MYD88 L265P Mutation Status Research Articles

Clinicopathological features and BRAF V600E and MYD88 L265P mutation status of nodal marginal zone lymphoma

Objective: To investigate the clinicopathological features as well as BRAF V600E and MYD88 L265P mutation status of nodal marginal zone B cell lymphoma (NMZL). Methods: Thirty-two cases of NMZL were diagnosed from September 2009 to February 2021 at the Henan Provincial People's Hospital and Peking University School of Basic Medical Sciences. The clinicopathologic characteristics were obtained and analyzed. BRAF V600E and MYD88 L265P mutation status were identified using PCR and Sanger sequencing, respectively. Results: There were 20 males and 12 females patients with a median age of 69 years (ranging 36-82 years). The most prevalent clinical manifestation was multiple lymph nodes enlargement in head and neck (22/32, 68.8%), followed by inguinal (12/32, 37.5%), axillary (11/32, 34.4%), mediastinum (5/32, 15.6%) and retroperitoneal lymph nodes (4/32, 12.5%). Most of the patients were in Ann Arbor stage Ⅰ/Ⅱ (21 cases). The morphologic features included diffuse (24/32, 75.0%), nodular (5/32, 15.6%), interfollicular (2/32,6.3%) and perifollicular (1/32,3.1%) types. The tumor cells showed monocyte-like, centrocyte-like, small lymphocyte-like and plasma cell-like differentiation. Immunophenotyping revealed diffuse expression of CD20 in all tumor cells, whereas CD43 (11/32, 34.4%), bcl-2 (20/32, 62.5%), MNDA (13/32, 40.6%) and CD5 (2/32, 6.3%) were partially expressed. Ki-67 proliferation index varied from 10% to 40%. BRAF V600E mutation was found in two cases (2/32, 6.3%), but MYD88 L265P mutation was not detected. Eighteen patients survived and three died at the end of follow-up period which ranged 6 to 110 months. Conclusions: The morphologic features of NMZL varies across individuals, it should be differentiated from various B-cell lymphomas; however immunological biomarkers with high specificity for NMZL are still lacking. No MYD88 L265P mutation is found in NMZL. Some cases may harbor BRAF V600E mutation and yet the prevalence remains indeterminate; further researches are warranted.

Current approach to Waldenström Macroglobulinemia

Waldenström Macroglobulinemia (WM) is a unique, low grade, IgM lymphoplasmacytic lymphoma with a heterogeneous clinical course. A paucity of high-grade evidence from large phase 3 trials remains a major issue in the field despite a rapidly expanding therapeutic armamentarium against WM. Prior knowledge of the patients’ MYD88L265P and CXCR4 mutation status aids in treatment decision making if Bruton's tyrosine kinase (BTK) inhibitor therapy is being considered. Head-to head comparative data to inform optimal approach are lacking, and a particularly vexing issue for the clinicians is choosing between fixed-duration bendamustine-rituximab (BR) therapy and an indefinite BTK inhibitor-based regimen, given that both approaches are well tolerated and effective, particularly for the patient population harboring MYD88L265P mutation. For the patients with MYD88WT genotype, chemo-immunotherapy such as BR is preferred, although zanubrutinib, a potent second generation BTK inhibitor, with its reduced off target effects and greater BTK occupancy compared to its predecessor, ibrutinib, has also recently shown activity in MYD88WT WM. This review summarizes the current literature pertaining to the diagnosis, prognosis, and the treatment of WM.

IBCL-085: Assessment of Fixed-Duration Therapies for Treatment-Naïve Waldenström Macroglobulinemia

