibrutinib in relapsed/refractory patients with Waldenström macroglobulinemia: a real-life, retrospective study on behalf of the "RTL" (regional Tuscan lymphoma network).

Ibrutinib represents the first approved treatment for patients with Waldenström macroglobulinemia (WM). There are very few published experiences outside of a clinical trial. In this study, we investigated treatment response, survival, and safety in a real life setting. We retrospectively analyzed 49 consecutive R/R WM patients, managed in 8 Tuscan onco-hematological centers, that received ibrutinib after its approval, at a maximum dose of 420mg once per day, until disease progression or unacceptable toxicity. Median age was 65years (range 32-86), and the median number of previous regimens was 2 (range 1-5). Overall and major response rate were 91.8% and 87.7%, respectively. At best response, median IgM level declined from 3,094 to 831mg/dl, and Hb level increased from 10.4 to 12.7g/dl. In an intention-to-treat analysis, 36/49 patients (73.5%) were still receiving treatment, while 13/49 (26.5%) had discontinued therapy. Six out of 49 cases (12.2%) relapsed after an initial response, and 13/49 (26.5%) had a dose reduction. Estimated 2-year PFS, DOR, and OS were 76.7%, 88.7%, and 84.1%, respectively. After a median follow-up of 18.3months, 43/49 patients (87.8%) were alive. The most frequent AE included atrial fibrillation or flutter (6/49 cases, 12.2%), bleeding (6/49 cases, 12.2%), arthralgia/myalgia (5/49 cases, 10.2%). Ibrutinib is a suitable treatment option for R/R WM patients and also suggested by ESMO, NCCN, and other societies. PFS and OS were durable, and DOR was sustained for responsive patients. Treatment toxicity is not negligible, but manageable in most cases without treatment discontinuation.

Dose reductions in patients with Waldenström macroglobulinaemia treated with ibrutinib.

Waldenström macroglobulinaemia (WM) is characterized by the presence of a MYD88L265P mutation. This mutation promotes growth and survival of malignant cells through Bruton tyrosine kinase (BTK) activation. Ibrutinib was the first BTK inhibitor approved for WM. Intolerance to ibrutinib frequently leads to dose reductions, though the impact of reducing ibrutinib dosing has not been systematically studied. We performed a retrospective study to determine the frequency and impact of reducing ibrutinib dosing in WM patients. With a median treatment time of 64 months, 96 (27%) of 353 WM patients required a dose reduction due to adverse events such as musculoskeletal symptoms, cardiac events, dermatologic symptoms, cytopenias, and gastrointestinal symptoms. The median time to initial dose reduction was 9.3months (range, 0.5-74). Dose reductions were more common in those 65 years of age or older versus under 65 [hazard ratio (HR) 2.46, 95% confidence interval (CI) 1.55-3.90; p < 0.001], and in females versus males (HR 2.20, 95% CI 1.41-3.28, p < 0.001). Most patients (65%) had improvement or resolution of adverse effects after initial dose reduction. With a median follow-up of three years from dose reduction, hematologic response sustained or deepened in 79% of patients. These data suggest that dose reduction of ibrutinib is a reasonable treatment approach for patients with intolerable side effects.

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

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

Survival trends in young patients with Waldenström macroglobulinemia: Over five decades of experience.

Waldenström macroglobulinemia (WM) is a rare, indolent lymphoma, that predominately affects the elderly. We report the outcomes of young WM patients, evaluated over five decades, compared to their older counterparts, matched for the time of diagnosis. Between January 1, 1960 and October 31, 2013, 140 (11.8%) WM patients were ≤50 years of age at diagnosis in our database, and their estimated 10-year overall survival (OS) was 74%, with death attributable to WM in a higher proportion of patients compared to their older (≥65 years) counterparts (91% vs. 58%, p= .0001). Young patients were grouped into three cohorts based on the timing of the initiation of therapy: Group 1 (1960-1977, n= 12), Group 2 (1978-1995, n= 48), and Group 3 (1996-2013, n= 74). Among young patients, there was no disease-specific survival (DSS) difference across the three periods, [median DSS at 13 years (95% CI 5-23), 16 years (95% CI 14-22), and 15 years (95% CI 10-NR; p= .41), respectively]. However, DSS for the older cohort incrementally improved (Group 1, median 5.2 years, Group 2: 9.6 years, Group 3: 12 years; p= .05) over these periods. The estimated average years-of-life lost for the young cohort was 11.2 years from diagnosis, based on the expected survival for a normal age- and sex-matched population. Despite a protracted disease course, nearly all young patients succumb to their disease. In contrast to the improved survival of the elderly patient population, the evolving treatment strategies in WM have not impacted the outcome of young patients; however, the impact of Bruton tyrosine kinase inhibitors on this unique patient population remains to be determined.

