Cutaneous lymphoplasmacytic lymphoma with MYD88 L265P mutation, bone marrow involvement, and paraproteinaemia

Abstract Lymphoplasmacytic lymphoma and marginal zone lymphoma in the bone marrow: paratrabecular involvement as an important distinguishing feature Assia Bassarova, Gunhild Tr¿en, Signe Spetalen, Francesca Micci, Anne Tierens, Delabie Abstract Lymphoplasmacytic lymphoma (LPL) is a neoplasm of small B-lymphocytes, lymphoplasmacytoid and plasma cells involving bone marrow and sometimes lymph nodes and spleen. Lymphoplasmacytic lymphoma is often accompanied by Waldenström macroglobulinemia. Since the original description, Waldenström macroglobulinemia has become recognized as a distinct clinicopathological entity defined by serum IgM paraprotein and bone marrow involvement by lymphoplasmacytic lymphoma. Since serum IgM paraprotein in itself is not specific and can be seen in a variety of small B-cell lymphoproliferative disorders, notable chronic lymphatic leukemia and marginal zone lymphoma, as well as in rare cases of myeloma, the diagnosis of Waldenström macroglobulinemia rests largely upon the proper diagnosis of LPL in the bone marrow. The differential diagnosis between bone marrow involvement by lymphoplasmacytic lymphoma (LPL) and marginal zone lymphoma (MZL) is challenging because histology and immunophenotype of both diseases overlap. The diagnosis may be helped by demonstrating the MYD88 L265P mutation, seen in most LPL. However, the mutation is also present in MZL, although at a lower frequency. To better define the distinguishing features of LPL we studied a series of bone marrow trephine biopsies of 59 patients with Waldenström's macroglobulinemia (WM) without extramedullary involvement and compared the findings with bone marrow biopsies from 23 patients with well-characterized MZL who also had bone marrow involvement. H&E and immunoperoxidase-stained sections of bone marrow trephine biopsies as well as flow cytometry and classical cytogenetics performed on aspirations were reviewed. The study was complemented with MYD88L265P mutation analysis on the bone marrow trephine biopsies of all patients. The features are summarized in Table 1. The most distinguishing features of LPL with respect to MZL were focal paratrabecular involvement (p<0.001), the presence of lymphoplasmacytoid cells (p<0.001), Dutcher bodies (p<0.001), increased numbers of mast cells (p<0.001) and the MYD88L265P mutation (p<0.001). Other features such as sinusoidal infiltration and immunophenotype were not distinguishing. Table 1. Summary of the pathology features of lymphoplasmacytic and marginal zone lymphoma in bone marrow trephine biopsies Lymphoplasmacytic lymphoma Marginal zone lymphoma p Infiltration pattern* Paratrabecular Nodular non-paratrabecular Paratrabecular and non-paratrabecular Intrasinusoidal Diffuse 37% (10/27) 0% (0/27) 56% (15/27) 37% (10/27) 0% (0/27) 0% (0/16) 75% (12/16) 0% (0/16) 37% (6/16) 25% (4/16) <0,001 <0,001 <0,001 1 0,015 Cytology Small lymphoid cells Plasmacytoid cells Plasma cells Dutcher nuclear inclusions Mast cells 100% (59/59) 100% (59/59) 93% (55/59) 90% (53/59) 87% (49/56) 100% (23/23) 0% (0/23) 78% (18/23) 0% (0/23) 9% (2/23) - <0,001 0,108 <0,001 <0,001 Immunophenotype of the lymphoma CD20 CD138 (plasma cells) CD5 CD23 IgK IgL IgM IgG Focal CD21+ or CD23+ follicular dendritic cell network in the stroma 100% (59/59) 88% (50/57) 21% (12/52) 29% (15/51) 81% (48/59) 19% (11/59) 97% (57/59) 3% (2/59) 20% (10/51) 100% (23/23) 80% (12/15) 0% (0/23) 13% (5/23) 26% (5/19) 10% (2/19) 64% (7/11) 0% (0/11) 48% (11/23) - - 0,014 0,580 - - - - 0,024 MYD88 L265P mutation 96% (45/47) 20% (3/15) 0,001 *the analysis was only performed on bone marrow trephine biopsies showing less than 66% lymphoma infiltration In conclusion, LPL can reliably be distinguished from MZL in the bone marrow by using a combination of pathology characteristics. In contrast to other studies, our findings stress the diagnostic importance of paratrabecular infiltration in LPL. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Response to ibrutinib of a refractory IgA lymphoplasmacytic lymphoma carrying the MYD88 L265P gene mutation

