Rare Circulating Cells in Familial Waldenstrom Macroglobulinemia Displaying the MYD88 L265P Mutation Are Enriched by Epstein-Barr Virus Immortalization.
ABSTRACT: The MYD88 L265P is a recurrent somatic mutation in neoplastic cells from patients with Waldenström Macroglobulinemia (WM). We identified the MYD88 L265P mutation in three individuals from unrelated families, but its presence did not explain the disease segregation within these WM pedigrees. We observed the mutation in these three individuals at high allele fractions in DNA extracted from EBV-immortalized Lymphoblastoid cell lines established from peripheral blood (LCL), but at much lower allele fractions in DNA extracted directly from peripheral blood, suggesting that this mutation is present in a clonal cell subpopulation rather than of germ-line origin. Furthermore, we observed that the MYD88 L265P mutation is enriched in WM families, detected in 40.5% of patients with familial WM or MGUS (10/22 WM, 5/15 MGUS), compared to 3.5% of patients with familial MM or MGUS (0/72 MM, 4/41 MGUS) (p = 10-7). The mutant allele frequency increased with passages in vitro after immortalization with Epstein-Barr virus (EBV) consistent with the MYD88 L265P described gain-of-function proposed for this mutation. The MYD88 L265P mutation appears to be frequently present in circulating cells in patients with WM, and MGUS, and these cells are amenable to immortalization by EBV.
Project description:We investigated the feasibility of using next-generation sequencing (NGS) technique using molecular barcoding technology to detect MYD88 L265P mutation in unselected peripheral blood mononuclear cells (PBMCs) in 52 patients with Waldenström's macroglobulinemia  and 21 patients with IgM-monoclonal gammopathy of undetermined significance (MGUS). The NGS technique successfully detected the MYD88 L265P in unselected PBMCs at a sensitivity of 0.02%, which was ×5 higher than that of AS-PCR. All the results between paired BM and PB samples from 2 IgM MGUS and 4 untreated WM patients matched completely. MYD88 L265P mutation was detected in 14/21 (66.7%), 14/19 (73.7%), and 10/33 (30.3%) with the median mutant allele burden of 0.36% (range, 0.06-2.85%), 0.48% (range, 0.02-32.3%), and 0.16% (range, 0.02-33.8%), in IgM-MGUS, untreated WM, and previously treated WM, respectively. Multiple linear regression analysis identified an absolute peripheral lymphocyte count as the positive predictor of PB mutant allele burden (R2 = 0,72, P<0.0001). Our non-invasive, simple NGS method has the potential to detect MYD88 L265P mutations in PBMCs of IgM MGUS and WM patients, which may especially utilized for monitoring minimal residual tumor burden after treatment.
Project description:MYD88 mutations are one of the most recurrent mutations in hematologic malignancies. However, recent mouse models suggest that MYD88<sub>L265P</sub> alone may not be sufficient to induce tumor formation. Interplay between MYD88<sub>L265P</sub> and other genetic events is further supported by the fact that TNFAIP3 (A20) inactivation often accompanies MYD88<sub>L265P</sub>. However, we are still lacking information about the consequence of MYD88<sub>L265P</sub> in combination with TNFAIP3 loss in human B cell lymphoma. Review of our genetic data on diffuse large B cell lymphoma (DLBCL) and Waldenstrom macroglobulinemia (WM), found that a large percentage of DLBCL and WM cases that have a MYD88 mutation also harbor a TNFAIP3 loss, 55% DLBCL and 28% of WM, respectively. To mimic this combination of genetic events, we used genomic editing technology to knock out TNFAIP3 in MYD88<sub>L265P</sub> non-Hodgkin's lymphoma (NHL) cell lines. Loss of A20 expression resulted in increased NF-?B and p38 activity leading to upregulation of the NF-?B target genes BCL2 and MYC. Furthermore, we detected the increased production of IL-6 and CXCL10 which led to an upregulation of the JAK/STAT pathway. Overall, these results suggest that MYD88<sub>L265P</sub> signaling can be enhanced by a second genetic alteration in TNFAIP3 and highlights a potential opportunity for therapeutic targeting.
Project description:Primary central nervous system lymphoma (PCNSL) patients have a poorer prognosis than systemic lymphoma. Gain-of-function <i>MYD88</i> c.794T?>?C (p. L265P) mutation and programed cell death-1 (PD-1) pathway alterations are potential targetable pathways. Our study objective was to determine the clinicopathologic correlates of <i>MYD88</i> mutation and PD-1 alterations in PCNSL and the impact of Epstein-Barr virus (EBV) infection. We studied 53 cases including 13 EBV-associated (EBV<sup>pos</sup>) PCNSL, 49% harbored <i>MYD88</i> mutation, none seen in EBV<sup>pos</sup> PCNSL. MYD88 protein expression did not correlate with <i>MYD88</i> mutation. T-cell and macrophage infiltration was common. All PD-L1-positive tumors were EBV<sup>pos</sup>. Two PD-L1 positive tumors showed 9p24.1/PD-L1 locus alterations by Fluorescence <i>In Situ</i> Hybridization. T cells and macrophages expressed PD-1 and/or PD-L1 in 98% and 83% cases, respectively. <i>MYD88</i> mutation or protein expression and PD-1 or PD-L1 expression did not predict outcome. We hypothesize that EBV<sup>pos</sup> PCNSL has a distinct activation mechanism, independent of genetic alterations.
Project description:The precise clinicopathologic significance of myeloid differentiation primary response gene (MYD88) L265P mutation in diffuse large B-cell lymphomas (DLBCLs) remains elusive. To investigate the frequency and clinicopathologic significance of the MYD88 L265P mutation in DLBCLs, we conducted a meta-analysis of 40 published studies on 2736 DLBCL patients. We collected relevant published research findings identified using the PubMed and Embase databases. The effect sizes of outcome parameters were calculated using a random-effects model. In this meta-analysis, the MYD88 L265P mutation in DLBCL showed a significant difference according to tumor sites. The overall incidence of the MYD88 L265P mutation in DLBCLs, excluding the central nervous system and testicular DLBCLs, was 16.5%. Notably, the MYD88 L265P mutation rates of CNS and testicular DLBCL patients were 60% and 77%, respectively. Interestingly, the MYD88 L265P mutation was more frequently detected in activated B-cell-like (ABC) or non-germinal center B-cell-like (GCB) than GCB subtype (OR?=?3.414, p?<?0.001). The MYD88 L265P mutation was significantly associated with old age and poor overall survival, but not with sex and clinical stage. This pooled analysis demonstrates that the MYD88 L265P mutation is significantly associated with the tumor sites and molecular subtypes in DLBCL patients.
Project description:MYD88 mutations in chronic lymphocytic leukemia (CLL) are not well characterized. Earlier reports yielded conflicting results in terms of clinicopathologic presentation and prognostic impact of MYD88 mutations in CLL patients. In addition, the morphological and immunophenotypic features of CLL cases carrying MYD88 mutations have not been explored. Finally, the clinical or biologic implications of the canonical L265P MYD88 mutation vs. mutations in other sites of MYD88 within the context of CLL are also unknown. In this study, a cohort of 1779 CLL patients underwent mutational analysis, and 56 (3.1%) cases were found to have MYD88 mutations, including 38 with L265P mutations (designated here as group A) and 18 with non-L265P mutations (group B). Cases with wild type MYD88 were included as controls. There was no morphological difference in cases with and without MYD88 mutations. Immunophenotypically, cases with mutated MYD88 (both groups A and B) more frequently had an atypical immunophenotype when compared to wild type cases. Group A patients were younger and were associated with variable favorable prognostic factors, including less elevated ?2-microglobulin level, negative CD38 and ZAP70, higher frequency of mutated IGHV and isolated del(13q14.3), and lower frequency of del(11q22.3) and mutations of NOTCH1 and SF3B1. In contrast, group B patients were more similar to CLL patients with wild type MYD88. There was no difference in time to first treatment when comparing MYD88-mutated vs. wild type CLL patients before and after stratification according to IGHV mutation status. In summary, MYD88 mutations are uncommon in CLL and cases with L265P mutation have distinctive clinical, immunophenotypic, cytogenetic, and molecular features. There is no significant impact of MYD88 mutations on time to first treatment in CLL.
Project description:CXCR4(WHIM) somatic mutations are distinctive to Waldenström Macroglobulinaemia (WM), and impact disease presentation and treatment outcome. The clonal architecture of CXCR4(WHIM) mutations remains to be delineated. We developed highly sensitive allele-specific polymerase chain reaction (AS-PCR) assays for detecting the most common CXCR4(WHIM) mutations (CXCR4(S338X C>A and C>G) ) in WM. The AS-PCR assays detected CXCR4(S338X) mutations in WM and IgM monoclonal gammopathy of unknown significance (MGUS) patients not revealed by Sanger sequencing. By combined AS-PCR and Sanger sequencing, CXCR4(WHIM) mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%) and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and marginal zone lymphoma patients, respectively, but no chronic lymphocytic leukaemia, multiple myeloma, non-IgM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4(S338X) mutations were primarily subclonal, with highly variable clonal distribution (median 35·1%, range 1·2-97·5%). Combined AS-PCR and Sanger sequencing revealed multiple CXCR4(WHIM) mutations in many individual WM patients, including homozygous and compound heterozygous mutations validated by deep RNA sequencing. The findings show that CXCR4(WHIM) mutations are more common in WM than previously revealed, and are primarily subclonal, supporting their acquisition after MYD88(L265P) in WM oncogenesis. The presence of multiple CXCR4(WHIM) mutations within individual WM patients may be indicative of targeted CXCR4 genomic instability.
Project description:Diffuse large B cell lymphoma (DLBCL) is associated with aggressive clinical cases and poor prognosis despite recent advances in disease treatment. In activated B-cell-like (ABC)-DLBCL, the most severe damaged signaling pathways converge to aberrantly activate the Toll-like receptor (TLR) 7/9/MyD88 pathways, leading to the avoidance of cell death and resistance to chemotherapy. A gain of function mutation in MyD88 (MyD88 L265P) enhanced the NF-?B and JAK-STAT signaling pathways and was associated with dysregulation of TLR signaling in the pathogenesis of ABC-DLBCL. Therefore, inhibition of the TLR signaling network may improve clinical outcomes. In this study, we designed a de novo synthesized oligodeoxynucleotide-based antagonist of TLR7 and TLR9, referred to as HJ901, which competitively binds to TLR7/9. We profiled HJ901 inhibition in various DLBCL cell lines and verified tumor suppression in a xenograft mouse model. We found that HJ901 treatment significantly reduced TLR7- and TLR9-mediated cell proliferation and cytokine production in a time- and dose-dependent manner in various DLBCL cell lines expressing the MyD88 L265P mutation. Moreover, HJ901 prevented tumor growth and downregulated the NF-?B and JAK2-STAT3 signaling pathways in a DLBCL xenograft mouse model with the MyD88 L265P mutation. These results reveal that the anti-tumor effects of the synthesized oligodeoxynucleotide-based antagonist, HJ901, which competitively binds to TLR7/9, may be associated with the downregulation of the NF-?B and JAK2-STAT3 signaling pathways and provide rationale for treating ABC-DLBCL patients with the MyD88 L265P mutation.
Project description:Recurrent mutations affecting MYD88 and CXCR4 gene nowadays form the basis for the diagnosis, risk stratification and use of inhibitors targeting these signalling pathways in LPL/WM which are rare B cell neoplasms. MYD88 L265P mutation analysis was performed on 33 cases of LPL/WM by AS-PCR (positivity-84.8%, n?=?28/33) and by Sanger sequencing (positivity-39.3%, n?=?13/33). We had only two cases with CXCR4 non-sense (NS) mutation (p.S338*) using Sanger sequencing. MYD88 (L265P) mutation detection by AS-PCR can form reliable biomarker for the diagnosis of LPL/WM in molecular labs. Although the cohort is small, still the CXCR4 mutation frequency in our study is low as compared to the published literature.
Project description:A highly recurrent somatic L265P mutation in the TIR domain of the signaling adapter MYD88 constitutively activates NF-?B. It occurs in nearly all human patients with Waldenström's macroglobulinemia (WM), a B cell malignancy caused by IgM-expressing cells. Here, we introduced an inducible leucine to proline point mutation into the mouse Myd88 locus, at the orthologous position L252P. When the mutation was introduced early during B cell development, B cells developed normally. However, IgM-expressing plasma cells accumulated with age in spleen and bone, leading to more than 20-fold elevated serum IgM titers. When introduced into germinal center B cells in the context of an immunization, the Myd88<sup>L252P</sup> mutation caused prolonged persistence of antigen-specific serum IgM and elevated numbers of antigen-specific IgM plasma cells. Myd88<sup>L252P</sup>-expressing B cells switched normally, but plasma cells expressing other immunoglobulin isotypes did not increase in numbers, implying that IgM expression may be required for the observed cellular expansion. In order to test whether the Myd88<sup>L252P</sup> mutation can cause clonal expansions, we introduced it into a small fraction of CD19-positive B cells. In this scenario, five out of five mice developed monoclonal IgM serum paraproteins accompanied by an expansion of clonally related plasma cells that expressed mostly hypermutated VDJ regions. Taken together, our data suggest that the Myd88<sup>L252P</sup> mutation is sufficient to promote aberrant survival and expansion of IgM-expressing plasma cells which in turn can cause IgM monoclonal gammopathy of undetermined significance (MGUS), the premalignant condition that precedes WM.
Project description:Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHLs) including the rare lymphoplasmacytic lymphoma, Waldenström's macroglobulinemia (WM). Using whole-exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B-cell survival, therapeutic targeting of MYD88 signaling pathways may be clinically useful. However, we are lacking a thorough characterization of the role of intermediary signaling proteins on the biology of MYD88L265P-expressing B cells. We report here that MYD88L265P signaling is constitutively active in both WM and diffuse large B-cell lymphoma cells leading to heightened MYD88L265P, IRAK and TRAF6 oligomerization and NF-?B activation. Furthermore, we have identified the signaling protein, TAK1, to be an essential mediator of MYD88L265P-driven signaling, cellular proliferation and cytokine secretion in malignant B cells. Our studies highlight the biological significance of MYD88L265P in NHL and reveal TAK1 inhibition to be a potential therapeutic strategy for the treatment of WM and other diseases characterized by MYD88L265P.