Dysfunctions of innate and adaptive immune tumor microenvironment in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is a rare subtype of non-Hodgkin lymphoma characterized by malignant lymphoplasmacytic cells in the bone marrow (BM). To dissect the pathophysiology of WM, we evaluated clonal cells by mapping of B cell lymphomagenesis with adaptive and innate immune tumor microenvironment (TME) in the BM of WM patients using mass cytometry (CyTOF). In-depth immunophenotypic profiling of WM cells exhibited profound expansion of clonal cells in both unswitched and switched memory B cells and also plasma cells with aberrant expression variations. WM B lymphomagenesis was associated with reduction of most B cell precursors assessed with the same clonally restricted light chain and phenotypic changes. The immune TME was infiltrated by mature monocytes, neutrophils and adaptive T cells, preferentially subsets of effector T helper, effector CTL and effector memory CTL cells that were associated with superior overall survival (OS), in contrast to progenitors of T cells and myeloid/monocytic lineage subsets that were suppressed in WM cohort. Moreover, decrease in immature B and NKT cells was related to worse OS in WM patients. Innate and adaptive immune subsets of WM TME were modulated by immune checkpoints, including PD-1/PD-L1&PD-L2, TIGIT/PVR, CD137/CD137-L, CTLA-4, BTLA and KIR expression. The response of ibrutinib treatment to the reduction of clonal memory B cell was associated with high levels of immature B cells and effector memory CTL cells. Our study demonstrates that CyTOF technology is a powerful approach for characterizing the pathophysiology of WM at various stages, predicting patient risk and monitoring the effectiveness of treatment strategies.

Molecular Clusters and Tumor-Immune Drivers of IgM Monoclonal Gammopathies.

IgM monoclonal gammopathy of undetermined significance (MGUS) and Waldenström macroglobulinemia (WM) represent a disease spectrum with highly varied therapeutic management, ranging from observation to chemoimmunotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. Further investigation is warranted to shed light on the biology that may account for the clinical differences. We used bone marrow (BM) clonal CD19+ and/or CD138+ sorted cells, matched BM supernatant, and peripheral blood serum from 32 patients (7 MGUS, 25 WM) to perform the first multi-omics approach including whole-exome sequencing, RNA sequencing, proteomics, metabolomics, and mass cytometry. We identified three clusters with distinct pathway activation, immune content, metabolomic, and clinical features. Cluster 1 included only patients with WM and was characterized by transcriptional silencing of genes involved in cell cycle and immune response, enrichment of mitochondrial metabolism, infiltration of senescent T effector memory cells, and aggressive clinical behavior. Genetic/structural alterations of TNFAIP3 were distinct events of this cluster. Cluster 2 comprised both MGUS and WM patients with upregulation of inflammatory response, senescence and glycolysis signatures, increased activated T follicular helper and T regulatory cells, and indolent clinical behavior. Cluster 3 also included both MGUS and WM patients and exhibited intermediate features, including proliferative and inflammatory signaling, as well as glycolysis and mitochondrial metabolism. We have identified three distinct molecular clusters, suggesting a potential biologic classification that may have therapeutic implications.