Mediterranean Journal of Hematology and Infectious Diseases ◽

10.4084/mjhid.2019.057 ◽

2019 ◽

Vol 11 (1) ◽

pp. e2019057 ◽

Cited By ~ 1

Author(s):

Francesca Maria Quaglia ◽

Gian Matteo Rigolin ◽

Elena Saccenti ◽

Massimo Negrini ◽

Eleonora Volta ◽

...

Keyword(s):

Computed Tomography ◽

Bone Marrow ◽

Ct Scan ◽

Progressive Disease ◽

Performance Status ◽

Lymphoplasmacytic Lymphoma ◽

Genetic Studies ◽

Transfusion Independence ◽

Myd88 L265p Mutation ◽

Myd88 L265p

In 2014 a 66 year-old woman presented with anemia and an IgAk monoclonal spike. Bone marrow (BM) biopsy showed 80% lymphocytes and lymphoplasmacytoid cells. Computed Tomography (CT) scan documented neither adenopathy nor splenomegaly. Diagnosis of IgA lymphoplasmacytic lymphoma was made. After three lines of treatment, progressive disease with adenopathies, splenomegaly and ascites were documented on a CT scan. Our patient developed thrombocytopenia, transfusion-dependent anemia and clinical deterioration. We performed genetic studies of peripheral blood lymphoctyes with NGS approach. Given the identification of MYD88 L265P mutation, in February 2018 our patient started ibrutinib. Hb and PLT improved from day +35. In July 2018 no ascites and 50% reduction of adenopathies and spleen were shown on a CT scan. In April 2019 the patient was still on ibrutinib with transfusion independence and good performance status.

Download Full-text

MYD88 Negative Waldenstrom Macroglobulinemia Has Distinct Clinical & Biological Features As Compared To Its MYD88 Mutant Counterpart

Blood ◽

10.1182/blood.v122.21.4299.4299 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4299-4299

Author(s):

Nikhil V Patkar ◽

Prashant Deshpande ◽

Russel Mascarenhas ◽

PG Subramanian ◽

Prashant Tembhare ◽

...

Keyword(s):

Bone Marrow ◽

Partial Response ◽

Conflicts Of Interest ◽

International Prognostic Index ◽

Post Treatment ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Myd88 L265p Mutation ◽