Context Waldenstrom macroglobulinemia (WM) is a rare lymphoma for which scant comparative data exist to guide frontline therapy. Objective To compare the efficacy of the most frequently used rituximab-based fixed-duration anti-WM regimens in routine practice [rituximab, bendamustine (R-Benda); dexamethasone, rituximab, cyclophosphamide (DRC); and bortezomib, dexamethasone, rituximab (BDR)] to inform clinicians about the benefits/limitations of choosing one regimen over the other. Design A retrospective cohort study. Participants Patients with active WM seen at Mayo Clinic Rochester, Arizona, and Florida campuses between 10/01/2000 and 10/31/2019 who received R-Benda, DRC, or BDR as primary therapy were included. Outcomes Measures Overall response rate (ORR), event-free survival (EFS), progression-free survival (PFS), overall survival (OS), time-to-next therapy (TTNT) and treatment related adverse-effects (AEs) based on the three treatment regimens. The MYD88L265P mutation status was assessed using allele-specific polymerase-chain-reaction (sensitivity 1%). Response rates were assessed by Consensus Criteria. Time-to-event analyses were performed from the frontline therapy, using Kaplan-Meier method. CTCAE v4.03 was used to grade toxicities. Results The study included 220 patients with active WM who were treated with R-Benda (n=83), DRC (n=92) or BDR (n=45). The median follow-up was 4.5 (95% CI: 4-5) years. The R-Benda cohort demonstrated superior ORR (98%), in comparison to DRC (78%) or BDR (84%) cohorts, p=0.003. Similarly, longer PFS was evident with R-Benda use [median 5.2 versus 4.3 (DRC) and 1.8 years (BDR), p=0.0003]. The TTNT favored R-Benda [median, not-reached, versus 4.4 (DRC) and 2.6 years (BDR), p=0.0002]. These endpoints were comparable between the DRC and BDR cohorts. Overall survival (OS) was similar across the three cohorts, p=0.77. In a subset analysis of 142 patients genotyped for MYD88L265P mutation, the ORR, PFS and TTNT were unaffected by the MYD88 signature within each cohort. Toxicity profiles across the three groups were comparable, barring a higher incidence of neutropenia and neuropathy in patients who received R-Benda and BDR regimens, respectively. Conclusions The ORR, PFS and TTNT with R-Benda are superior compared to DRC or BDR in treatment-naive patients with active WM. The patient outcomes with any one of these three regimens are unaffected by the MYD88L265P mutation status.

Nodal lymphoplasmacytic lymphoma/Waldenstrom's macroglobulinemia: a clinicopathological and prognostical study

Objective: To study the clinicopathological features and prognosis of nodal lymphoplasmacytic lymphoma/Waldenstrom's macroglobulinemia (n-LPL/WM). Methods: A total of 19 cases of n-LPL/WM were collected from May 2009 to January 2020 at First Affiliated Hospital of Zhengzhou University. The clinicopathologic features, immunophenotype, Ig gene rearrangement (BIOMED-2), MYD88 L265P mutation status (by Sanger sequencing) and follow-up data (by telephone) were analyzed. Results: There were 15 males and 4 females with a median age of 61 years (range 43 to 82 years). There were 14 WM and five LPL. The most common symptoms were weakness, fatigue (9/19) and B symptoms (11/19). Majority of the patients (16/18) presented with systemic multiple lymphadenopathies. Eighteen patients presented at advanced stages (Ⅲ/Ⅳ stage). Serum M protein status was IgM (15 cases), IgG (1 case), IgA (1 case) and no-secretory type (2 cases). Seventeen patients had bone marrow involvement. Morphologically, all 19 cases were divided into two groups: typical group (9 cases) or atypical group (10 cases). In the typical group, the structures of the lymph nodes were preserved; the neoplastic cells were predominantly plasmacytoid lymphocytes or mixed small lymphocytes, plasmacytoid lymphocytes and plasma cells, without proliferation of FDC network and follicular implantation. In the atypical group, the tumor showed effaced nodal architecture (5 cases), mainly proliferation of small lymphocytes (6 cases), FDC proliferation and/or follicular implantation (6 cases), marginal zone B cell differentiation (4 cases) and diffuse amyloidosis (1 case). Hemosiderin deposition (19 cases), infiltration of fatty tissue (19 cases) and interstitial sclerosis (9 cases) were commonly seen in both groups. Immunohistochemically, the neoplastic B cells expressed CD20 and CD79α, and the neoplastic plasma cells were positive for CD38, CD138 and MUM-1; eight cases showed light chain restriction; of the seven detected cases, five expressed IgM and the other two expressed IgG and IgA respectively; four cases expressed CD23 weakly, Ki-67 index was 10%-30%. MYD88 L265P mutation was seen in 18/18 cases. There was no significant difference in clinicopathologic features and prognosis between the two groups (P>0.05). The median follow-up time was 61 months, 11 patients were alive, while eight died; the 5-year survival rate was 21.1%. Conclusions: n-LPL/WM is rare, but patients usually present in advanced stages. It is easily confused with other small B-cell lymphomas with plasma cell differentiation, especially basing on morphologic features alone; thus the accurate diagnosis of n-LPL/WM requires a combination of clinical features, serum M protein, immunohistochemistry, bone marrow morphology,flow cytometry and MYD88 L265P mutation status etc. The prognosis of n-LPL/WM may be not very good, and further studies with more cases are needed.