Single-Cell Transcriptome Analysis Reveals Stem Cell-like Subsets in the Progression of Waldenström's Macroglobulinemia

Waldenström's macroglobulinemia (WM) is an uncommon lymphoproliferative disorder characterized by monoclonal immunoglobulin M protein production and bone marrow infiltration by lymphoplasmacytoid cells. It remains an incurable disease. IgM monoclonal gammopathy of undetermined significance (MGUS) is the premalignant condition and is associated with the risk of developing WM. MYD88L265P is a recurrent mutation found in more than 90% of WM patients, and this genetic event is detectable in at least half of IgM MGUS patients. However, the precise cellular landscape and the mechanisms of progression from IgM MGUS to WM remain unclear. Single-cell RNA sequencing (scRNA-seq) provides us a powerful approach to explore tumor heterogeneity and identify rare malignant cells. Anti-CD38 monoclonal antibodies have been introduced into the therapeutic arsenal for plasma cell diseases. Therefore, we analyzed CD38+ immune microenvironment together with B cells and plasma cells to illustrate the first cellular landscape of IgM MGUS and WM in single cell resolution. We isolated bone marrow CD19+ and CD19-CD38+ cells from WM patients(n=3), IgM MGUS patients(n=3) and healthy donors(n=3) and employed the 10x Genomics platform to perform single-cell transcriptomic sequencing. After quality control, our dataset contained a total of 73024 cells. We used UMAP to visualize the cell superclusters and identified clusters including B cells, plasma cells, CD3+CD20+ (CD19+) cells, T cells and NK cells based on the expression of canonical lineage markers and cluster-specific markers. We found that healthy donors had the highest percentage of B cells (72.7% in healthy donors, 19.0% in IgM MGUS patients, 60.5% in WM patients; p<0.001) and that IgM MGUS and WM patients had more NK and T cells than healthy controls (0.06% and 17.7%, respectively, in healthy donors; 24.1% and 20.5% in IgM MGUS patients; 8.5% and 24.0% in WM patients; p<0.001). We also noticed that CD3+CD20+(CD19+) cells were present in both IgM MGUS and WM patients. First, we investigated the cell clusters correlated with WM pathology including B cells and plasma cells. We compared the B-cell gene expression profiles after removing the early B cells. Compared with those of IgM MGUS patients, mature B cells of WM patients showed upregulation of PIM1, DUSP22, HES1, NEAT1, RGS1, RGS16, and GADD45B. Light chain restriction and higher CNV levels were found in B cells of WM patients. SCENIC analysis showed that FOS, FOSB, EGR1 and JUNB were downregulated in B cells from WM and IgM MGUS patients. REL, a proto-oncogene promoting the survival and proliferation of B lymphocytes, was upregulated in B cells of WM patients, and IRF8 and ELF1 were upregulated in B cells of IgM MGUS patients. Nevertheless, plasma cells of WM patients, IgM MGUS patients and healthy donors were colocalized in one cluster, which might indicate their similar gene expression patterns. Next, we further determined the characteristics of B-cell subpopulation with aberrant T-cell antigen expression. Trajectory analysis revealed that CD3+CD20+(CD19+) cells and pro-B cells were located at the same end of the branched structure and were then directed toward mature B cell and plasma cell fates, respectively (Figure. 1A). This inferred developmental trajectory suggested that CD3+CD20+(CD19+) cells may be tumor stem cell-like subset. Using flow cytometry, we validated that CD3 was expressed in a fraction of CD19+ cells (mean: 3.69%, range: 0.28%-15.10%) of bone marrow samples from 8 WM patients. In addition, targeted gene sequencing was performed on CD3+CD19+ and CD3-CD19+ cells from 6 WM patients. MYD88 were proved to be the most frequently mutated gene in this cell subset. And we inferred that MYD88 mutation might be the early events in tumorigenesis by VAF analysis (Figure. 1B). Additional subclonal hits, such as CXCR4 and MAP2K1 mutations, could be acquired during tumor progression. In conclusion, our study provides comprehensive insights into mechanisms of progression from IgM MGUS to WM. We identified the rare CD19+ cell subpopulation with T-cell antigen expression in WM patients. It will be interesting to explore novel therapeutic strategies targeting rare potential cancer-initiating cells in future. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Determinants of Overall Survival (OS) of Waldenstrom Macroglobulinemia (WM): A National Cancer Database (NCDB) Analysis of over 14,000 Patients Treated between Years 2004 and 2017