Myd88 L265p

Abstract Introduction Waldenstrom Macroglobulinemia (WM) harbors a mutation in MYD88 gene (MYD88L265P) with frequencies varying from 67% to 91%. Although of diagnostic use its clinical significance in terms of prognosis and treatment response is unclear. We retrospectively analyzed WM for MYD88 L265P mutation, immunogenetic profile (presence of somatic hypermutations and biased gene usage) & correlated these with standard clinical variables including prognosis and patient outcome. Patients & Methods 32 cases WM (diagnosed as per WHO 2008/2001 criteria) were retrospectively accrued from 2007-2013. Genomic DNA extracted from bone marrow aspirate smears was subjected to an allele specific oligonucleotide PCR to detect the MYD88L265P mutation using fluorescently labeled primers followed by capillary electrophoresis. Immunogenetics was assessed in 29 patients. Clonal FR1/FR2 regions of the VH gene were amplified & sequenced. Sequence data was compared to the closest germline sequences on NCBI & IMGT databases. Laboratory variables (Hb, WBC, platelet, M Protein concentration, S. IgM, b2M level, S. Globulin, LDH, %of lymphoplasmacytic lymphocytes) were evaluated at baseline along with the International Prognostic Index (ISSWM). Response evaluation was done as per VIth International Consensus guidelines after treatment as well as at last follow up. 2-tailed Student's t-Test & Chi squared test were applied for statistical analysis. Results Median age was 60 years (range: 46-77), male predominant (87.5%).Majority of patients had cytopenia (90.6%) of one or more blood lineages. Median IWSSM was 3 (n=26). The median follow up was 21.5 months (range: 1 week to 82 months). Majority of patients were treated with cyclophosphamide/vincristine/prednisone ± rituximab (55.1%), followed in others by bendamustine/rituximab(13.8%) or fludarabine/cyclophosphamide/rituximab,(13.8%) or cyclophosphamide/thalidomide/dexamethasone (10.3%). MYD88 L265P mutation was found in 84.3% (27/32) of patients. The immunogenetic results here pertain only to samples with productive IGHV gene rearrangement [22/29 (∼76%) cases]. 96% of cases revealed somatic hypermutations. 59% of cases showed a biased use for the VH3 gene followed by VH4 (22.7%) and VH1 (18.18%). The commonest gene used was IGHV3-7 (27.3%) followed by IGHV1-18 (18.2%). Clinical features separating MYD88 negative from MYD88 mutated WM are seen in Table 1. MYD88 negative WM presented with lower number of infiltrating tumor cells in the bone marrow (p=0.05), older age (p=0.02) and had a lower IWSSM score at presentation (p=0.03) as compared to mutated WM. Majority of the MYD88 negative group were in VGPR,(very good partial response) or CR (complete response) (75%:VGPR/CR) post treatment as compared to MYD88 mutated patients [21%: VGPR/CR, 31.6%: PR (partial response): 26.3%, SD (stable disease):15.8%, PD (progressive disease):6.3%]. At the last follow up 44.4% of MYD88 mutated WM had PD where as no patient in MYD88 WT had changed their initial post treatment status. Two patients with MYD88 mutation died due to disease related complications. Conclusion Our data indicates that WM is a biologically heterogeneous subset dichotomized by MYD88 mutations. WM patients with MYD88 mutations present at younger age with high tumor burden in the bone marrow, high risk of progression and poor therapeutic response. Although limited in number, MYD88 negative WM patients were not associated with PD as compared to the mutated group. Overall MYD88mutation may be considered as an adverse prognostic factor in WM. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

MYD88 L265P mutation analysis helps define nodal lymphoplasmacytic lymphoma

Modern Pathology ◽

10.1038/modpathol.2014.120 ◽

2014 ◽

Vol 28 (4) ◽

pp. 564-574 ◽

Cited By ~ 42

Author(s):

Fatima Hamadeh ◽

Stephen P MacNamara ◽

Nadine S Aguilera ◽

Steven H Swerdlow ◽

James R Cook

Keyword(s):

Mutation Analysis ◽

Lymphoplasmacytic Lymphoma ◽

Myd88 L265p Mutation ◽

Myd88 L265p

Download Full-text

Disruption of MYD88 Pathway Signaling Leads to Loss of Constitutive IRAK1, NF-κβ and JAK/STAT Signaling and Induces Apoptosis of Cells Expressing the MYD88 L265P Mutation in Waldenstrom's Macroglobulinemia

Blood ◽

10.1182/blood.v118.21.597.597 ◽

2011 ◽

Vol 118 (21) ◽

pp. 597-597 ◽

Cited By ~ 1

Author(s):

Guang Yang ◽

Yangsheng Zhou ◽

Xia Liu ◽

Yang Cao ◽

Zachary Hunter ◽

...