Assessment of fixed-duration therapies for treatment-naïve Waldenström macroglobulinemia.

Comparative data guiding initial therapy for Waldenström macroglobulinemia (WM), an infrequently encountered non-Hodgkin lymphoma, are sparse. We evaluated three commonly used rituximab-based frontline regimens: rituximab-bendamustine (R-Benda); dexamethasone, rituximab, cyclophosphamide (DRC); and bortezomib, dexamethasone, rituximab (BDR) in 220 treatment-naïve patients with WM, seen at Mayo Clinic between November 1, 2000 and October 31, 2019. The median follow-up was 4.5 (95%CI: 4-5) years. The R-Benda cohort (n = 83) demonstrated superior overall response rate (ORR: 98%), in comparison to DRC (n=92, ORR: 78%) or BDR (n=45, ORR: 84%) cohorts, p=0.003. Similarly, longer progression-free survival (PFS) was evident with R-Benda use [median 5.2 vs. 4.3 (DRC) and 1.8 years (BDR), p < 0.001]. The time-to-next therapy (TTNT) favored R-Benda [median, not-reached, 4.4 (DRC) and 2.6 years (BDR), p < 0.001). These endpoints were comparable between the DRC and BDR cohorts. Overall survival (OS) was similar across the three cohorts, p=0.77. In a subset analysis of 142 patients genotyped for MYD88L265P mutation, the ORR, PFS and TTNT were unaffected by the patients' MYD88 signature within each cohort. In conclusion, ORR, PFS and TTNT with R-Benda are superior compared to DRC or BDR in treatment-naïve patients with active WM. The patient outcomes with any one of these three regimens are unaffected by the MYD88L265P mutation status.

Clinicopathological features and MYD88 L265P mutation status of intravascular large B cell lymphoma

Objective: To study the clinicopathologic features and MYD88 L265P mutation status of intravascular large B cell lymphoma (IVLBCL). Methods: Fourteen cases of IVLBCLs were diagnosed from March 2014 to December 2019 at the First Affiliated Hospital of Zhengzhou University. The clinicopathologic features and prognosis were analyzed. Epstein-Barr virus encoded RNAs and MYD88 L265P mutation status were detected using in situ hybridization and Sanger sequencing, respectively. The follow-up data were obtained by telephone interview. Results: There were 6 males and 8 females with a median age of 62 years (range: 48-73 years). The involved anatomic locations were demonstrated by positron emission tomography-computed tomography, including adrenal gland (7/14), bone (6/14), central nerve system (4/14), skin (3/14), female reproductive system (3/14), local lymph nodes (3/14), prostate (2/14), liver and spleen (2/14), sphenoid sinus (1/14), penis (1/14), bladder (1/14), and right lung (1/14). Fever was the most common symptom (7/14), followed by neurologic symptoms and lower abdominal pain (2/14 each). The reminder symptoms included rash with edema, legs weakness and numbness, or postmenopausal bleeding (1/14 each). Eleven cases were at Lugano stage Ⅳ. Four cases were associated with the hemophagocytic syndrome, while 6 cases with bone marrow involved. Microscopically, the tumor cells were generally concentrated within the small-to-medium vascular lumens or sinusoids; they had centroblast-like appearance and showed large round or oval nuclei with slightly irregularities, coarse chromatin and 1-3 distinct nucleoli. One exception was the one case with an embryoid nuclei, reminiscent of anaplastic large cell lymphoma. The mitosis was not uncommon. Extravascular neoplastic cells were seen in two cases. The neutrophils could be appreciable in most of the cases (10/14). Immunophenotyping showed that CD20 and CD79α were diffusely and strongly positive in 14 cases; 12 cases were classified as the non-GCB subtype; 6 out of the 11 cases were double expressor lymphoma; 7 out of the 12 cases were CD5-positive. Twelve cases were EBER negative. The MYD88 L265P mutation was detected in 1 case (1/10). The duration of the follow-up ranged from 0.5 to 24.0 months, and 11 patients survived and 3 died. Conclusions: IVLBCL is rare. The most common type of IVLBCL in China is Asian type with scant tumor cells. Combination of clinical and immunohistochemical features can avoid most, if not all, misdiagnoses and missed diagnoses. Some IVLBCL cases may harbor the MYD88 L265P mutation, but the prevalence of MYD88 L265P mutation in the population still warrants additional studies.