Introduction: Real world outcome data for Waldenstrom Macroglobulinemia (WM) is limited due to relatively rare and indolent nature of this B cell Non-Hodgkin lymhoplasmacytic lymphoma. In this retrospective analysis, we utilized National Cancer Data Base (NCDB) to identify variables contributing to the overall survival (OS) of over 14,000 WM patients treated at Commission on Cancer (CoC)-accredited cancer centers across USA. Methods: From NCDB, 14,295 WM patients diagnosed and treated during 2004-2017 were identified and included in the analysis after excluding entries with missing relevant values. For OS, univariate analysis was used first to summarize and assess the potential survival factors individually, and multivariate Cox regression analysis with backward elimination was utilized to identify the most important survival factors. In addition, Kaplan-Meier survival curves of patient cohort were also produced for OS. SAS version 9.4 was used to analyze the data. Results: The baseline characteristics of the patients are shown in Table 1A. Multivariate Cox regression analysis with backward elimination method revealed that there were 10 significant independent survival factors including age, sex, race, Charlson comorbidity score (CC), income, facility type, insurance, chemotherapy, sequence number, and year-of-diagnosis (Table 1B). Older patients were more likely to die compared to younger patients with age <55 (55≤age<65: HR=1.43, p<0.0001; 65≤age<75: HR=1.89, p<0.0001; and age≥75: HR=4.29, p<0.0001); male were more likely to die compared to female (HR=1.17, p<0.0001); blacks were more likely to die compared to white (HR=1.28, p<0.0001); patients with CC ≥1 had higher chance to suffer death compared to patients with CC =0 (HR=1.72, p<0.0001); patients at lower income area were more likely to die compared to patients at ≥$63,000 income area ($48,000 - $62,999: HR=1.16, p<0.0001; $38,000 - $47,999: HR=1.23, p<0.0001; and <$38,000: HR=1.30, p<0.0001); patients at other facility type were more likely to die compared to patients at Academic/Research program (HR=1.09, p=0.0019); and patients with other insurance were more likely to die compared to patients with private insurance (PI) with (HR=1.31, p<0.0001), patients with insurance unknown status were also more likely to pass away compared to patients with PI (HR=1.26, p=0.0207); there was no significant difference in survival between patients with multi-agent chemotherapy and with single/unknown agent (HR=1.02, p=0.6436), and patients without chemotherapy were less likely to die compared to patients with single/unknown agent (HR=0.83, p<0.0001); patients with sequence number of not-zero (defined as > 1 malignant sites) were more likely to die compared to patients with sequence number of zero (single malignant site) with (HR=1.19, p<0.0001). And finally, patients diagnosed between 2004 and 2010 were more likely to die compared to patients diagnosed between 2011 and 2017 (HR=1.25, P<0.0001). Kaplan-Meier estimate (Figure 1A) showed that OS rate at 1, 3, and 5 years were 88.0%, 76.3%, and 65.8%, respectively. Figure 1B shows that the OS was worse for those with co-morbidities as reflected by CC ≥1. Conclusion: In this large real-world retrospective analysis, we demonstrate racial, demographics and socioeconomic differences impacting the outcomes of WM patients. Older WM patients, male sex, black race, higher CC and lower income were significantly associated with lower OS. Patients with access to PI and academic centers had better OS but those with non-PI and treated at non-academic centers had inferior OS. In terms of disease specific factors, WM patients with higher tumor burden (>1 malignant sites) and those needing therapy had worse OS compared to patients with lower tumor burden and those observed without therapy. Interestingly, patients diagnosed between 2011 and 2017 had superior OS compared to the patients diagnosed between 2004 and 2010 likely due to advancement in therapy over time. Cancer disparity data specific to WM are lacking and our study highlights the importance of socioeconomic, demographics and racial factors in the prognosis of patients with WM in addition to the standard disease and treatment specific prognostic variables. Since the social determinants of health can greatly impact outcomes in WM, improving access to high quality care could potentially help overcome the observed disparity in the outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Second Primary Malignancy in Waldenström Macroglobulinemia: Insights from a Population-Based Analysis

