L265P Mutation Research Articles

Tre-P-28 - Primary cutaneous B-cell lymphoma leg-type related to an anti-TNF-alpha therapy

<h3>Introduction:</h3> Drug-induced primary cutaneous B-cell lymphomas are rare. <h3>Observation:</h3> A 63 year old woman was referred for a lumbar cutaneous lesion which had been rapidly growing for 1 month. Her past medical history included multiple allergies and a rheumatoid arthritis which was treated with oral methotrexate 10 mg per week and adalimumab. Methotrexate had been taken for 8 years, while adalimumab had been initiated 6 months earlier. Physical examination revealed a solitary thick, violaceous plaque of 6 X 4 cm in diameters, without enlarged lymph nodes. No fatigue, fever or pruritus were present. The first clinical hypothesis was a cutaneous B-cell lymphoma. The histological examination of a skin biopsy specimen showed a monomorphous dermal proliferation of large centroblast-like cells with large round, sometimes irregular, nuclei. Only rare reactive small cells were present. The infiltrate spared the epidermis, with a Grenz zone. The tumor cells were positive for CD45, CD20, bcl2, bcl6, MUM1, highly proliferating (Ki67 95%), and negative for CD3, CD30, CD10 and EBV/EBER. No CD21 or CD23 positive follicular dendritic cell network was identified. Fluorescence in situ hybridization showed no BCL2, BCL6 or CMYC rearrangement, while high resolution melting identified the MYD88 L265P mutation. A B-cell rearrangement was detected in the lesion. Whole body FDG-PET/CT showed a single hypermetabolic activity corresponding to the cutaneous lombar lesion, without distant tumours. Although the Borrelia burgdorferi serology was negative, a 15 days course of doxycyline 200 mg daily was given. Once the diagnosis of primary cutaneous large B-cell lymphoma, leg type (PCLBCL-LT) was made, adamimumab (but not methotrexate) was discontinued. The tumor completely disappeared within 4 months. No other TNF inhibitors were later given. No recurrence was observed after a follow-up time of 4 years. <h3>Discussion:</h3> PCDLBCL-LT are aggressive B-cell lymphomas mainly (but not exclusively) located on the leg, characterized by a monomorphous proliferation of large CD20+, Bcl2+, Mum1+, CD10 - B cells, frequently exhibiting the MYD88 L265P mutation, as observed in our patient. In contrast with indolent subtypes of primary cutaneous B-cell lymphomas, spontaneous regressions in PCLBCL-LT are exceptional, and constantly followed by recurrences. Progression of undiagnosed cutaneous lymphomas after anti-TNF-alpha therapy (mainly cutaneous T-cell lymphomas misdiagnosed as psoriasis or eczema) have been reported [1]. However, the role of anti-TNF-alpha in the occurrence of new lymphoproliferative disorders remains controversial [2,3]. To our knowledge, no PCLBCL-LT related to TNF-alpha inhibitors has been reported to date. Although methotrexate may play a role as a cofactor for the development of this B-cell malignancy, the negative EBV staining and the long-term complete remission after adalimumab withdrawal while methotrexate was continued, strongly argue for a predominant role of the anti-TNF-alpha in the occurrence of this lymphoma.

OAB-039: Distinct immunoglobulin heavy-chain variable gene repertoire and its clinical relevance in Waldenström macroglobulinemia/lymphoplasmacytic lymphoma

<h3>Background</h3> Waldenström macroglobulinemia/lymphoplasmacytic lymphoma (WM/LPL) is a heterogeneous disease whose role of immunoglobulin heavy chain variable gene (IGHV) usage remains unknown. <h3>Methods</h3> To determine the clinical relevance of IGHV repertoire in WM/LPL treated with standard immunochemotherapy, we performed the immunoglobulin gene rearrangements and complementarity determining region 3 (CDR3) analysis on 136 patients with WM/LPL. <h3>Results</h3> The IGHV gene repertoire was remarkably biased in WM/LPL. IGHV3-23, IGHV4-34, IGHV3-30, IGHV3-7 and IGHV3-74 accounted for half of the whole cohort. Most of the cases were mutated (97.0%) using a 98% IGHV germline homology cutoff. IGHV3-30 was associated with long heavy chain CDR3, indicating the specific antigen selection in WM/LPL. Patients with IGHV3-7 were significantly more likely to harbor 6q deletion (p<0.001) and complex karyotype (p=0.002). The IGHV hypermutation rate in patients with MYD88 L265P mutation was significantly higher than wildtype patients (p=0.009). IGHV3-23 and IGHV3-74 segments were more frequently detected in MYD88 mutated WM/LPL patients (p=0.025). When we looked into the five predominant IGHV segments mentioned above, we found a trend that IGHV3-23 and IGHV3-74 segments were more frequently detected in MYD88 mutated WM/LPL patients (25.7% vs. 4.3%, p=0.025). Besides, we found IGHV3-7 and IGHV4-59 were represented more in MYD88 wildtype patients compared with MYD88 mutated patients (30.4% vs. 8.9%, p=0,005). IGHV4 segments were higher expressed in MYD88 wildtype patients compared with MYD88 L265P mutated patients (39.1% vs. 21.8%, P=0.083). Moreover, Patients with IGHV4 especially IGHV4-34 had higher level of LDH. IGHV4 was a predictive marker of shorter progression-free-survival. <h3>Conclusion</h3> WM/LPL appears to be composed of different subgroups based on the IGHV repertoire. The mutational status and the IGHV CDR3 length indicated the role for antigen selection in WM/LPL development. The presence of IGHV4 genes proved to be a potential risk factor associated with outcome which deserved further study.

IgM Plasma Cell Myeloma.