Pleural fluid MYD88 L265P mutation supporting diagnosis and decision to treat extramedullary Waldenstrom\u2019s macroglobulinemia: a\xa0case report

BackgroundOur case of a patient with untreated lymphoplasmacytic lymphoma/Waldenstrom’s macroglobulinemia with extramedullary pleural effusion is the first documented case of pleural fluid MYD88 L265P mutation status in a community hospital setting. Our patient was intolerant to 420 mg ibrutinib, but still achieved a lasting complete remission, as defined by National Comprehensive Cancer Network guidelines, with a dose reduction to 240 mg of ibrutinib.Case presentationA 72-year-old Caucasian (white) man diagnosed with monoclonal immunoglobin M kappa lymphoplasmacytic lymphoma/Waldenstrom’s macroglobulinemia monitored without treatment for 2 years, presented with dyspnea and a left pleural effusion. At presentation, computed tomography scans of his chest, abdomen, and pelvis showed layering left pleural effusion and para-aortic lymphadenopathy. Pleural fluid cytology demonstrated B-cell lymphoma of the lymphoplasmacytic subtype, with monoclonal kappa B-cell population on flow and a positive MYD88 L265P mutation. The pleural effusion recurred post-thoracentesis and he achieved a lasting complete remission as defined by National Comprehensive Cancer Network guideline with 240 mg ibrutinib.ConclusionsOur discussion details a comprehensive literature review of extramedullary pulmonary involvement in Waldenstrom’s macroglobulinemia. Establishing a malignant etiology for pleural effusion in Waldenstrom’s macroglobulinemia can be challenging, as standard techniques may be insensitive. Allele-specific polymerase chain reaction for detecting MYD88 L265P mutations is more sensitive for confirming lymphoplasmacytic lymphoma/Waldenstrom’s macroglobulinemia in pleural fluid. Extramedullary pulmonary involvement usually presents post-diagnosis of Waldenstrom’s macroglobulinemia and responds well to Waldenstrom’s macroglobulinemia-directed treatment regimens. Allele-specific polymerase chain reaction is a sensitive assay for detecting MYD88 L265P mutations in pleural fluid to support the diagnosis of malignant pleural effusion in the setting of Waldenstrom’s macroglobulinemia and helps guide the treatment decision to use ibrutinib. Although intolerant of ibrutinib 420 mg, our patient achieved complete and sustained remission of pleural effusion with a dose of 240 mg with progression free survival of over 30 months.

Effectiveness of digital PCR for MYD88L265P detection in vitreous fluid for primary central nervous system lymphoma diagnosis.

Primary central nervous system lymphoma (PCNSL) is a rare type of primary extranodal lymphoma (PEL). MYD88L265P mutation has been observed in up to 75% of PCNSL cases, however, the validity and sensitivity of digital PCR in detecting this mutation remains to be elucidated. A total of 44 PCNSL patients, 15 diffuse large B-cell lymphoma not otherwise specified (DLBCL-NOS) patients and 13 other PEL patients were enrolled in the present study. The abilities of reverse transcription quantitative PCR (RT-qPCR) and droplet digital PCR (ddPCR) to detect the MYD88L265P mutation in cerebral spinal fluid (CSF) samples were compared. The results suggested that ddPCR showed superior mutation detection sensitivity when compared with RT-qPCR (58 vs. 15%; P<0.05). The MYD88L265P mutation was significantly associated with increased MYD88 protein overexpression in PCNSL brain tissue samples (P<0.05). Analysis of MYD88L265P mutation status in CSF and vitreous fluid samples using ddPCR may be a promising technique for minimally invasive confirmation of PCNSL diagnosis.