INTRODUCTION: Waldenström Macroglobulinemia (WM), is a hyper-viscosity syndrome characterized by unrestricted proliferation of lymphoplasmacytic precursors in the bone marrow and monoclonal IgM gammopathy. The development of secondary primary malignancy (SPMs) in WM is a multifactorial phenomenon; the indolent nature of the disease, associated immune dysregulation, environmental factors and therapeutic measures, are responsible for high incidence of SPM in WM. This study was conducted to analyse the site-specific incidence of SPM in WM using data from the US Surveillance, Epidemiology and End Results (SEER) cancer registries. METHODS: A retrospective study was conducted using the SEER-18 dataset. WM patients who were diagnosed between 2000 and 2018, according to the ICD-O-3 = 9761/3 were included. Patients diagnosed on autopsy or through death certificates, and patients with incomplete follow-up data were excluded. SPM was defined as other malignancy appearing at least 1 year after WM diagnosis. Latency was defined as interval between WM and SPM diagnosis. Standardized incidence ratios (SIRs) and absolute excess risks (AERs) were calculated using SEER*Stat software (version 8.3.9). P-values and 95% confidence intervals (CI) were generated assuming Poisson distribution of observed incidence of SPM. RESULTS: A total of 4112 WM patients were identified. SPM was reported in 699 (17.0%) patients. The overall risk of developing SPM was significantly higher in WM patients as compared to the general population (SIR= 1.53, 95% CI=1.42-1.65, p <0.05) with an AER of 102.69 per 10,000 population. The risk of developing second primary solid tumors was significantly greater compared to the general population (SIR = 1.12, 95% CI= 1.02-1.23, p >0.05), but significantly increased risks were reported for secondary tumours of the lung and bronchus (SIR = 1.44), skin melanoma (SIR = 1.88), mouth (SIR 3.28), brain (SIR 2.73) and non-melanotic skin tumors (SIR = 4.09). The overall risk of developing secondary hematological malignancies was significantly increased (SIR = 4.95, 95% CI 4.31-5.66, p <0.05). Significantly increased risks were reported for nodal non-Hodgkin lymphoma (NHL) (SIR = 6.62), extranodal NHL (SIR = 11.3), acute lymphocytic leukemia (ALL) (SIR 8.04), acute non-lymphocytic leukemia (ANLL) (SIR = 4.2) and acute myeloid leukemia (AML) (SIR 4.22). Increased risk of SPMs was noted for patients with age 50-74 years (SIR= 1.65, 95%CI=1.44-1.89, p value <0.05) or > 75 years (SIR= 1.3, 95% CI= 1.13-1.49, p value <0.05), but not for patients younger than 50 years. Patients aged 50-74 years had a significantly increased risk of developing SPMs of lungs and bronchus (SIR 2.23), skin (SIR 3.09), vulva (SIR 8.5), thyroid (SIR 3.59), nodal NHL (SIR 6.06), extranodal NHL (SIR 11.87), ANLL (SIR 5.77) and AML (SIR 6.37). Patients aged >75 years had a significantly increased risk of developing SPMs of cecum (SIR 2.50), colon (SIR 1.62), soft tissues (SIR 5.25), nodal NHL (SIR 2.38), extranodal NHL (SIR 7.51), ANLL (SIR 3.69) and AML (SIR 3.63). Risk of SPM was significantly increased in both males (SIR 1.39) and females (SIR 1.59). On subsite evaluation, males had a significantly greater risk of SPMs involving lung and bronchus (SIR 1.71), soft tissues (SIR 4.52), skin (SIR 1.88), nodal NHL (SIR 3.85), extra-nodal NHL (SIR 6.9), ANLL (SIR 5.4) and AML (SIR 5.55). Females reported a significantly increased risk of SPM involving cecum (SIR 3.88), skin (SIR 2.87), vagina (SIR 11.51) nodal NHL (SIR 3.86) and extranodal NHL (SIR 13.57). The risk of SPM was significantly greater in Caucasians (SIR 1.46) but not African Americans or other races. The risk of SPM was significantly greater in patients at all latencies. The risk of SPM of cecum (SIR 2.76) and non-melanotic skin cancer (SIR 5.52) was significantly greater in patients with latency of 12-59 months, but not at subsequent latencies. The risk of melanotic skin cancer (SIR 2.61) was significantly greater in patients with latency of 60-119 months but not other latencies. The risk of thyroid cancer (SIR 6.43) was significantly increased for patients with latency 120+ months only. CONCLUSION: This study showed that 17% of WM patients developed SPM. WM patients were at a 53% greater risk of developing SPM compared to the general population. Caucasians and patients aged greater than 50 years were at particularly high risk of developing SPM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Increased Tumor-Associated CD66b+ Myeloid-Derived Suppressor Cells in Waldenstrom Macroglobulinemia Inhibit T-Cell Immune Function