Immunoglobulin M plasma cell myeloma (IgMPCM) is a rare entity that is difficult to distinguish from other IgM-related neoplasms. The study aims to characterize the clinicopathologic features of IgMPCM, including MYD88 L265P and CXCR4 mutations. From our institutional archives, bone marrow biopsy specimens from January 1, 2008, to December 1, 2018, with monotypic plasma cells (PCs) expressing IgM that met current International Myeloma Working Group/World Health Organization criteria for PCM were included. Sanger sequencing was used to test for MYD88 L265P and WHIM-like CXCR4 mutations. Nine cases of IgMPCM were identified. Serum IgM paraproteins were detected in eight cases. CD138-positive PC burden averaged 41.9% (5%-80%). In four cases, PCs had lymphoplasmacytic morphology with cyclin D1 expression by immunohistochemistry. Three of four tested cases were positive for t(11;14) by fluorescence in situ hybridization, one with monosomy 13. The remaining case was positive for del13q14. All were negative for MYD88 L265P and WHIM-like CXCR4 mutations. Eight patients received immunochemotherapy, with four receiving autologous hematopoietic stem cell transplant. Median follow-up was 61 months (range, 11-120). All patients were alive except one. Distinguishing IgMPCM from other IgM-related disorders requires correlation with clinical, laboratory, and radiologic findings. Exclusion of MYD88 L265P and WHIM-like CXCR4 mutations may be useful to diagnose IgMPCM.

Waldenström's Macroglobulinemia: An Exploration into the Pathology and Diagnosis of a Complex B-Cell Malignancy.

After 77 years since the initial description, Waldenström macroglobulinemia (WM) remains as a bone marrow neoplastic disorder with lymphoplasmacytic differentiation oversecreting a monoclonal immunoglobulin M (IgM). However, many biological and genetic aspects of this entity have been unraveled and it is now easy to correctly diagnose patients with this illness. The diagnosis requires the presence of a monoclonal IgM component and bone marrow lymphoid infiltration must be demonstrated. In addition, other small B-cell lymphoid neoplasms with plasma cell differentiation must be discarded. Although the clinical picture is highly heterogeneous, the diagnosis is much easier today compared to the past, since now we can demonstrate the presence of somatic mutations, especially the L265P mutation in the MYD88 gene, highly characteristic of WM (>90% of the patients), followed by the WHIM-like mutations in the CXCR4 gene (~35%). The identification of these mutations is very important, because they can modulate the response to new treatments with Bruton’s tyrosine kinase (BTK) inhibitors. Thus, the conventional prognostic factors that predict the outcome of these patients (anemia, thrombopenia, high M component, high B2M, and advanced age), must be complemented with the genetic evaluation of the patient, that can help us in the prediction of the risk of transformation from asymptomatic to symptomatic forms (Del6q) and/or from indolent forms of the disease to aggressive lymphomas (CD79b mutations).

Characteristics of PD-L1 expression in tumor cells and tumor microenvironment of DLBCL with MYD88 L265P mutation

Objective: To study the effect of MYD88 L265P mutation on the expression of PD-L1 in tumor cells and tumor microenvironment in diffuse large B-cell lymphoma (DLBCL), and to provide theoretical basis for immunotherapy for patients. Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to detect the frequency of MYD88 L265P mutation in 72 cases of DLBCL diagnosed by pathologists in Cancer Hospital of Chinese Academy of Medical Sciences from August 2008 to May 2010. Expression of PD-L1 in tumor cells and tumor microenvironment in all samples was evaluated using PD-L1 (22C3) and PD-L1 (SP142) with Ventana automatic immunohistochemical (IHC) platform. The relationship between MYD88 L265P mutation and the expression of PD-L1 in DLBCL tumor cells and tumor microenvironment was assessed. Results: Of the 72 cases of DLBCL, MYD88 L265P mutation was detected in 15 (20.8%) cases. Nine cases with JAK2 amplification were excluded, and the remaining 63 cases of DLBCL were divided into MYD88 L265P mutant group (n=14) and MYD88 L265P wild-type group (n=49). IHC results showed that among the 14 cases of MYD88 L265P mutant groups, PD-L1 (22C3) was positive in 7 cases (7/14) of tumor cells and PD-L1 (SP142) was positive in 4 cases (4/14) of tumor microenvironment. Among the 49 cases of MYD88 L265P wild-type group, 9 cases (18.4%) were positive for PD-L1 (22C3) in tumor cells, and 38 cases (77.6%) were positive for PD-L1(SP142) in tumor microenvironment. In addition, among the 16 cases with PD-L1(22C3) expression in tumor cells, only 2 of the 7 cases with MYD88 L265P mutation were positive for PD-L1 (SP142) in tumor microenvironment. All 9 cases with wild-type MYD88 L265P were positive for PD-L1 (SP142) in tumor microenvironment. Statistical analysis showed that the expression level of PD-L1 (22C3) in tumor cells in the MYD88 L265P mutant group was significantly higher than that in the MYD88 L265P wild-type group (P=0.017). The expression level of PD-L1 (SP142) in tumor microenvironment in the MYD88 L265P mutant group was significantly lower than that in the MYD88 L265P wild-type group (P=0.001). Conclusions: MYD88 L265P mutation may play an important role in the regulation of PD-L1 expression in DLBCL tumor cells and tumor microenvironment. Further studies will provide a theoretical basis for immunotherapy of DLBCL patients with MYD88 L265P mutation.

High-affinity T-cell receptor specific for MyD88 L265P mutation for adoptive T-cell therapy of B-cell malignancies

BackgroundAdoptive transfer of engineered T cells has shown remarkable success in B-cell malignancies. However, the most common strategy of targeting lineage-specific antigens can lead to undesirable side effects. Also, a...

Molecular Characterization of Waldenström's Macroglobulinemia Patients Using Peripheral Blood Cell-Free Tumor DNA

BackgroundMutational characterization of Waldenström's macroglobulinemia (WM) currently relies on DNA from CD19-selected cells derived from bone marrow (BM) aspirates, which is an invasive technique, associated with significant patient discomfort. Peripheral blood cell-free tumour DNA (cf or ctDNA) has been recently shown to be a powerful non-invasive molecular biomarker in monitoring tumor status in several cancers. In this study, we aimed to characterize the mutational status of WM/IgM MGUS patients by using peripheral blood plasma-derived cfDNA and using matched tumor DNA (tDNA) from BM-CD19+ selected cells as a control, in order to determine whether peripheral blood , by means of cfDNA, can be used as an additional diagnostic tool in identifying the mutational profile of WM.MethodsA total of 30 consecutive patients with IgM monoclonal gammopathies were included in this study. All patients gave a written informed consent for sample collection and analysis. Peripheral blood (10-12mL) was collected in EDTA tubes and DNA was extracted using the MagMax cell free DNA isolation kit (ThermoFisher Scientific). cfDNA concentration was measured using the Qubit fluorometer. The quantities of cfDNA varied between 1.5 ng/μL and 8 ng/μL. BM aspirates were collected at the same time with peripheral blood, and were processed for CD19 enrichment, using CD19 magnetic beads (Miltenyi Biotech), followed by DNA extraction (Invitrogen). The paired tDNA and cfDNA samples from the 30 patients were analyzed for the MYD88 L265P and for CXCR4 mutations. The presence of L265P mutation was initially assessed by Allele-Specific PCR (AS-PCR) and then confirmed with direct sequencing. The presence of CXCR4 mutations was assessed with direct sequencing.ResultsThe study included patients with symptomatic WM before initiation of any therapy (N=4) , with WM under therapy (N=4) or after completion of therapy (N=12), with asymptomatic WM (N=8) and with IgM MGUS (N=2). The median age of the patients was 70 years (range 22-87). The median bone marrow infiltration of the tested samples was 28% (range 5 to 90%) and median IgM levels were 1510 mg/dL (range 80 to 7310 mg/dL).In all, but one patient, (29/30) the L265P mutation was detected in both cfDNA and tDNA (100% concordance). This mutation was initially detected by AS-PCR which was validated by direct sequencing in all patients. Assessment of CXCR4 mutation status was feasible in 29 patients; in one patient CXCR4 sequencing was unsuccessful in both cfDNA and tDNA. Mutations in CXCR4 were detected in 24% (7/29) of patients. The pathogenic mutation S338X (rs104893626) was present in 2 (7%) patients and was identified in both cfDNA and tDNA. In one patient the L50X (1/29) truncating mutation was found in tDNA but not in cfDNA. Two patients harbored the F29L (2/29) mutation and another two (2/29) the P31T mutation, both in cfDNA and in tDNA; these two mutations have not been previously reported. Synonymous mutations were also found in 10% of the tested patients in both cfDNA and tDNA. Thus, the concordance for CXCR4 mutations identified by direct sequencing of cfDNA and tDNA was 97% (in 28/29 patients).ConclusionsOur study demonstrates that in patients with IgM monoclonal gammopathies peripheral blood cell-free DNA can be used as a non-invasive method to detect the MYD88 L265P mutation with excellent concordance to BM CD19+ derived tumor DNA. Furthermore, CXCR4 mutations could also be detected with high concordance to tDNA by direct sequencing. This method could be more cost effective, as it avoids the expenses and time needed for CD19 selection, either BM or peripheral blood derived. Peripheral blood cfDNA could be used as an initial test to assess the presence of MYD88 L265P and CXCR4 mutations, so that BM testing can be limited to patients with indeterminate results. Updated results will be presented in the meeting. Further investigation is ongoing in order to assess tumor dynamics using peripheral blood. DisclosuresTerpos:Janssen: Honoraria, Research Funding; Genesis/Celgene: Honoraria, Other: DMC member, Research Funding; Abbvie: Honoraria; Takeda: Honoraria, Other: SC member; Amgen: Honoraria, Other: SC member, Research Funding; GSK: Honoraria; BMS: Honoraria. Kastritis:Prothena: Honoraria; Janssen: Honoraria; Genesis pharma: Honoraria; Takeda: Honoraria; Amgen: Honoraria, Research Funding. Dimopoulos:Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology: Consultancy, Honoraria, Other: Advisory Committee: Amgen Inc, Celgene Corporation, Janssen Biotech Inc, Onyx Pharmaceuticals, an Amgen subsidiary, Takeda Oncology; Novartis: Consultancy, Honoraria; Genesis Pharma: Research Funding.

Detection of MYD88 L265P Mutation By Next Generation Deep Sequencing in Peripheral Blood Mononuclear Cells of Waldenstrom Macroguloblinemia and IgM Monoclonal Gammopathy of Undetermined Significance

Introduction: Whole genome sequencing has recently revealed MYD88 L265P somatic mutations in Waldenstrom macroguloblinemia (WM) tumor cells. Allele-specific polymerase chain reaction (AS-PCR) using bone marrow (BM) aspirate and peripheral blood (PB) is the most frequently used technique to detect the specific mutation. In search for a non-invasive, simple, and more sensitive detection of MYD88 L265P mutation, we performed next generation deep sequencing (NGS) technique using unselected peripheral blood mononuclear cells (PBMCs) collected from WM and IgM monoclonal gammopathy of undetermined significance (IgM MGUS) patients. The main purpose of this study is to assess the clinical implication of MYD88 L265P mutation burden detected by NGS technique using unselected PBMCs.Patients and methods: The study was approved from the Institutional Review Board. After informed consent was obtained in accordance with the Declaration of Helsinki, PBMCs were collected from 38 patients with WM and 20 patients with IgM MGUS. The median time from diagnosis to PBMC collection was 2.2 years (range, 0-25 years). Twenty-nine (76.3%) of WM patients were previously treated. The overall response (CR+PR+MR) rate at the time of analysis was 75.8% (22/29). Genomic DNA was extracted from unselected PBMCs and 300 ng of DNA was used for NGS analysis. Variation rates of MYD88 at the nucleotide position corresponding to L265P were measured with an Illumina MiSeq DNA sequencer by molecular barcoding technology essentially as previously reported (Nature Protocols 12, 664:2017). PCR products were sequenced on a MiSeq DNA sequencer with a Reagent kit v2. for 300 cycles. Serial dilution assessment was carried out to investigate the sensitivity of the method by analyzing the DNA of BM sample from a MYD88 mutation positive patient diluted in DNA from a healthy control.Results: Dilution assessment demonstrated that NGS technique was detectable to a sensitivity of 0.02%, ×5 higher than previously reported sensitivity by AS-PCR. MYD88 L265P mutation was detected from 6/9 (66.7%), 8/29 (27.6%), and 11/20 (55%) in patients with untreated WM, previously treated WM, and IgM MGUS, respectively. Among the patients who were positive forMYD88 L265P mutations, the median percentage of mutant allele relative to WT was 3.0% (0.14-32.3%), 0.24% (0.02-33.8%), and 0.55% (0.06-2.85%), in untreated WM, previously treated WM, and IgM MGUS, respectively. Patients with Untreated WM and those with lymphadenopathy showed a trend toward higher mutant allele burden in PBMCs, but the difference was not statistically significant. Among 26 previously treated WM patients, those with at least minimal response showed significantly lower detection rate of MYD88 L265P mutation (13.6 vs. 75.0%, P=0.02) and mutant allele burden (median, 0.00% vs. 0.23%, P=0.01), compared to patients with less than minimal response.Conclusion: This study is the first to report NGS analysis of MYD88 L265P mutation using unselected PBMCs of WM and IgM MGUS patients. The sensitivity for MYD88 L265P mutation in unselected PBMCs from IgM MGUS patients was comparable with that detected by AS-PCR using CD19-selected PB cells (Leukemia, 2014;28:1698-704). Although its specificity is in need for further investigations, NGS technique successfully detected the mutation carried by minimal tumor cells included in unselected PBMCs with high sensitivity as AS-PCR using CD19-selected PB cells. The mutation detection rate of previously treated WM patients was lower than that of untreated patients in our study and previously reported cohort; however, this in turn may potentially indicate that NGS detection of MYD88 L265P mutation in PBMC be clinically utilized in assessing treatment response by monitoring tumor burden during patients' clinical course. DisclosuresNo relevant conflicts of interest to declare.

Mast Cell Density and Its Clinical Relevance in Waldenstrom's Macroglobulinemia

Background. Waldenström's macroglobulinemia (WM) is characterized by a lymphoplasmacytic infiltration of the bone marrow (BM) along with a serum monoclonal IgM and includes MYD88 L265P and CXCR4 mutations in around 90% and 25% respectively (Treon, NEJM 2012; Hunter, Blood 2014). CXCR4WHIMmutations confer worse outcome and are associated with larger BM involvement and less adenopathies (Treon, Blood 2014). One hallmark and histological criteria for WM diagnosis is the presence of numerous mast cells (MC) segregating with tumor cells in the BM (Waldenström, Acta Haematologica 1958). MC have been shown to support lymphoplasmacytic cell growth, through CD154/CD40 signaling (Tournilhac, Ann Oncol 2006), but there is so far no clear demonstration of the prognostic impact of BM MC density in WM.Aim. We proposed to investigate BM MC density by using sensitive and specific digital quantification, allowing the analysis of large tumor cell infiltrated BM area, in order to assess its clinical relevance in WM.Methods. A total of 65 WM patients were investigated, including 54 pts at diagnosis and 11 at relapse. Mutational status could be determined in 35 pts: allele specific PCR found MYD88 L265P mutation in 32/35 pts (91%) and deep next-generation analysis found CXCR4WHIMmutation in 7/35 (20%) pts. Diagnostic and treatment criteria for WM fulfilled recommendations of the 8th international WM workshop (Castillo, BJH 2016 ; Leblond, Blood 2016). MC density was explored using a digital tool previously used for quantifying immune cells infiltrates on tumor tissues sections (Galon, Science 2006). Tryptase and CD20 immunohistochemisty stainings were done on contiguous sections of deparaffinized BM trephine biopsies. After numerization of each section, BM surface area was manually marked out, excluding bone framework and adipocytes, in order to limit subsequent analyses to hematopoietic tissue. We then defined optimal thresholds for horseradish peroxidase signal detection. MC were counted up with “Immunoscore module” (Definiens Developer XT), on the hematopoietic tissue of the whole BM section : MC density was automately recorded as the number of tryptase positive cells per unit of selected bone marrow tissue surface area.Results. MC density was found to be heterogeneous over BM, with a mean of 106 MC.mm-2 of hematopoietic tissue (Figure 1). Higher MC density (> 100 MC.mm-2) was associated with larger BM involvement by WM cells (BM deep infiltration (p=0.039) and diffuse tumor pattern (p=0.003)) and with less frequent spleen involvement (p=0.011) and a trend towards less lymph node (p=0.072) and liver (p=0.054) involvement. Higher MC density was also associated with features of advanced disease such as anemia (<115g.L-1 ; p = 0.041), thrombocytopenia (<100G.L-1 ; p=0.01) and lower albumin rate (<35g.L-1 ; p=0.03)). Furthermore MC density was 194 MC.mm-2 in patients with high IPSS, 82.4 MC.mm-2 in those with intermediate IPSS and 67.9 MC.mm-2 in those with low IPSS (p=0.0003). Regarding outcome, patients with higher MC density had a shorter median OS (56.5 months vs non reached in patients with low MC density, p = 0.0139)) (Figure 2). Among the pts scheduled to receive a 1st line treatment for an active disase (n=43) and molecularly characterized (n=20), those with CXCR4WHIM mutations had a significantly higher MC density (240.7 MC.mm-2vs 90.9 MC.mm-2 for CXCR4 WT pts ; p=0.0221).Conclusion. By using specific digital tool on well-outlined hematopoietic tissue surface, MC density can be accurately measured in WM patients. High MC density is associated with aggressive features, poor clinical outcome and possibly with CXCR4WHIM mutation. [Display omitted] DisclosuresYsebaert:Janssen: Consultancy, Research Funding, Speakers Bureau. Leblond:Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Honoraria; Novartis: Honoraria; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hermine:Hybrigenics: Research Funding; INatherys: Equity Ownership, Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; Novartis: Research Funding; Celgene: Research Funding. Guieze:GILEAD: Other: Educational Presentation; ABBVIE: Other: Educational Presentation; JANSSEN: Other: Educational Presentation. Tournilhac:Janssen: Honoraria, Other: travel funding; AMGEN: Other: Travel funding, Research Funding; ROCHE: Honoraria, Other: Travel funding, Research Funding; GILEAD: Honoraria, Other: Travel Funding, Research Funding; Abbvie: Honoraria, Other: Travel funding.

Characterization of Chromosome 6q Deletions in MYD88 Mutant and Wild-Type Waldenström's Macroglobulinemia

Introduction: MYD88 L265P mutations have been described in more than 90% of Waldenström's Macroglobulinemia (WM) patients. MYD88 wild-type (wt) WM represent less than 10% of WM, and associate with shorter overall survival versus MYD88 mutated (mut) WM, as well as with lack of major responses and inferior progression-free survival to ibrutinib. Mutated MYD88 triggers WM cells growth and survival through NF-kB activation via IRAK1/4, BTK and HCK transactivation. CXCR4 mutations promote pro-survival AKT and ERK1/2 signaling and are associated with delayed response to ibrutinib. Deletions involving the long arm of chromosome 6 (Del6q) are highly recurrent in various B-cell malignancies, particularly those that are driven by mutated MYD88, and have been observed in up to half of WM patients. Chr6q genomic loss in WM affect important modulators of NFkB (TNFAIP3, HIVEP2), BCL2 protein family (BCLAF1), apoptosis and IGF1/PI3K/AKT signaling (FOXO3), and BTK (IBTK). The frequency and impact of Del6q on the different patient genotype remains to be clarified.Patients and Methods: Based on location and known regulation of key pathways in WM, copy number alterations (CNA) and gene expression level were assessed for IBTK, FOXO3, BCLAF1, TNFAIP3 and HIVEP2 genes, along with CXCR4 transcriptional levels. DNA and RNA from CD19+ sorted BM lymphoplasmacytic cells from 32 untreated WM and one IgM and IgG-secreting lymphoplasmacytic lymphoma patients were analyzed. Paired CD19-depleted peripheral-blood mononuclear cells (PBMCs) were used as germline controls. Paired CD19+ and CD19- PBMCs from 6 healthy donors were analyzed to rule out possible B-cell specific findings. CNA were measured in quadruplicate and gene expression in triplicate using TaqMan real-time polymerase chain reaction (RT-PCR) assays. Deletions affecting less than 20% of WM cells were considered to be beneath the RT-PCR detection threshold. The cohort included 33 patients (21 males, 12 females), with a median age of 62 (range 35-91) years, BM involvement of 60% (range 2.5-90%), serum IgM levels of 3010 (range 257-6910) mg/dl, and hemoglobin of 10.9 (range 8.4-14.4) g/dl. MYD88 L265P mutations were detectable in 25 (76%) patients, 11 of whom (44%) also carried CXCR4WHIM mutations. Eight patients (24%) were MYD88 and CXCR4 wt.Results: Somatic Del6q were observed in 4/8 (50%) and in 20/25 (80%) patients in the MYD88 wt and mut population, respectively (p=NS). In contrast, no CNA were present in the HDs CD19+/CD19- compartments. IBTK remained intact in all 8 MYD88 wt WM, whereas resulted deleted in 13/25 (52%) MYD88 mut patients (p=0.01). BCLAF1 deletion frequency was significantly different among the two subgroups, with only 2/8 deleted MYD88wt (25%) and 19/25 (76%) deleted MYD88 mut patients (p=0.02; Figure 1). Eight MYD88 mut WM (32%) showed contiguous fully clonal Del6q, that spanned all studied genes in 7/8 (87.5%) cases; in contrast, contiguous subclonal deletions occurred only in 3/12 (25%) cases (p=0.02). All 8 fully clonal Del6q patients were CXCR4 wt; in contrast, CXCR4 mut were detected in 9/12 (75%) patients with subclonal Del6q (p=0.001; Figure 2). Unlike the MYD88 mut population, all 4 MYD88 wt patients presented with non-contiguous subclonal deletions (p=NS). Fully clonal deletions of IBTK, BCLAF1 and HIVEP2 significantly reduced the respective gene transcriptional levels (p= 0.03, 0.01 and 0.01, respectively), while CNA in TNFAIP3 and FOXO3 did not affect their expression. The MYD88 wt patients showed significant decreases in the transcriptional levels of BCLAF1 related to gene subclonal deletions, and reduced CXCR4 levels compared to MYD88 mut WM (p=0.02 and p=0.02, respectively).Conclusions: Our findings demonstrate that gene loss of IBTK, FOXO3, BCLAF1, TNFAIP3 and HIVEP2 occurs in most WM patients, but at a lower frequency in MYD88 wt. Unlike the MYD88 mut patients, those MYD88 wt showed no contiguous fully clonal deletions. The latter correlate with CXCR4 wt and reduce only IBTK, BCLAF1 and HIVEP2 levels, suggesting that regulatory mechanisms may compensate for FOXO3 and TNFAIP3 gene loss. Significant variation in IBTK and BCLAF1 deletion rate suggest that BCR and BCL2 signaling may play a different role in MYD88 mut and wt WM. The findings provide valuable insights into MYD88 wt WM pathogenesis, and may be relevant to understanding therapeutic outcome with agents that target MYD88, CXCR4, BTK and BCL2. [Display omitted] DisclosuresCastillo:Millennium: Research Funding; Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Arcaini:Pfizer, Celgene, Bayer, Roche: Membership on an entity's Board of Directors or advisory committees; Celgene, Roche, Sandoz: Consultancy; Gilead: Research Funding. Varettoni:Jannsen: Consultancy, Other: travel expenses. Treon:Pharmacyclics: Consultancy, Research Funding.

Differential Diagnosis of Suspected MYD88 Wild-Type Waldenstrom's Macroglobulinemia Includes Other Malignant and Non-Malignant IgM Secreting Entities, Including Frequent Cases of IgM Myeloma

MYD88 mutations are present in 95% of patients with Waldenstrom's Macroglobulinemia (WM), and permit diagnostic discrimination from other overlapping IgM-secreting B-cell malignancies. However, such diagnostic discrimination can often be difficult among suspected WM patients with wild-type MYD88 (MYD88WT), and can result in either diagnostic misclassification or mistreatment. We therefore examined a relatively large cohort of patients with suspected MYD88WT WM, and performed a systematic review of their clinical, pathologic and laboratory studies. Sixty-four patients with suspected MYD88 wild-type WM were identified. All had a monoclonal IgM protein, and bone marrow (BM) disease involvement. AS-PCR testing for MYD88 L265P mutation, and exclusion of non-L265P mutations by Sanger sequencing was performed to confirm MYD88WT status utilizing CD19-selected and unselected BM mononuclear cells (Xu et al, Blood 2013). WHO and WM consensus guidelines were used to establish clinicopathological diagnosis.Forty-six (71.8%) patients fulfilled WHO and WM consensus criteria for WM. Their median BM involvement was 37.5% (range 5-95%), serum IgM was 2,980 (range 160-9,000 mg/dL), and hemoglobin was 11 (range 4-15.5 g/dL). Sixteen (35%) and 13 (28%) had adenopathy and splenomegaly, respectively. Principal reasons for diagnosis included anemia (50%), and/or other cytopenias (13%), peripheral neuropathy (13%), adenopathy (8%), symptomatic hyperviscosity (5%), and renal failure (5%). At a median follow-up of 5.4 years, 31 (67%) patients required therapy. Notably, 7 (15.2%) MYD88WT patients were diagnosed with diffuse large B-cell lymphoma (DLBCL) during the follow-up period. Eleven (24%) patients died, 4 due to DLBCL transformation. Among the other MYD88WT patients, 7 (10.9%) had IgM multiple myeloma (IgM MM). Their median BM disease involvement was 60% (range 10%-80%), with a predominant plasma cell infiltrate. In 3(43%) patients, both clonal B-cells and plasma cells were detected, while clonal plasma cells alone were observed in the other 4 patients. FISH testing of BM mononuclear cells showed t(11;14) in 5 patients; t(14;16) in one patient; and normal cytogenetics in one patient. The median serum IgM level was significantly higher at 8,375 (range 2,530-12,000 mg/dL) versus MYD88WT WM patients (p=0.016). One (14%) patient had both adenopathy and splenomegaly. Principal reasons for diagnosis included anemia (100%), and symptomatic hyperviscosity (43%). With a median follow-up of 2.4 years, all patients required treatment and one died of myeloma. Findings consistent with marginal zone lymphoma (MZL) were observed in 6 (9%) patients. Their median BM involvement was 10% (range 5-25%), and three (50%) had detectable clonal B-cells by BM flow cytometry. Their median serum IgM level was 1,642 (95-2,800 mg/dL), and was not significantly different versus MYD88WT WM patients. Adenopathy and splenomegaly were present in 4 (67%) and 2 (33%) patients. With a median follow-up of 3.8 years, 4 (67%) required therapy, and all remain alive. For 3 (4.6%) patients, the findings favored a diagnosis of IgM MGUS with plasma cell infiltration. The median BM involvement for these patients was 5% (range 5-8%) with flow cytometry demonstrating clonal plasma cells. The median serum IgM level for these patients was 1,846 (range 1,262-2,390 mg/dL), and did not differ versus MYD88WT WM patients. With a median follow-up of 2.5 years, none progressed or received treatment, and are alive. In one patient, the findings favored the diagnosis of CLL while another patient had intravascular DLBCL. The serum IgM level was 1,822 and 355 mg/dL respectively. The BM disease burden was 5% in both patients. The CLL patient remains alive without treatment, while the patient with DLBCL required therapy and remains alive. No DLBCL transformation was observed among non-WM cases. Our findings show that up to 30% of suspected MYD88WT WM cases have an alternative clinicopathological diagnosis that includes both malignant and non-malignant entities. IgM MM was frequently present among suspected MYD88WT WM cases, and presented with very high serum IgM levels, symptomatic hyperviscosity and chr. 14 translocations. Patients with MYD88WT WM disease had a high incidence of DLBCL transformation that contributed to a third of deaths. The findings highlight the importance of diagnostic discrimination among suspected MYD88WT WM cases. DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding. Castillo:Abbvie: Research Funding; Millennium: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding.

Somatic Variants Associated with MDS/AML Are Present in MYD88 Mutated Waldenstrom's Macroglobulinemia

Background: The Rapid Heme Panel (RHP) is a targeted amplicon-based custom next-generation sequencing used as a clinical assay to identify mutations in 95 genes related to hematological malignancies that include acute leukemias (AML, ALL), myelodyplastic syndromes (MDS), and B-cell lymphoproliferative disorders. Clonal hematopoiesis of indeterminate potential (CHIP) has recently been shown to be an age-related precursor to MDS when blast or dysplasia criteria are not met for a formal MDS diagnosis. The RHP is used to identify the top somatic mutated genes closely associated with CHIP or MDS, and includes MYD88 and CXCR4 variant testing for Waldenstrom's Macroglobulinemia (WM). Materials and Methods: Sample processing and sequencing of bone marrow (BM) samples from WM patients was used for isolation of genomic DNA for RHP. Amplicons were designed to cover 250bp and 95 genes. Analyses were performed by identifying and separating somatic variants from those seen as germline. Variants with read percentage depth of 50% were presumed to be of germline origin. Variants with read percentages greater than the mutated MYD88 read percentage depth were identified as potential CHIP or MDS clonal variants. These variants were validated using Sanger sequencing using CD19-selected and CD19-depleted BM mononuclear cells. Mutant variants were annotated against Ensembl Variant Effect Predictor (VEP) database. Results: Three-hundred thirty-six WM patients were included in this analysis. The median age for these patients was 66.7 (range 31-93 years). The median BM disease involvement for these patients was 35%. RHP identified the MYD88 L265P mutation in 267 (80%) patients. The remaining patients were either unmutated (n=34), or lacked MYD88 reads (n=35). Therefore the percentage of patients with MYD88 L265P among those with MYD88 reads was 88.7%. CXCR4 C-terminal domain mutations were also observed in 55/267 (20.6%) WM patients with MYD88 L265P mutations. In addition to MYD88 and CXCR4, variants in 66 other genes contained within the RHP profile were identified that included variants in genes associated with MDS/AML in 9 (3.3%) patients. The median age for these patients was 64 (range 44-80 years) and included variants at: EP300S1400N, a histone acetyltransferase regulating transcription (n=1); TET2T1270fs, a methylcytosine dioxygenase that converts methylcytosine to 5-hydroxymethylcytosine (n=1); and DNMT3AV338fs, DNMT3AY584fs, DNMT3AG587fs, and DNMT3AL754R, a DNA methyltransferase that represses transcription (n=4). Variants in DNMT3BR97C, DNMT3BE39Q, and DNMT3BA384T (n=3). Sanger sequencing of BM mononuclear cells confirmed the somatic, non-CD19 origin for DNMT3A and TET2. Four of 9 patients were previously untreated, and two received alkylator based therapy previously. None exhibited BM morphological evidence of MDS or AML. Conclusions: Infrequently variants related to MDS/AML are present in BM of patients with WM, including previously untreated patients. These variants occur in the absence of BM morphological evidence of MDS, and may be consistent with CHIP. DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding.

TP53 mutations Are Associated with Mutated MYD88 and CXCR4, and Confer an Adverse Outcome in Waldenström Macroglobulinemia

Background: Whole genome sequencing identified highly recurrent somatic mutations in Waldenström macroglobulinemia (WM). Activating somatic mutations in MYD88 and CXCR4 are present in 90-95% and 30-40% of WM patients, respectively, and impact disease presentation, treatment outcome, and overall survival. In contrast, somatic mutations in the tumor suppressor gene TP53 occur less frequently (7%), and the clinical significance remains to be delineated. TP53 aberrations confer an adverse prognosis in related B-cell malignancies, such as chronic lymphocytic leukemia and multiple myeloma, predicting for treatment resistant and inferior survival. As such, we sought to characterize the clinical implications, as well as the clonal architecture, of somatic TP53 mutations in WM. Methods: We searched our database for WM patients with a TP53 mutation identified by a clinical next generation sequencing (NGS) assay using unsorted bone marrow (BM) samples. To validate the findings, CD19+ cells from BM aspirates were isolated, and DNA was extracted for mutational analysis. CD19-depleted peripheral blood (PB) mononuclear cells were used as normal paired samples. All samples were screened for MYD88, CXCR4, and TP53 mutations by Sanger sequencing, and zygosity was determined by establishing the ratio of mutant versus wile-type (WT) allele expression. TP53 copy number was determined using TaqMan Copy Number Assays (Applied Biosystems, Grand Island, NY, USA). Medical files were manually reviewed to gather pertinent clinical data. Results: Fourteen WM patients (14/265; 5.3%) had a TP53 mutation detected by a clinical NGS assay. Sanger sequencing identified somatic TP53 mutations within the WM clone in six patients, including one patient with two somatic mutations. Three patients had a TP53 mutation identified in both CD19+ and CD19- sorted samples, 4 patients were WT, and 1 patient did not have DNA for validation. No recurrent variants were identified. Among the six patients with validated somatic TP53 mutations, four (67%) had biallelic inactivation of TP53 . Three patients had a homozygous TP53 mutation determined by Sanger sequencing and one patient had deletion of TP53 . All six patients harbored both a CXCR4 mutation (4 nonsense, 2 frameshift) and the MYD88 L265P mutation, of which 4 patients (67%) had mutated homozygous MYD88 . At the time the TP53 mutation was detected, the median BM involvement and hemoglobin level was 80% and 9.2 g/dl, respectively. The median serum IgM level was 2,508 mg/dl, and two patients had symptomatic hyperviscosity. Five patients (83%) were high-risk according to the International Prognostic Scoring System for WM. Three patients were untreated, 2 patients were refractory to their most recent therapy (i.e. bortezomib, dexamethasone, rituximab), and 1 patient was relapsing. Four patients went on to receive systemic therapy, to which two patients were refractory. Two patients were treated with ibrutinib and both obtained a major response. After a median follow-up 7.7 months, 2 patients (33%) have died due to progressive disease; both patients had biallelic inactivation of TP53 . Conclusion: Somatic TP53 mutations occur concurrently with both MYD88 and CXCR4 mutations in WM patients and appear to confer an aggressive disease course. Our findings highlight the need to understand the cell-specific origin of mutations detected by clinical NGS assays. DisclosuresCastillo:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

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.

Relationship between Polymorphism of miR-155 and Its Target Gene MyD88 and Clinicopathological Features of Diffuse Large B-cell Lymphoma

To investigate the relationship between the polymorphism of miR-155 and its target gene MyD88 and clinicopathological features of diffuse large B-cell lymphoma (DLBCL). 135 cases of DLBCL patients in our hospital from March 2015 to August 2017 were selected, and 90 cases of reactive hyperplasia of lymph nodes were selected as the control group. The relative expression of miR-155 and MyD88 gene polymorphism were detected in the two groups, and the relationship between miR-155 and MyD88 gene polymorphism and clinicopathological characteristics of DLBCL was analyzed. The relative expression of miR-155 in DLBCL patients was significantly higher than that in the control group (P<0.05). The mutation rate of MyD88 L265P in DLBCL group was significantly higher than that in control group (P<0.05). The relative expression of miR-155 in patients with MyD88 L265P mutation was significantly higher than that in patients with wild-type DLBCL (P<0.05). The relative expression of miR-155 and the polymorphism of MyD88 L265P were associated with lesion location, stage, BCL-2 protein expression and MyD88 protein expression in DLBCL patients (t=7.461、8.804、6.487、10.812； χ2=10.681、8.599、7.251、23.008；P<0.05). The survival of DLBCL patients with low miR-155 expression was significantly better than that with high miR-155 expression (P<0.05). The survival of wild-type DLBCL patients with MyD88 L265P locus was significantly better than that of mutant DLBCL patients (P<0.05). There was a significant positive correlation between the relative expression of miR-155 and the expression of MyD88 protein (r=0.428, P=0.000). The abnormal expression of miR-155 and the mutation rate of MyD88 gene in DLBCL patients are increased, and the expression of miR-155 and the mutation of MyD88 gene affect the disease progression and prognosis of patients, which may be potential biological indicators for the diagnosis, treatment and prognosis of DLBCL.

Integrative Statistics, Machine Learning and Artificial Intelligence Neural Network Analysis Correlated CSF1R with the Prognosis of Diffuse Large B-Cell Lymphoma

Tumor-associated macrophages (TAMs) of the immune microenvironment play an important role in the Diffuse Large B-cell Lymphoma (DLBCL) pathogenesis. This research aimed to characterize the expression of macrophage colony-stimulating factor 1 receptor (CSF1R) at the gene and protein level in correlation with survival. First, the immunohistochemical expression of CSF1R was analyzed in a series of 198 cases from Tokai University Hospital and two patterns of histological expression were found, a TAMs, and a diffuse B-lymphocytes pattern. The clinicopathological correlations showed that the CSF1R + TAMs pattern associated with a poor progression-free survival of the patients, disease progression, higher MYC proto-oncogene expression, lower MDM2 expression, BCL2 translocation, and a MYD88 L265P mutation. Conversely, a diffuse CSF1R + B-cells pattern was associated with a favorable progression-free survival. Second, the histological expression of CSF1R was also correlated with 10 CSF1R-related markers including CSF1, STAT3, NFKB1, Ki67, MYC, PD-L1, TNFAIP8, IKAROS, CD163, and CD68. CSF1R moderately correlated with STAT3, TNFAIP8, CD68, and CD163 in the cases with the CSF1R + TAMs pattern. In addition, machine learning modeling predicted the CSF1R immunohistochemical expression with high accuracy using regression, generalized linear, an artificial intelligence neural network (multilayer perceptron), and support vector machine (SVM) analyses. Finally, a multilayer perceptron analysis predicted the genes associated with the CSF1R gene expression using the GEO GSE10846 DLBCL series of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP), with correlation to the whole set of 20,683 genes as well as with an immuno-oncology cancer panel of 1790 genes. In addition, CSF1R positively correlated with SIRPA and inversely with CD47. In conclusion, the CSF1R histological pattern correlated with the progression-free survival of the patients of the Tokai series, and predictive analytics is a feasible strategy in DLBCL.

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.

Waldenström macroglobulinemia: 2021 update on diagnosis, risk stratification, and management.

Waldenström macroglobulinemia (WM) is a lymphoplasmacytic lymphoma with immunoglobulin M (IgM) monoclonal protein. Clinical features include anemia, thrombocytopenia, hepatosplenomegaly, lymphadenopathy, and rarely hyperviscosity. Presence of IgM monoclonal protein associated with ≥10% clonal lymphoplasmacytic cells in bone marrow confirms the diagnosis. The L265P mutation in MYD88 is detectable in more than 90% of patients and is found in the majority of IgM MGUS patients. Age, hemoglobin level, platelet count, β2 microglobulin, LDH and monoclonal IgM concentrations are characteristics that are predictive of outcomes. Not all patients who fulfill WM criteria require therapy; these patients can be observed until symptoms develop. Rituximab-monotherapy is inferior to regimens that combine it with bendamustine, an alkylating agent, a proteosome inhibitor, or ibrutinib. Purine nucleoside analogues are active but usage is declining in favor of less toxic alternatives. The preferred Mayo Clinic induction is rituximab and bendamustine. Bortezomib, fludarabine, thalidomide, everolimus, Bruton Tyrosine Kinase inhibitors, carfilzomib, lenalidomide, and bendamustine have all been shown to have activity in relapsed WM. Given WM's natural history, reduction of therapy toxicity is an important part of treatment selection.

Lymphoplasmacytic lymphoma and a CD5+ lymphoproliferative process presenting as composite disease in the bone marrow: a report of two cases

Correct subclassification of B cell lymphoma requires integration of histologic and immunohistochemical findings in addition to results of flow cytometric immunophenotyping, cytogenetic analysis, and molecular studies. Evaluation for the MYD88 L265P mutation and immunohistochemical staining for LEF1 can be helpful in the workup of small B cell lymphomas. In this case report of two diagnostically difficult cases, we present two patients with lymphoplasmacytic lymphoma (LPL) and a CD5+ lymphoproliferative process presenting as composite disease within the bone marrow. In both cases, integration of ancillary studies and results of biopsy at other sites of disease in addition to a careful histologic review and an expanded immunohistochemistry panel were important for the correct final classification.

MYD88 L265P mutation in primary central nervous system lymphoma is associated with better survival: A single-center experience.

BackgroundThe myeloid differentiation primary response gene (MYD88) mutation in primary central nervous system lymphomas (PCNSL) may be associated with unfavorable prognosis; however, current evidence remains limited. We aimed to characterize PCNSLs by integration of clinicopathological, molecular, treatment, and survival data.MethodsWe retrospectively identified and validated 57 consecutive patients with PCNSLs according to the 2017 WHO classification of lymphoid neoplasms over 13 years. Formalin-fixed paraffin-embedded tumor samples underwent polymerase chain reaction assay to detect MYD88 mutation. We used Cox regression for survival analysis, including age, treatment, and MYD88 as covariates. We searched the literature for studies reporting demographics, treatment, MYD88, and survival of PCNSL patients and incorporated individual patient data into our analyses.ResultsThe median age was 66 years and 56% were women. All 57 patients had PCNSL of non-germinal center cell subtype and the majority (81%) received either single or combined therapies. There were 46 deaths observed over the median follow-up of 10 months. MYD88 mutation status was available in 41 patients of which 36 (88%) were mutated. There was an association between MYD88 mutation and better survival in the multivariable model (hazard ratio [HR] 0.277; 95% confidence interval [CI]: 0.09-0.83; P = .023) but not in a univariable model. After incorporating additional 18 patients from the literature, this association was reproducible (HR 0.245; 95% CI: 0.09-0.64; P = .004).ConclusionsAdjusting for confounders, MYD88-mutant PCNSL appears to show improved survival. While further validation is warranted, detection of MYD88 mutation will aid the identification of patients who may benefit from novel targeted therapies.