Clinical Relevance of the High Prevalence of MYD88 L265P Mutated Vitreoretinal Lymphoma Identified by Droplet Digital Polymerase Chain Reaction

ABSTRACT Purpose: To investigate the frequency and clinical relevance of missense mutation at position 265 changing leucine to proline in the myeloid differentiation factor 88 gene (MYD88 L265P) in the vitreous of Chinese patients with vitreoretinal lymphoma (VRL) using droplet digital polymerase chain reaction (ddPCR). Methods: Vitreous fluid (VF) from 29 eyes of 20 VRL patients at the North Huashan Hospital were included. MYD88 L265P analysis of VF was performed using ddPCR. Associations between clinicopathologic characteristics and MYD88 mutation were analyzed using t-test or Fisher’s exact test. Results: MYD88 L265P mutations were detected in 22 of 29 samples from 14 patients with diffuse large B-cell lymphomas and one patient with lymphoplasmacytoid lymphoma. However, no significant associations were found between MYD88 L265P mutation status and age, sex, lymphoma subtype or location of the primary lesion. Conclusion: The high prevalence of MYD88 L265P identified by ddPCR suggests that this method of evaluating the frequency of MYD88 L265P is a promising tool for accurate diagnosis of VRL.

Prognosis of Patients with Waldenström Macroglobulinemia: A Simplified Model

Prognosis of Patients with Waldenström Macroglobulinemia: A Simplified Model

Impact of MYD88L265P mutation Status on Histological Transformation of Waldenstrom Macroglobulinemia

Impact of MYD88L265P mutation Status on Histological Transformation of Waldenstrom Macroglobulinemia

MYD88 mutation status does not impact overall survival in Waldenström macroglobulinemia.

Waldenström macroglobulinemia (WM) is an immunoglobulin M-associated lymphoma, with majority of cases demonstrating MYD88 locus alteration, most commonly, MYD88L265P . Owing to low prevalence of the wild-type (WT) MYD88 genotype in WM, clinically relevant data in this patient population are sparse, with one study showing nearly a 10-fold increased risk of mortality in this subgroup compared to patients with MYD88L265P mutation. We studied a large cohort of patients with MYD88L265P and MYD88WT WM, evaluated at Mayo Clinic, Rochester, between 1995 and 2016, to specifically assess the impact of these genotypes on clinical course. Of 557 patients, MYD88L265P mutation status, as determined by allele-specific polymerase chain reaction, was known in 219, and 174 (79%) of those exhibited MYD88L265P , 157 of 174 patients had active disease. Of 45 (21%) patients with MYD88WT genotype, 44 had active disease. The estimated median follow-up was 7.0 years; median overall survival was 10.2 years (95% CI: 8.4-16.5) for MYD88L265P versus 13.9 years (95% CI: 6.4-29.3) for the MYD88WT (P = 0.86). The time-to-next therapy from frontline treatment and the presenting features were similar in the two patient populations. For patients with smoldering WM at diagnosis, the median time-to-progression to active disease was 2.8 years (95% CI: 2.2-3.8) in the MYD88L265P cohort and 1.9 years (95% CI: 0.7-3.1) in the MYD88WT cohort (P = 0.21). The frequency of transformation to high-grade lymphoma, or the development of therapy-elated myelodysplastic syndrome was higher in the MYD88WT cohort (16% versus 4% in the MYD88L265P , P = 0.009). In conclusion, MYD88L265P mutation does not appear to be a determinant of outcome, and its presence may not be a disease-defining feature in WM. Our findings warrant external validation, preferably through prospective studies.

Dexamethasone, rituximab and cyclophosphamide for relapsedand/or refractory and treatment-naïve patients with Waldenstrom macroglobulinemia.

The management of Waldenström macroglobulinaemia (WM) relies predominantly on small trials, one of which has demonstrated activity of dexamethasone, rituximab and cyclophosphamide (DRC) in the frontline setting. We report on the efficacy of DRC, focusing on relapsed/refractory (R/R) patients. Ibrutinib, a recently approved agent in WM demonstrated limited activity in patients with MYD88WT genotype. Herein, we additionally report on the activity of DRC based on the MYD88L265P mutation status. Of 100 WM patients evaluated between January 2007 and December 2014 who received DRC, 50 had R/R WM. The overall response rate (ORR) was 87%. The median progression-free survival (PFS) and time-to-next-therapy (TTNT) were 32 (95% confidence interval [CI]: 15-51) and 50 (95% CI: 35-60) months, respectively. In the previously untreated cohort (n=50), the ORR was 96%, and the median PFS and TTNT were 34months (95% CI: 23-not reached [NR]) and NR (95% CI: 37-NR), respectively. Twenty-five (86%) of 29 genotyped patients harbored MYD88L265P . The response rates and outcomes were independent of MYD88 mutation status. Grade ≥3 adverse effects included neutropenia (20%), thrombocytopenia (7%) and infections (3%). Similar to the frontline setting, DRC is an effective and well-tolerated salvage regimen for WM. In contrast to ibrutinib, DRC offers a less expensive, fixed-duration option, with preliminary data suggesting efficacy independent of the patients' MYD88 status.

Bendamustine and Rituximab Versus Dexamethasone, Rituximab and Cyclophosphamide in Patients with Waldenstrom Macroglobulinemia (WM)

The treatment approaches for Waldenstrom macroglobulinemia (WM) are largely based upon information from single-arm phase II trials, without comparative data. We compared the efficacy of two commonly used regimens in routine practice (bendamustine-rituximab (BR) and dexamethasone, rituximab plus cyclophosphamide (DRC)) and evaluated their activity with respect to the patients’ MYD88L265P mutation status. Of 160 consecutive patients, 60 received BR (43 with relapsed/refractory WM) and 100 received DRC (50 had relapsed/refractory WM). In the treatment-naive setting, overall response rate (ORR) was 93% with BR versus 96% with DRC (p = 0.55). Two-year progression-free survival (PFS) with BR and DRC was 88 and 61%, respectively (p = 0.07). In salvage setting, ORR was 95% with BR versus 87% with DRC, p = 0.45; median PFS with BR was 58 versus 32 months with DRC (2-year PFS was 66 versus 53%; p = 0.08). Median disease-specific survival was not reached with BR versus 166 months with DRC (p = 0.51). The time-to-event endpoints and depth of response were independent of the MYD88 mutation status. Grade ≥ 3 adverse events of both regimens were comparable. A trend for longer PFS was observed with BR although the regimens have comparable toxicities. The activity of BR and DRC appears to be unaffected by patients’ MYD88 mutation status.

Clone-specific MYD88 L265P and CXCR4 mutation status can provide clinical utility in suspected Waldenström macroglobulinemia/lymphoplasmacytic lymphoma

MYD88 L265P, a diagnostic marker for lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) can also be detected in other hematopoietic malignancies. We demonstrate a novel approach to increase the specificity of this marker for WM/LPL diagnosis by combining flow cytometric cell sorting with molecular analysis.Clonal B-lymphocyte and co-occurring clonal plasma cell populations of low-grade B-cell lymphomas were sorted by flow cytometry and analyzed for immunoglobulin gene rearrangements (PCR), and for MYD88 and CXCR4 mutations.Identical clonal origin was confirmed by PCR for 21 LPL/WM cases and MYD88 L265P was detected in both B-cell and plasma cell fractions. 9/20 other B-cell lymphomas with identical light chain restriction on B-cells and plasma cells were genotypically identical by PCR and MYD88 L265P was detected in both cell fractions in 7/9 whereas in 11/20 specimens with different clonal origin, MYD88 L265P was absent (5/11), or only found in B-lymphocytes (4/11), or plasma cells (2/11). CXCR4 mutations were detected in 17/39 cases, but missed in 63% of these without cell sorting.Confirming MYD88L265P in both B-cells and plasma cell fractions can provide a novel and powerful discriminator to distinguish LPL/WM from phenotypically similar disorders. Furthermore, this approach significantly increases CXCR4 detection sensitivity.

Lymphoplasmacytic Lymphoma With a Non-IgM Paraprotein Shows Clinical and Pathologic Heterogeneity and May Harbor MYD88 L265P Mutations.

Lymphoplasmacytic lymphoma (LPL) with non-immunoglobulin M (IgM) paraproteinemia remains poorly understood. The goal of this study was to investigate the clinicopathologic features of LPL in the bone marrow in patients with immunoglobulin G (IgG) or immunoglobulin A (IgA) paraproteins and evaluate MYD88 L265P mutation status to determine the relationship of these cases to Waldenström macroglobulinemia (WM). Bone marrows from LPL cases with IgG or IgA paraproteins diagnosed between January 1, 2007, and June 30, 2014, were retrieved from the clinical archive. Clinicopathologic features were retrospectively reviewed. MYD88 L265P mutation status was assessed by allele-specific polymerase chain reaction prospectively on all cases. Of 27 cases, four were reclassified as multiple myeloma, all MYD88 mutation negative. MYD88 L265P mutations were present in 10 (43%) of 23 remaining cases. No association between MYD88 status and bone marrow morphologic or phenotypic features, including the presence of Dutcher bodies, mast cells, expression of CD19 by plasma cells, or hemosiderin, was identified, although these features were present in a subset of cases, similar to WM. Clinical features of WM such as hyperviscosity were uncommon in this group and did not correlate with MYD88 status. Non-IgM LPLs are a clinically and pathologically heterogeneous group and often harbor MYD88 L265P mutation, albeit at a lower rate than classic WM. MYD88 status does not correlate with any specific pathologic or clinical manifestations.

MYD88 L265P Mutation Detection: Analysis of Flow Cytometry Sorted Plasma and Lymphoid Cell Clones Improves Sensitivity and Specificity for WM/LPL Diagnosis

Background:Waldenström’s macroglobulinemia (WM) and lymphoplasmacytic lymphoma (LPL) are lymphoproliferative disorders with bone marrow infiltration by clonal lymphoplasmacytic cells (Treon et al., 2003, 2005). The somatic point mutation L265P in the myeloid differentiation primary response gene 88 (MYD88) has been reported in more than 90% of WM patients (Treon et al., 2012). Therefore MYD88 mutation analysis has been implemented in clinical practice to support the diagnosis of LPL/WM.After the implementation of MYD88 L265P assays with increased detection sensitivity, a substantial portion of patients with IgM monoclonal gammopathy of undetermined significance (IgM-MGUS) was also reported MYD88 L265P positive. Splenic marginal zone lymphoma (SMZL), B-cell chronic lymphoproliferative disorders (B-CLPD) and diffused large B-cell lymphoma (DLBCL) have been found positive with much lower incidence rates (Varettoni et al., 2013; Xu et al., 2013).Hence the remaining need to differentiate WM/LPL from other lymphoproliferative disorders with co-occurring plasma cells with high confidence.Patients and Methods:In this study, flow cytometric cell sorting was utilized to isolate clonal plasma and B lymphoid cell fractions as previously described (Zehentner et al., 2011). 69 patient specimen fractions with a clinical history of WM /LPL, multiple myeloma, CLL and lymphoma were analyzed for MYD88 L265P mutation using Sanger sequencing. Furthermore, the Biomed-2 primer sets for the immunoglobulin heavy (IgH) and/or the immunoglobulin kappa light chain region (IgK) were used to compare B cell clonality profiles in the lymphoid versus plasma cell compartments.Results:MYD88 L265P mutation was detected in all specimens with confirmed Waldenström’s macroglobulinemia (17/17, 100%). Of these 16/17 (94%) revealed MYD88 L265P as well as identical monoclonality profile by gene rearrangement analysis in both the plasma and the B lymphoid cell collections. In 47% (8/17), the mutation was only detected in the plasma and B cell fractions, but not in the whole bone marrow specimens.21 patient specimens with a known clinical history of LPL and co-occurring clonal plasma cells were tested. 9 of 21 (43%) were categorized with identical B cell clonality profile when comparing plasma and B lymphoid cells; whereas 12 (57%) had unrelated clonality profiles. 7 of the 9 (78%) specimens in the identical clonality group tested positive for MYD88 L265P in both the plasma and B lymphoid clone. None of the unrelated clonality group specimens carried the mutation in both cell fractions; for 7/12 (58%) MYD88 L265P was found in either the plasma (2) or the B-cell fraction (5) whereas 5/12 (42%) tested negative.11 bone marrow aspirate specimen with known presence of lymphoma (including splenic marginal zone (SMZL), mantle cell and marginal zone) were analyzed. 10/11 (90%) tested negative for MYD88 L265P, with the exception of one SMZL specimen. Furthermore, 12 known myeloma, 5 CLL and 3 healthy donor specimens were analyzed, all tested negative.Conclusions:In this study, we developed and tested a novel approach to assess MYD88 L265P mutation status in order to assist WM/LPL diagnosis. Flow cytometric cell sorting for clonal plasma and B cell populations was combined with molecular analysis. Subsequently, MYD88 L265P mutation as well as B-cell clonality profile was compared in both cell fractions.Our study postulates a significant improvement in sensitivity and most importantly specificity when applying MYD88 L265P mutation status in combination with cell sorting for WM/LPL diagnostic decisions. 47% WM patients (8/17) and 44% LPL patients (4/9) were positive for the MYD88 mutation exclusively in both flow cytometry sorted cell fractions but not in whole bone marrow specimens. 94% (16/17) WM as well as 78% (7/9) LPL specimens with identical plasma and B-cell clonality profile revealed the presence of the MYD88 L265P mutation in both the plasma cell and the B lymphoid cell clones. Whereas LPL specimens with unrelated clonality profile of the plasma and lymphoid cell fractions as well as other control specimens (lymphoma, myeloma, CLL and healthy) either tested negative or positive only in one of the sorted cell fractions.We therefore conclude that confirming the presence of MYD88 L265P in both B-lymphoid and plasma cell fraction is an important prerequisite to distinguish LPL/WM from related disorders with high confidence. DisclosuresWang:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Burnworth:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Wentzel:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Ghirardelli:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Wells:HematoLogics Inc.: Employment, Equity Ownership. Loken:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.

Carfilzomib, rituximab, and dexamethasone (CaRD) treatment offers a neuropathy-sparing approach for treating Waldenström's macroglobulinemia

AbstractBortezomib frequently produces severe treatment-related peripheral neuropathy (PN) in Waldenström's macroglobulinemia (WM). Carfilzomib is a neuropathy-sparing proteasome inhibitor. We examined carfilzomib, rituximab, and dexamethasone (CaRD) in symptomatic WM patients naïve to bortezomib and rituximab. Protocol therapy consisted of intravenous carfilzomib, 20 mg/m2 (cycle 1) and 36 mg/m2 (cycles 2-6), with intravenous dexamethasone, 20 mg, on days 1, 2, 8, and 9, and rituximab, 375 mg/m2, on days 2 and 9 every 21 days. Maintenance therapy followed 8 weeks later with intravenous carfilzomib, 36 mg/m2, and intravenous dexamethasone, 20 mg, on days 1 and 2, and rituximab, 375 mg/m2, on day 2 every 8 weeks for 8 cycles. Overall response rate was 87.1% (1 complete response, 10 very good partial responses, 10 partial responses, and 6 minimal responses) and was not impacted by MYD88L265P or CXCR4WHIM mutation status. With a median follow-up of 15.4 months, 20 patients remain progression free. Grade ≥2 toxicities included asymptomatic hyperlipasemia (41.9%), reversible neutropenia (12.9%), and cardiomyopathy in 1 patient (3.2%) with multiple risk factors, and PN in 1 patient (3.2%) which was grade 2. Declines in serum IgA and IgG were common. CaRD offers a neuropathy-sparing approach for proteasome inhibitor-based therapy in WM. This trial is registered at www.clinicaltrials.gov as #NCT01470196.

Detection of MYD88 L265P in peripheral blood of patients with Waldenström’s Macroglobulinemia and IgM monoclonal gammopathy of undetermined significance

MYD88 L265P is highly prevalent in Waldenstrom's Macroglobulinemia (WM) and IgM monoclonal gammopathy of unknown significance (MGUS). We investigated whether MYD88 L265P could be identified by peripheral blood (PB) allele-specific PCR. MYD88 L265P was detected in untreated WM (114/118; 96.6%); previously treated WM (63/102; 61.8%); and IgM MGUS (5/12; 41.7%) but in none of 3 hyper-IgM or 40 healthy individuals. Median PB MYD88 L265P ΔCt was 3.77, 7.24, 10.89, 12.33 and 14.07 for untreated WM, previously treated WM, IgM MGUS, hyper-IgM and healthy individuals, respectively (P<0.0001). For the 232 IgM MGUS and WM patients, PB MYD88 L265P ΔCt moderately correlated to bone marrow (BM) disease (r=-0.3553; P<0.0001), serum IgM (r=-0.3262; P<0.0001) and hemoglobin (r=0.3005; P<0.0001) levels. PB MYD88 L265P ΔCt and serum IgM correlated similarly with BM disease burden. For positive patients, PB MYD88 L265P ΔCt was <6.5 in 100/114 (88%) untreated WM, and >6.5 in 4/5 (80%) IgM MGUS patients (P=0.0034). Attainment of a negative PB MYD88 L265P mutation status was associated with lower BM disease (P=0.001), serum IgM (P=0.019) and higher hemoglobin (P=0.004) levels in treated patients. These studies show the feasibility for detecting MYD88 L265P by PB examination, and the potential for PB MYD88 L265P ΔCt use in the diagnosis and management of WM patients.