Waldenstrom macroglobulinemia (WM) is a discrete clinicopathologic entity resulting from the accumulation of lymphoplasmacytic lymphoma (LPL) cells in the bone marrow (BM) that secrete a monoclonal immunoglobulin (Ig)M protein. MYD88 (>90%) and CXCR4 (30-35%) mutations are the most commonly identified mutations in this disease. While much is known about the genomics of WM, far less is known about the role of the BM microenvironment in this disease. Emerging evidence has attested to the role of myeloid-derived suppressor cells (MDSCs) and their potent immunosuppressive activity, however, there is limited information about these cells in WM and monoclonal gammopathy of undetermined significance (MGUS). This study was therefore designed to explore the phenotype of MDSCs and their interaction with other cells in the BM in WM. A multiparametric approach was combined with single-cell genomic and proteomic tools to better understand MDSC immunology and the antitumor immune response in WM. In the present study, using flow cytometry, we first analyzed the number of MDSCs (by gating on Lin- HLA-DR- CD11b+ CD33+) in normal BM (NBM; n=11) and WM symptomatic /MGUS (n=18) and found an increased number of MDSCs in WM specimens compared to controls (p=0.005). We also found that most MDSCs in WM specimens had a granulocytic phenotype (Lin- HLA-DR- CD11b+ CD33+ CD15+). A detailed phenotypic study using CyTOF (mass spectrometry) was performed on MDSCs from NBM (n=4) and WM (n=8). The CyTOF analysis identified 3 subtypes of MDSCs including CD66b+ MDSCs with a granulocytic phenotype (G-MDSCs) and confirmed the expansion of CD66b+ MDSCs in WM symptomatic patients when compared to smoldering WM and normal controls. Our previous studies have shown that granulocyte colony-stimulating factor (G-CSF) expression is increased in the bone marrow of WM (Jalali et al., 2018). Therefore, to determine whether G-CSF accounted for the expansion of CD66b+ G-MDSCs, we treated WM-BM MDSCs with G-CSF and found that the CD66b+ G-MDSC population substantially increased (p=0.0017). Further, to establish a better understanding of the transcriptome profile and a unique phenotype of CD66b+ G-MDSCs, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) on WM (n=3) and NBM (n=2) specimens. A significantly high expression of NLRP12, IGFBP7, CXCR4, IL2, CD55, SOX4, and FoxP1 genes was observed in CD66b+ MDSCs as compared to other MDSC populations. This gene expression signature suggested upregulation of inflammatory pathways potentially affecting immune function. Therefore, to evaluate the effect of CD66b+ G-MDSCs on the immune function of other cells in the BM, CD66b+ and CD66b- MDSCs were co-cultured with activated autologous T-cells. We found that while all populations of MDSCs suppressed T-cell activation, CD66b+ G-MDSCs had the greatest suppressive effect. In conclusion, this study has shown that CD66b+ G-MDSCs are expanded in WM patients, exhibit a unique inflammatory transcriptome, and substantially inhibit T-cell activation and proliferation. Furthermore, CD66b+ G-MDSCs are expanded due to increased G-CSF in the BM microenvironment in WM. Clinical strategies to inhibit the expansion of CD66b+ G-MDSCs may therefore enhance the efficacy of immunological therapies in patients with WM.

