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Featured Impact Articles

In this paper, we describe the feasibility of testing for MYD88 L265P mutation by peripheral blood testing. MYD88 L265P is a highly prevalent mutation in Waldenstrom's Macroglobulinemia (WM) and its precursor condition IgM monoclonal gammopathy of unknown significance (MGUS). Using allele-specific PCR technology which is very sensitive for detecting single point mutations, B-cells from the peripheral blood of patients with WM and IgM MGUS were examined for the MYD88 L265P mutation. MYD88 L265P was detected in untreated WM patients (114/118; 96.6%); previously treated WM patients (63/102; 61.8%); and IgM MGUS patients (5/12; 41.7%) but it was not detected in 3 patients with the non-malignant hyper-IgM syndrome or 40 healthy individuals. The level of detection of the MYD88 L265P mutation (ΔCt) helped distinguish those patients with WM versus IgM MGUS, and also correlated with underlying disease burden. This study shows the feasibility for detecting MYD88 L265P by PB examination, and the potential for PB MYD88 L265P ΔCt use in the diagnosis and management of WM patients.

In this paper, we describe the feasibility of testing for MYD88 L265P mutation by using allele-specific PCR (AS-PCR) technology which is very sensitive for detecting single point mutations.  B-cells from bone marrow biopsy samples of patients with WM, as well as other malignancies which are often overlapping with WM such as marginal zone lymphomas, IgM myeloma. CLL, and IgM MGUS were examined. MYD88 L265P was detected in 97 of 104 (93%) WM and 13 of 24 (54%) IgM MGUS patients and was either absent or rarely expressed in samples from marginal zone lymphoma (2/20; 10%), CLL (1/26; 4%), multiple myeloma (including IgM cases, 0/14), and immunoglobulin G MGUS (0/9) patients as well as healthy donors. AS-PCR assay identified IgM MGUS patients progressing to WM and showed a high rate of concordance between MYD88 L265P levels (ΔCT) and BM disease involvement in WM patients undergoing treatment. These studies identify MYD88 L265P as a widely present mutation in WM and IgM MGUS patients using AS-PCR assays with potential use in diagnostic discrimination and/or response assessment. The finding of this mutation in many IgM MGUS patients suggests that MYD88 L265P may be an early oncogenic event in WM pathogenesis.

In Waldenström macroglobulinaemia (WM), the mechanism(s) responsible for repression of B-cell differentiation remains unknown. Normally B-cells are supposed to differentiate to plasma cells, but with WM, the B-cells are stuck at an earlier differentiation state. This means they cannot grow old and die but are stuck at an earlier stage. We found that expression of SPIB and ID2 were significantly increased and decreased, respectively, in WM lymphoplasmacytic cells (LPC) isolated from bone marrow biopsy samples. We used a viral system to express high levels of SPIB in healthy donor B-cells and showed that it blocked their ability to differentiate to plasma cells. In primary WM cells, knock-down of SPIB induced plasmacytic differentiation. Given that SPIB is a direct target of POU2AF1 (OBF1) in complex with POU2F2 or POU2F1, we next examined their expression in WM LPC. POU2F2 transcription, as well as POU2F2 and POU2AF1 protein expression was higher in WM LPC. When we over expressed POU2F2 in healthy donor CD19+ cells we observed increased transcription of SPIB. Chromatin immunoprecipitation analysis in BCWM.1 WM cells confirmed binding of POU2F2 and POU2AF1 in SPIB regulatory region. These findings establish an important role for SPIB in arresting plasmacytic differentiation in WM.

Myeloid differentiation factor 88 (MYD88) L265P somatic mutation is highly prevalent in Waldenström macroglobulinemia (WM) and supports malignant growth through the transcription factor nuclear factor κB (NF-κB). The signaling cascade(s) by which MYD88 L265P promotes NF-κB activation in WM remain unclear. By use of viral mediated strategy to knock down MYD88 expression or use of a MYD88 inhibitor, we showed decreased activation of the NF-κB gatekeeper IκBα and survival occurred in MYD88 L265P-expressing WM cells. Conversely, WM cells engineered to overexpress MYD88 L265P showed enhanced survival. Coimmunoprecipitation studies identified Bruton tyrosine kinase (BTK) complexed to MYD88 in L265P-expressing WM cells, with preferential binding of MYD88 to the activated “phosphorylated” form of BTK (pBTK). Increased pBTK was also observed in WM cells made to overexpress L265P vs wild-type (native) MYD88. Importantly, MYD88 binding to BTK was blocked following treatment of MYD88 L265P-expressing cells with ibrutinib, a BTK kinase inhibitor. Inhibition of BTK or interleukin-1 receptor-associated kinase 1 and 4 (IRAK-1 and -4) kinase activity induced death of WM cells, and their combination resulted in more robust inhibition of NF-κB signaling and synergistic WM cell killing. The results establish BTK as a downstream target of MYD88 L265P signaling, and provide a framework for the study of BTK inhibitors alone, and in combination with IRAK inhibitors for the treatment of WM.