Yang Cao

MYD88 L265P Somatic Mutation in Waldenström's Macroglobulinemia

BACKGROUND Waldenströms macroglobulinemia is an incurable, IgM-secreting lymphoplasmacytic lymphoma (LPL). The underlying mutation in this disorder has not been delineated. METHODS We performed whole-genome sequencing of bone marrow LPL cells in 30 patients with Waldenströms macroglobulinemia, with paired normal-tissue and tumor-tissue sequencing in 10 patients. Sanger sequencing was used to validate the findings in samples from an expanded cohort of patients with LPL, those with other B-cell disorders that have some of the same features as LPL, and healthy donors. RESULTS Among the patients with Waldenströms macroglobulinemia, a somatic variant (T→C) in LPL cells was identified at position 38182641 at 3p22.2 in the samples from all 10 patients with paired tissue samples and in 17 of 20 samples from patients with unpaired samples. This variant predicted an amino acid change (L265P) in MYD88, a mutation that triggers IRAK-mediated NF-κB signaling. Sanger sequencing identified MYD88 L265P in tumor samples from 49 of 54 patients with Waldenströms macroglobulinemia and in 3 of 3 patients with non-IgM-secreting LPL (91% of all patients with LPL). MYD88 L265P was absent in paired normal tissue samples from patients with Waldenströms macroglobulinemia or non-IgM LPL and in B cells from healthy donors and was absent or rarely expressed in samples from patients with multiple myeloma, marginal-zone lymphoma, or IgM monoclonal gammopathy of unknown significance. Inhibition of MYD88 signaling reduced IκBα and NF-κB p65 phosphorylation, as well as NF-κB nuclear staining, in Waldenströms macroglobulinemia cells expressing MYD88 L265P. Somatic variants in ARID1A in 5 of 30 patients (17%), leading to a premature stop or frameshift, were also identified and were associated with an increased disease burden. In addition, 2 of 3 patients with Waldenströms macroglobulinemia who had wild-type MYD88 had somatic variants in MLL2. CONCLUSIONS MYD88 L265P is a commonly recurring mutation in patients with Waldenströms macroglobulinemia that can be useful in differentiating Waldenströms macroglobulinemia and non-IgM LPL from B-cell disorders that have some of the same features. (Funded by the Peter and Helen Bing Foundation and others.).

Ibrutinib in Previously Treated Waldenström's Macroglobulinemia

BACKGROUND MYD88(L265P) and CXCR4(WHIM) mutations are highly prevalent in Waldenströms macroglobulinemia. MYD88(L265P) triggers tumor-cell growth through Brutons tyrosine kinase, a target of ibrutinib. CXCR4(WHIM) mutations confer in vitro resistance to ibrutinib. METHODS We performed a prospective study of ibrutinib in 63 symptomatic patients with Waldenströms macroglobulinemia who had received at least one previous treatment, and we investigated the effect of MYD88 and CXCR4 mutations on outcomes. Ibrutinib at a daily dose of 420 mg was administered orally until disease progression or the development of unacceptable toxic effects. RESULTS After the patients received ibrutinib, median serum IgM levels decreased from 3520 mg per deciliter to 880 mg per deciliter, median hemoglobin levels increased from 10.5 g per deciliter to 13.8 g per deciliter, and bone marrow involvement decreased from 60% to 25% (P<0.01 for all comparisons). The median time to at least a minor response was 4 weeks. The overall response rate was 90.5%, and the major response rate was 73.0%; these rates were highest among patients with MYD88(L265P)CXCR4(WT) (with WT indicating wild-type) (100% overall response rate and 91.2% major response rate), followed by patients with MYD88(L265P)CXCR4(WHIM) (85.7% and 61.9%, respectively) and patients with MYD88(WT)CXCR4(WT) (71.4% and 28.6%). The estimated 2-year progression-free and overall survival rates among all patients were 69.1% and 95.2%, respectively. Treatment-related toxic effects of grade 2 or higher included neutropenia (in 22% of the patients) and thrombocytopenia (in 14%), which were more common in heavily pretreated patients; postprocedural bleeding (in 3%); epistaxis associated with the use of fish-oil supplements (in 3%); and atrial fibrillation associated with a history of arrhythmia (5%). CONCLUSIONS Ibrutinib was highly active, associated with durable responses, and safe in pretreated patients with Waldenströms macroglobulinemia. MYD88 and CXCR4 mutation status affected responses to this drug. (Funded by Pharmacyclics and others; ClinicalTrials.gov number, NCT01614821.).

MYD88 L265P in Waldenström macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction

By whole-genome and/or Sanger sequencing, we recently identified a somatic mutation (MYD88 L265P) that stimulates nuclear factor κB activity and is present in >90% of Waldenström macroglobulinemia (WM) patients. MYD88 L265P was absent in 90% of immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) patients. We therefore developed conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays for more sensitive detection and quantification of MYD88 L265P. Using either assay, 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 splenic 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 (0/40; P < 1.5 × 10(-5) for WM vs other cohorts). Real-time AS-PCR identified IgM MGUS patients progressing to WM and showed a high rate of concordance between MYD88 L265P ΔCT and BM disease involvement (r = 0.89, P = .008) in WM patients undergoing treatment. These studies identify MYD88 L265P as a widely present mutation in WM and IgM MGUS patients using highly sensitive and specific 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.

A mutation in MYD88 (L265P) supports the survival of lymphoplasmacytic cells by activation of Bruton tyrosine kinase in Waldenström macroglobulinemia

Myeloid differentiation factor 88 (MYD88) L265P somatic mutation is highly prevalent in Waldenström macroglobulinemia (WM) and supports malignant growth through nuclear factor κB (NF-κB). The signaling cascade(s) by which MYD88 L265P promotes NF-κB activation in WM remain unclear. By lentiviral knockdown or use of a MYD88 inhibitor, decreased phosphorylation 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 phosphorylated BTK (pBTK). Increased pBTK was also observed in WM cells transduced to overexpress L265P vs wild-type MYD88. Importantly, MYD88 binding to BTK was abrogated following treatment of MYD88 L265P-expressing cells with a BTK kinase inhibitor. Inhibition of BTK or interleukin-1 receptor-associated kinase 1 and 4 (IRAK-1 and -4) kinase activity induced apoptosis 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.

The genomic landscape of Waldenström macroglobulinemia is characterized by highly recurring MYD88 and WHIM-like CXCR4 mutations, and small somatic deletions associated with B-cell lymphomagenesis

The genetic basis for Waldenström macroglobulinemia (WM) remains to be clarified. Although 6q losses are commonly present, recurring gene losses in this region remain to be defined. We therefore performed whole genome sequencing (WGS) in 30 WM patients, which included germline/tumor sequencing for 10 patients. Validated somatic mutations occurring in >10% of patients included MYD88, CXCR4, and ARID1A that were present in 90%, 27%, and 17% of patients, respectively, and included the activating mutation L265P in MYD88 and warts, hypogammaglobulinemia, infection, and myelokathexis-syndrome-like mutations in CXCR4 that previously have only been described in the germline. WGS also delineated copy number alterations (CNAs) and structural variants in the 10 paired patients. The CXCR4 and CNA findings were validated in independent expansion cohorts of 147 and 30 WM patients, respectively. Validated gene losses due to CNAs involved PRDM2 (93%), BTG1 (87%), HIVEP2 (77%), MKLN1 (77%), PLEKHG1 (70%), LYN (60%), ARID1B (50%), and FOXP1 (37%). Losses in PLEKHG1, HIVEP2, ARID1B, and BCLAF1 constituted the most common deletions within chromosome 6. Although no recurrent translocations were observed, in 2 patients deletions in 6q corresponded with translocation events. These studies evidence highly recurring somatic events, and provide a genomic basis for understanding the pathogenesis of WM.

Somatic mutations in MYD88 and CXCR4 are determinants of clinical presentation and overall survival in Waldenstrom's Macroglobulinemia.

Whole genome sequencing has revealed activating somatic mutations in MYD88 (L265P) and CXCR4 in Waldenström macroglobulinemia (WM). CXCR4 somatic mutations in WM are the first ever reported in human cancer and are similar to nonsense (NS) and frameshift (FS) germline mutations found in warts, hypogammaglobulinemia, infections and myelokathexis (WHIM) syndrome. We genotyped lymphoplasmacytic cells from 175 WM patients and observed significantly higher bone marrow (BM) disease involvement, serum immunoglobulin-M levels, and symptomatic disease requiring therapy, including hyperviscosity syndrome in those patients with MYD88(L265P)CXCR4(WHIM/NS) mutations (P < .03). Patients with MYD88(L265P)CXCR4(WHIM/FS) or MYD88(L265P)CXCR4(WILDTYPE (WT)) had intermediate BM and serum immunoglobulin-M levels; those with MYD88(WT)CXCR4(WT) showed lowest BM disease burden. Fewer patients with MYD88(L265P) and CXCR4(WHIM/FS or NS) vs MYD88(L265P)CXCR4(WT) presented with adenopathy (P < .01), further delineating differences in disease tropism based on CXCR4 status. Neither MYD88 nor CXCR4 mutations correlated with SDF-1a (RS1801157) polymorphisms in 54 patients who were genotyped for these variants. Unexpectedly, risk of death was not impacted by CXCR4 mutation status, but by MYD88(WT) status (hazard ratio 10.54; 95% confidence interval 2.4-46.2, P = .0018). Somatic mutations in MYD88 and CXCR4 are important determinants of clinical presentation and impact overall survival in WM. Targeted therapies directed against MYD88 and/or CXCR4 signaling may provide a personalized treatment approach to WM.

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

Bortezomib frequently produces severe treatment-related peripheral neuropathy (PN) in Waldenströms macroglobulinemia (WM). Carfilzomib is a neuropathy-sparing proteasome inhibitor. We examined carfilzomib, rituximab, and dexamethasone (CaRD) in symptomatic WM patients naïve to bortezomib and rituximab. Protocol therapy consisted of intravenous carfilzomib, 20 mg/m2 (cycle 1) and 36 mg/m(2) (cycles 2-6), with intravenous dexamethasone, 20 mg, on days 1, 2, 8, and 9, and rituximab, 375 mg/m(2), on days 2 and 9 every 21 days. Maintenance therapy followed 8 weeks later with intravenous carfilzomib, 36 mg/m(2), and intravenous dexamethasone, 20 mg, on days 1 and 2, and rituximab, 375 mg/m(2), on day 2 every 8 weeks for 8 cycles. Overall response rate was 87.1% (1 complete response, 10 very good partial responses, 10 partial responses, and 6 minimal responses) and was not impacted by MYD88(L265P) or CXCR4(WHIM) mutation status. With a median follow-up of 15.4 months, 20 patients remain progression free. Grade ≥2 toxicities included asymptomatic hyperlipasemia (41.9%), reversible neutropenia (12.9%), and cardiomyopathy in 1 patient (3.2%) with multiple risk factors, and PN in 1 patient (3.2%) which was grade 2. Declines in serum IgA and IgG were common. CaRD offers a neuropathy-sparing approach for proteasome inhibitor-based therapy in WM. This trial is registered at www.clinicaltrials.gov as #NCT01470196.

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

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

CXCR4 WHIM-like frameshift and nonsense mutations promote ibrutinib resistance but do not supplant MYD88L265P-directed survival signalling in Waldenström macroglobulinaemia cells

CXCR4WHIM frameshift and nonsense mutations follow MYD88L265P as the most common somatic variants in Waldenström Macroglobulinaemia (WM), and impact clinical presentation and ibrutinib response. While the nonsense (CXCR4S338X) mutation has been investigated, little is known about CXCR4 frameshift (CXCR4FS) mutations. We engineered WM cells to express CXCR4FS mutations present in patients, and compared their CXCL12 (SDF‐1a) induced signalling and ibrutinib sensitivity to CXCR4wild‐type (WT) and CXCR4S338X cells. Following CXCL12 stimulation, CXCR4FS and CXCR4S338X WM cells showed impaired CXCR4 receptor internalization, and enhanced AKT1 (also termed AKT) and MAPK1 (also termed ERK) activation versus CXCRWT cells (P < 0·05), though MAPK1 activation was more prolonged in CXCR4S338X cells (P < 0·05). CXCR4FS and CXCR4S338X cells, but not CXCR4WT cells, were rescued from ibrutinib‐triggered apoptosis by CXCL12 that was reversed by AKT1, MAPK1 or CXCR4 antagonists. Treatment with an inhibitor that blocks MYD88L265P signalling triggered similar levels of apoptosis that was not abrogated by CXCL12 treatment in CXCR4WT and CXCR4WHIM cells. These studies show a functional role for CXCR4FS mutations in WM, and provide a framework for the investigation of CXCR4 antagonists with ibrutinib in CXCR4WHIM‐mutated WM patients. Direct inhibition of MYD88L265P signalling overcomes CXCL12 triggered survival effects in CXCR4WHIM‐mutated cells supporting a primary role for this survival pathway in WM.

Clonal architecture of CXCR4 WHIM-like mutations in Waldenström Macroglobulinaemia

CXCR4WHIM somatic mutations are distinctive to Waldenström Macroglobulinaemia (WM), and impact disease presentation and treatment outcome. The clonal architecture of CXCR4WHIM mutations remains to be delineated. We developed highly sensitive allele‐specific polymerase chain reaction (AS‐PCR) assays for detecting the most common CXCR4WHIM mutations (CXCR4S338X C>A and C>G) in WM. The AS‐PCR assays detected CXCR4S338X mutations in WM and IgM monoclonal gammopathy of unknown significance (MGUS) patients not revealed by Sanger sequencing. By combined AS‐PCR and Sanger sequencing, CXCR4WHIM 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 CXCR4S338X 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 CXCR4WHIM mutations in many individual WM patients, including homozygous and compound heterozygous mutations validated by deep RNA sequencing. The findings show that CXCR4WHIM mutations are more common in WM than previously revealed, and are primarily subclonal, supporting their acquisition after MYD88L265P in WM oncogenesis. The presence of multiple CXCR4WHIM mutations within individual WM patients may be indicative of targeted CXCR4 genomic instability.