VEGF and IL-6 As Markers of Severity and Response to Treatment in Waldenstrom Macroglobulinemia and IgM-Monoclonal Gammopathy of Undetermined Significance: A Monocentric Experience

Background: Angiogenesis plays an important role in the development and the maintenance of hematolymphoid malignancies. Serum levels of angiogenic cytokines, such as vascular endothelial growth factor (VEGF) and interleukin 6 (IL-6), are known to be increased in patients with Waldenstrom Macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (MGUS). Even if the role of IL-6 and VEGF as markers of disease status and severity has been recognized, only a limited number of studies have addressed the role of cytokines in WM and there is no proof about any correlation between their serum levels in WM and IgM-MGUS patients. Nevertheless, their dosage at various stages of the disease aims to understand if they have a comparable pattern, and therefore they could be used in clinical practice for disease monitoring. Aims: This study aims to find a correlation of serum levels of VEGF and IL-6 with the burden of disease in our patients with IgM-MGUS and WM and to evaluate a possible relationship between the two angiogenic cytokines, in an attempt to identify potential markers of severity. Methods: We performed a retrospective monocentric study of 18 patients (ten with WM and eight with IgM-MGUS) with a median age of 73 years (range: 52-90), whose 66% were male. Patients with WM were at different stages of disease (four were naive, four in remission, two relapsed/refractory). A total of 18 controls with a similar age and gender distribution were also tested. Controls were selected among patients with neuropathies in the absence of any hematological condition. We used the 1:1 ratio for the selection of controls: for every male or female WM patient, one control of the same gender and of similar age was included. In order to find a relationship of VEGF and IL-6 with a laboratory parameter, IgM levels dosage for each patient have been measured at the same time-point of the measurement of the cytokines. In all patients and controls, we evaluated serum levels of VEGF and IL-6 using Immunoassay panels (i.e., anti-IL-6 and anti-VEGF ELISA kit). Results: All patients showed elevated values of angiogenic cytokines when compared with controls (P value < 0.05). Both VEGF and IL-6 serum levels (median values: 623,5 pg/mL and 4,5 pg/mL, respectively; ranges: 113,4-1981 pg/mL and 0,3-21,9 pg/mL, respectively) showed a positive correlation with IgM serum levels (median value: 2055 mg/dL, range: 300-6123 pg/mL) (Fig. 1a and 1b). Furthermore, the relationship between IL-6 and VEGF was statistically significant and could be explained by a linear model (P value = 0,007) (Fig. 1c). No statistically significant differences were observed between the different phases of WM. Conclusions: Evidence from our sample shows that serum levels of angiogenic cytokines as VEGF and IL-6 have a comparable pattern with IgM levels. Therefore, it seems reasonable to suggest that they could be used in clinical practice as markers of severity of disease and response to treatment. Moreover, since the cytokines profiles are comparable, IL-6 can be used interchangeably with VEGF, allowing the monitoring of the disease with easily availability and unexpensive techniques. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multi-Omic Analysis of 253 Untreated Patients with Waldenström's Macroglobulinemia Reveals Clinically and Genomically Distinct Disease Subtypes and a Model for Disease Progression