Keyword(s):

Bone Marrow ◽

B Cells ◽

Healthy Donor ◽

Kinase Activity ◽

Conflicts Of Interest ◽

Signaling Proteins ◽

Downstream Signaling ◽

Myd88 L265p Mutation ◽

Myd88 Pathway ◽

Myd88 L265p

Abstract Abstract 597 Introduction: Through whole genome sequencing of bone marrow lymphoplasmacytic cells (LPCs) from WM patients, we recently identified a broadly expressed somatic variant (MYD88 L265P) which was present in 90% of WM patients, as well as in BCWM.1 and MWCL-1 WM cells. The presence of this variant has been reported to confer oncogenic activity in cell lines derived from patients with DLBCL ABC subtype lymphomas by induction of IRAK 1/4 kinase activity, and downstream signaling via NF-κβ (Ngo et al, Nature 2011, 470:115-119). We therefore sought to delineate the activity of agents which either disrupt MYD88 homodimerization, a prerequisite to IRAK 1/4 auto-phosphorylation, or by direct inhibition of IRAK 1/4 kinase activity in WM. Patients and Methods: Primary WM LPCs were isolated from bone marrow biopsy specimens from WM patients, and CD19+ B-cells were isolated from the peripheral blood of healthy donors. The mutational status of MYD88 was identified by Sanger sequencing, which confirmed the presence of the L265P mutation in BCWM.1 and MWCL-1 cells, and absent in Ramos and MM1S cells. Western blot and phospho-flow studies using phospho-specific antibodies were performed for signaling proteins downstream of MYD88, cell killing evaluated by Annexin V/PI staining and AlamarBlue, and IgM and IL-6 release determined after treatment with and without inhibitors of MYD88 homodimerization (IMG-2005-5, IMGENEX, San Diego CA), and IRAK 1/4 kinase function (407601, EMD, Gibbstown, USA). Results: Phosphorylation of the MYD88 downstream signaling proteins IRAK1, IκBα, NFκβ-p65 and STAT3 was greater in MYD88 L265P expressing BCWM.1 and MWCL-1 cells, and primary WM patient LPC, versus MYD88 wild type expressing Ramos, MM1S, and healthy donor CD19+ B-cells. Inhibition of MYD88 homodimerization and IRAK 1/4 kinase activity led to decreased phosphorylation of IRAK1, IκBα, NFκβ-p65 and STAT3 in BCWM.1 and MWCL-1 cells. Importantly, both inhibitors induced robust apoptosis of BCWM.1 and MWCL-1 cells, and primary WM LPCs bearing the MYD88 L265P mutation in comparison to Ramos, MM1S, or healthy donor CD19+ B-cells. Activation of caspase-3 and PARP, decreased BCL-2 expression, and decreased release of IL-6 and IgM accompanied treatment of WM cells following treatment with inhibitors of MYD88 homodimerization and/or IRAK 1/4 kinase activity. Conclusion: Disruption of MYD88 pathway signaling leads to loss of constitutive IRAK1, NF-κβ and JAK/STAT activation and induces apoptosis of cells expressing the MYD88 L265P mutation in WM. The results of this study provide novel insights into the pathogenesis of WM, and provide a framework for the clinical investigation of inhibitors of the MYD88 pathway in WM. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Fast Detection Of MYD88-L265P Mutation By PCR-RFLP In Chronic Lymphoproliferative Disorders

Blood ◽

10.1182/blood.v122.21.5076.5076 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5076-5076

Author(s):

Nikoletta Argentou ◽

George Vassilopoulos ◽

Maria Ioannou ◽

Anastasios Germenis ◽

Matthaios Speletas

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Direct Sequencing ◽

Genetic Defect ◽

Lymphoproliferative Disorders ◽

Neoplastic Disease ◽

Reliable Technique ◽

Pcr Rflp ◽

Myd88 L265p Mutation ◽

Myd88 L265p

Abstract Introduction Waldenstrom’s macroglobulinemia (WΜ) is an incurable disorder characterized by bone marrow infiltration from neoplastic lymphoplasmacytic B-lymphocytes and monoclonal IgM production. Recent data suggest a possible causal relationship of MYD88-L265P mutation with the pathogenesis of the disease (Treon Sp et al. N Eng J Med 2012;367:826-33). Additional studies have conformed that MYD88-L265P is characteristic of WΜ, but it is also rarely present in other chronic lymphoproliferative disorders (LPDs) (Jiménez C et al. Leukemia 2013,doi: 10.1038/leu.2013.62; Varettoni M et al. Blood 2013;121:2522-8). The purpose of this study was to analyse the prevalence of the aforementioned mutation in patients with WM and other LPDs, using a fast and reliable polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol. Methods Genomic DNA was extracted from the bone marrow of 11 patients with WΜ, 12 patients with B-cell chronic lymphocytic leukemia (CLL), 8 with splenic lymphoma with villous lymphocytes (SLVL). Furthermore, consecutive samples of peripheral blood and bone marrow isolated CD19+ cells, derived from a patient with monoclonal IgM gammopathy of undetermined significance (MGUS-IgM), were also retrospectively analysed for the presence of MYD88-L265P defect. The detection of the mutation was performed by PCR amplification of the MYD88 exon 5 region, followed by RFLP analysis, since the presence of the mutation results in the generation of BsiEI restriction enzyme site. PCR-RFLP results were also confirmed by direct sequencing of purified CD19+cells. Results The MYD88-L265P mutation was detected in 10 patients with WΜ (90.9%), in 1 patient with SLVL with markers of lymphoplasmatocytoid differentiation (12.5%) and was absent in all CLL patients. Interestingly, our PCR-RFLP protocol exhibited a greater sensitivity than direct sequencing, when applied to total bone marrow cells (12.5% vs 25%). Moreover, the patient with MGUS-IgM displayed the MYD88-L265P mutation in isolated CD19+cells of both bone marrow and peripheral blood in all consecutive samples and remains in a stable condition for the last 7 years. Conclusion PCR-RFLP is a rapid, sensitive and reliable technique for the detection of MYD88-L265P mutation in patients with WM and lymphomas with lymphoplasmatocytoid differentiation. Additionally, the presence of MYD88-L265P mutation in MGUS-IgM in the absence of disease progression for many years, suggests that this genetic defect alone is not sufficient to lead to overt neoplastic disease. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Detection of the MYD88 L265P mutation in Waldenström’s macroglobulinemia using a highly sensitive allele-specific PCR assay.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.8042 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 8042-8042

Author(s):

Lian Xu ◽

Zachary R Hunter ◽

Guang Yang ◽

Yang Cao ◽

Xia Liu ◽

...

Keyword(s):

Bone Marrow ◽

Sanger Sequencing ◽

Pcr Assay ◽

Specific Pcr ◽

Allele Specific ◽

Myd88 L265p Mutation ◽

Specific Pcr Assay ◽

Allele Specific Pcr ◽

Waldenström's Macroglobulinemia ◽

Myd88 L265p

8042 Background: Waldenström’s macroglobulinemia (WM) is a B-cell malignancy characterized by bone marrow (BM) infiltration with lymphoplasmacytic cells and production of an IgM paraprotein. By whole genome sequencing, we recently identified a somatic mutation (L265P) in the MYD88 gene in 27/30 (90%) WM patients (Treon et al, ASH 2011). To expand this finding for possible diagnostic testing, we developed an allele-specific PCR assay for MYD88 L265P and evaluated this assay in a large cohort of WM patients. Methods: An allele-specific PCR assay was developed with a threshold of detection of 0.125% for MYD88 L265P. DNA from bone marrow aspirates from 99 patients with the clinicopathological diagnosis of WM was used for assessment of MYD88 L265P expression by both allele-specific PCR and Sanger sequencing. Findings were correlated with clinical parameters using ANOVA. Results: We observed that 85/99 (86%) WM patients were positive for MYD88 L265P using the allele-specific PCR assay. Of the 85 allele-specific PCR positive patients, 81 demonstrated a detectable mutation peak by Sanger sequencing. All 14 allele-specific PCR negative patients remained negative by Sanger sequencing. By the allele-specific PCR assay, MYD88 L265P positive patients showed greater bone marrow involvement, higher serum IgM and lower serum IgA and IgG levels versus MYD88 L265P negative patients (p<0.008). Conclusions: MYD88 L265P is highly expressed in BM samples of WM patients using an allele-specific PCR assay, and is associated with greater bone marrow disease burden and serum IgM levels. Use of allele-specific PCR provides a simple and sensitive diagnostic tool for detection of the MYD88 L265P mutation.

Download Full-text

Lymphoplasmacytic Lymphoma and Waldenström Macroglobulinemia

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2012-0034-rs ◽

2013 ◽

Vol 137 (4) ◽

pp. 580-585 ◽

Cited By ~ 30

Author(s):

Nadia Naderi ◽

David T. Yang

Keyword(s):

Bone Marrow ◽

B Cell ◽

Bone Marrow Involvement ◽

Accurate Diagnosis ◽

Immunoglobulin M ◽

Low Grade ◽

Lymphoplasmacytic Lymphoma ◽

Waldenström Macroglobulinemia ◽

Waldenstrom Macroglobulinemia ◽

Definition Of

Lymphoplasmacytic lymphoma (LPL) is a low-grade, B-cell neoplasm composed of small lymphocytes, plasmacytoid lymphocytes, and plasma cells that typically involve the bone marrow, and it is associated with an immunoglobulin M (IgM) gammopathy. The definition of Waldenström macroglobulinemia (WM) and its relationship to LPL has been confusing in the past. In addition, the diagnosis of LPL itself can be challenging because LPL lacks disease-specific morphologic, immunophenotypic, and genetic features to differentiate it from other mature B-cell neoplasms. Accurate diagnosis of LPL/WM rests on recognition of the differential diagnostic features between LPL and other diagnostic possibilities and the use of the recently refined definition of WM and its relationship with LPL: The presence of an IgM monoclonal gammopathy of any level in the setting of bone marrow involvement by LPL. This review summarizes the clinical, laboratory, and histologic features of LPL/WM, with particular emphasis on unique aspects of LPL/WM that may aid in accurate diagnosis.

Download Full-text

MYD88 L265P mutation analysis is a useful diagnostic adjunct for lymphoplasmacytic lymphoma with pleural effusion

Pathology International ◽

10.1111/pin.12854 ◽

2019 ◽

Vol 69 (10) ◽

pp. 601-607

Author(s):

Shien‐Tung Pan ◽

Ren Ching Wang ◽

Chun‐Chi Kuo ◽

Yen‐Chuan Hsieh ◽

Ying‐Zhen Su ◽

...

Keyword(s):

Pleural Effusion ◽

Mutation Analysis ◽

Lymphoplasmacytic Lymphoma ◽

Myd88 L265p Mutation ◽

Myd88 L265p

Download Full-text

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

Blood ◽

10.1182/blood.v124.21.1645.1645 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1645-1645

Author(s):

Zhixing Wang ◽

Wayne Fritschle ◽

Richard Bennington ◽

Bettina Burnworth ◽

Angela Bennington ◽

...

Keyword(s):

Bone Marrow ◽

B Cell ◽

Lymphoid Cell ◽

Employment Equity ◽

Myd88 L265p Mutation ◽

Equity Ownership ◽

B Lymphoid ◽

Cell Fractions ◽

Myd88 L265p ◽

Cell Clonality

Abstract 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. Disclosures Wang: 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.

Download Full-text