Background: Waldenström's Macroglobulinemia (WM) is characterized by recurring mutations in MYD88 (95-97%) and CXCR4 (40%). While these genomic insights have heralded new insights into disease pathogenesis and treatment, a multi-omic approach in a large population of WM patients may further clarify underlying disease biology. Methods: Stranded 150bp paired end RNA sequencing was performed on CD19+ bone marrow (BM) biopsies from 253 treatment-naive WM patients along with paired CD19+CD27- and CD19+CD27+ peripheral blood (PB) cells from 13 healthy donors. For 216 patients, whole exome sequencing of CD19+ BM WM cells with CD19- PB controls was also performed. All p-values were subjected to correction using the false discovery rate. Results: Baseline characteristics were typical of treatment-naive patients. Their median age was 66 years; BM disease involvement 50% ; and serum IgM 3,108 mg/dL. The median follow-up was 7.5 years. MYD88 mutations were identified in 250 (98.8%) patients. Exploratory analysis of MYD88 mutated patients revealed three distinct groups of patients that were observed in a hierarchical clustering analysis shown in Fig 1A. Gene set enrichment analysis revealed distinct B-cell Like (BCL) and Plasma Cell Like (PCL) patient groupings and a third non-BCL/PCL patient group that clustered independently. Non-BCL/PCL patients were more likely to be asymptomatic (36/84; 43%) versus those in the BCL (62/96; 64.6%) and PCL (48/68; 70.6%) groups (p<0.005). Among BCL and PCL grouped patients, important differences in disease presentation were noted including BM involvement (50% vs. 70%; p<0.001), adenopathy (18.6% vs. 48.4%; p<0.002), CD10 (1.1% vs. 13.4%; p=0.012), CD23 (39.4% vs. 22.4%; p=0.09) and CD5 (19.1% vs. 6%; p=0.09) expression, respectively. CXCR4 mutations were identified in 76/97 (78.4%) of BCL versus 6/69 (8.7%) of PCL patients (p<0.001). Amplifications in chr18q amplifications were also more prevalent in BCL (14/83; 16.9%) versus PCL (1/64; 1.6%) grouped patients (p=0.035). chr6q deletions were observed in 24/83 (29%) of BCL versus 30/64 (47%) PCL patients (p=0.11). BCL patients also exhibited fewer chr6p amplifications, i.e. 3/83 (3.6%) versus 12/64 (18.8%) of PCL patients (p=0.036). Differences in deletions in chr17p (0/83; 0%) vs (6/64; 9.4% (p=0.036); as well as NOTCH1 (0/82; 0%) vs. (6/64; 9.4%) (p=0.084) and EP300 3/82; 3.7%) vs. (11/64; 17.2%) (p=0.084) mutations were also identified in BCL vs PCL patients, respectively. NOTCH pathway mutations were also found in fewer BCL (17/82; 20.7%) versus PCL (30/64; 46.9%) patients (p=0.01). Diffusion mapping and pseudo-time analysis were performed on the top 1,000 high variance genes which bifurcated patients into two distinct populations over time as shown in Fig. 1B, with the point size scaled for BM involvement. By diffusion pseudo-time (DPT), patients were stratified into early- and late-stage WM, the former showing lower median BM involvement (30% vs. 70%; p<0.001) and serum IgM (2,666 vs. 3713; p=0.133); higher hemoglobin (12.1g/dL vs. 10.65g/dL; p<0.001), serum IgA (56.5mg/dL vs. 41mg/dL; p=0.018) and IgG (629mg/dL vs. 498mg/dL; p=0.015), and less splenomegaly (3.7% vs. 17.2%; p=0.006). A longer time to first treatment (5.3 vs 0.9 months; (p<0.001) was also observed in those with early- versus late- stage WM, respectively. DPT showed that the non-BCL/PCL patients were more likely to be early stage (68/84; 81%) versus BCL (51/97; 53%) and PCL (30/69; 43%) grouped patients (p<0.001). In addition, hierarchical clustering delineated the non-BCL/PCL group into two subgroups with CXCR4 mutations rates of 30/57 (52.6%) and 2/27 (7.4%), and gene expression similarities that aligned with either the BCL or PCL subtypes, respectively. Taken together, these findings suggest that the non-BCL/PCL group represents an earlier evolving stage of WM that may progress into BCL and PCL like WM disease. Conclusions: In this largest multi-omic investigation undertaken into WM, we identified three distinct WM subtypes with distinct clinical and genomic features. Central these findings were the identification of BCL, PCL, and an earlier non-BCL/PCL evolving stage recognized through DPT modeling. The discovery of the DPT model for WM disease progression provides insight into the biology and targeting of WM progression. Our findings provide a critical framework for multi-omic based study and targeted treatment approach to WM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal