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Activating MYD88 mutations promote pro-survival signaling through BTK and HCK, both targets of ibrutinib. Despite high response rates, complete responses to ibrutinib are lacking, and other MYD88 triggered pro-survival pathways may contribute to primary drug resistance. B-cell receptor (BCR) signaling has been observed in lymphomas driven by mutated MYD88, even without activating the BCR pathway mutations. We identified activated SYK (p-SYK), a component of BCR in complex with MYD88 in MYD88-mutated WM and ABC DLBCL lymphoma cells. Confocal microscopy confirmed co-localization of MYD88 with SYK in MYD88-mutated cells. Knockdown of MYD88 or use of a MYD88 signaling inhibitor abrogated SYK activation, while expression of mutated but not wild-type MYD88 amplified p-SYK in MYD88-mutated and wild-type lymphoma cells. Knockdown of SYK or use of inhibitors targeting SYK blocked p-STAT3 and p-AKT signaling in MYD88-mutated cells. Cell viability analysis showed that combining ibrutinib and SYK inhibitors triggered synthetic killing of MYD88-mutated lymphoma cells. Our findings extend the spectrum of mutated MYD88 pro-survival signaling to include SYK directed BCR cross talk in MYD88-mutated lymphomas. Targeting SYK in combination with ibrutinib produces synthetic lethality, providing a framework for the clinical investigation of ibrutinib with SYK inhibitors in MYD88-mutated lymphomas.

Little is known about TP53 mutations in Waldenström Macroglobulinaemia (WM). We evaluated 265 WM patients for TP53 mutations by next-generation sequencing, and validated the findings by Sanger sequencing. TP53 mutations were identified and validated in 6 (2·6%) patients that impacted the DNA-binding domain. All six were MYD88- and CXCR4-mutated. Ibrutinib showed activity in patients carrying all three mutations. With a median follow-up of 18 months, 2 (33%) with biallelic TP53 inactivation died of progressive disease. TP53 mutations are rare in WM, and associate with MYD88 and CXCR4 mutations. WM patients with TP53 mutations show response to ibrutinib., (© 2018 British Society for Haematology and John Wiley & Sons Ltd.)

Activating MYD88 mutations are present in 95% of Waldenström macroglobulinemia (WM) patients, and trigger NF-κB through BTK and IRAK. The BTK inhibitor ibrutinib is active in MYD88- mutated (MYD88 MUT ) WM patients, but shows lower activity in MYD88 wild-type ( MYD88 WT ) disease. MYD88 WT patients also show shorter overall survival, and increased risk of disease transformation in some series. The genomic basis for these findings remains to be clarified. We performed whole exome and transcriptome sequencing of sorted tumor samples from 18 MYD88 WT patients and compared findings with WM patients with MYD88 MUT disease. We identified somatic mutations predicted to activate NF-κB ( TBL1XR1 , PTPN13, MALT1 , BCL10 , NFKB2 , NFKBIB , NFKBIZ, and UDRL1F ), impart epigenomic dysregulation ( KMT2D , KMT2C, and KDM6A) , or impair DNA damage repair ( TP53 , ATM , and TRRAP ). Predicted NF-κB activating mutations were downstream of BTK and IRAK, and many overlapped with somatic mutations found in diffuse large B-cell lymphoma. A distinctive transcriptional profile in MYD88 WT WM was identified, although most differentially expressed genes overlapped with MYD88 MUT WM consistent with the many clinical and morphological characteristics that are shared by these WM subgroups. Overall survival was adversely affected by mutations in DNA damage response in MYD88 WT WM patients. The findings depict genomic and transcriptional events associated with MYD88 WT WM and provide mechanistic insights for disease transformation, decreased ibrutinib activity, and novel drug approaches for this population., (© 2018 by The American Society of Hematology.)

Purpose Ibrutinib is active in previously treated Waldenström macroglobulinemia (WM). MYD88 mutations ( MYD88 MUT ) and CXCR4 mutations ( CXCR4 MUT ) affect ibrutinib response. We report on a prospective study of ibrutinib monotherapy in symptomatic, untreated patients with WM, and the effect of CXCR4 MUT status on outcome. Patients and Methods Symptomatic, treatment-naïve patients with WM were eligible. Ibrutinib (420 mg) was administered daily until progression or unacceptable toxicity. All tumors were genotyped for MYD88 MUT and CXCR4 MUT . Results A total of 30 patients with WM received ibrutinib. All carried MYD88 MUT , and 14 (47%) carried a CXCR4 MUT . After ibrutinib treatment, median serum IgM levels declined from 4,370 to 1,513 mg/dL, bone marrow involvement declined from 65% to 20%, and hemoglobin level rose from 10.3 to 13.9 g/dL ( P < .001 for all comparisons). Overall (minor or more than minor) and major (partial or greater than partial) responses for all patients were 100% and 83%, respectively. Rates of major (94% v 71%) and very good partial (31 v 7%) responses were higher and time to major responses more rapid (1.8 v 7.3 months; P = 0.01) in patients with wild-type CXCR4 versus those with CXCR4 MUT , respectively. With a median follow-up of 14.6 months, disease in two patients (both with CXCR4 MUT ) progressed. The 18-month, estimated progression-free survival is 92% (95% CI, 73% to 98%). All patients are alive. Grade 2/3 treatment-related toxicities in > 5% of patients included arthralgia (7%), bruising (7%), neutropenia (7%), upper respiratory tract infection (7%), urinary tract infection (7%), atrial fibrillation (10%), and hypertension (13%). There were no grade 4 or unexpected toxicities. Conclusion Ibrutinib is highly active, produces durable responses, and is safe as primary therapy in patients with symptomatic WM. CXCR4 MUT status affects responses to ibrutinib.

Ibrutinib is the first approved therapy for symptomatic patients with Waldenström macroglobulinemia (WM). The reasons for discontinuing ibrutinib and subsequent outcomes have not been previously evaluated in WM patients. We therefore conducted a retrospective review of 189 WM patients seen at our institution who received treatment with ibrutinib, of whom 51 (27%) have discontinued therapy. Reasons for discontinuation include: disease progression (n = 27; 14%), toxicity (n = 15; 8%), nonresponse (n = 5; 3%), and other unrelated reasons (n = 4; 2%). The cumulative incidence of ibrutinib discontinuation at 12, 24, 36, and 48 months from treatment initiation was 22%, 26%, 35%, and 43%, respectively. A baseline platelet count ≤100 K/µL and presence of tumor CXCR4 mutations were independently associated with 4-fold increased odds of ibrutinib discontinuation. An IgM rebound (≥25% increase in serum IgM) was observed in 37 patients (73%) following ibrutinib discontinuation and occurred within 4 weeks for nearly half of patients. The response rate to salvage therapy was 71%; responses were higher in patients without an IgM rebound and when salvage therapy was initiated within 2 weeks of stopping ibrutinib. Patients who discontinued ibrutinib due to disease progression versus nonprogression events had significantly shorter overall survival (21 versus 32 months; P = .046). Response to salvage therapy was associated with an 82% reduction in the risk of death following ibrutinib discontinuation. WM patients who discontinue ibrutinib require close monitoring, and continuation of ibrutinib until the next therapy should be considered to maintain disease control., (© 2017 Wiley Periodicals, Inc.)

Purpose: Proteasome inhibition is of proven efficacy in patients with Waldenström macroglobulinemia (WM). However, WM remains incurable with standard treatments. Novel agents, safe and effective, are needed. Patients and Methods: We designed a prospective phase II study evaluating the combination of ixazomib, dexamethasone, and rituximab (IDR) as primary therapy in symptomatic patients with WM. Protocol therapy consisted of oral ixazomib, 4 mg, with intravenous or oral dexamethasone, 20 mg, on days 1, 8, and 15 every 4 weeks for induction cycles 1 and 2, and in combination with intravenous rituximab, 375 mg/m 2 , on day 1, every 4 weeks for cycles 3 to 6. Maintenance therapy followed 8 weeks later with IDR given every 8 weeks for 6 cycles. Results: Twenty-six patients were enrolled. All patients had the MYD88 L265P mutation, and 15 patients (58%) had a CXCR4 mutation. The median time to response was 8 weeks, which was longer (12 weeks) in WM patients with CXCR4 mutations ( P = 0.03). The overall response rate was 96%, and the major response rate was 77%. With a median follow-up of 22 months, the median progression-free survival was not reached. Grade ≥2 adverse events reported in >1 patient included infusion-related reactions (19%), rash (8%), and insomnia (8%). Conclusions: IDR offers a highly effective and well tolerated, neuropathy-sparing regimen for primary therapy in patients with WM. This trial is registered at www.clinicaltrials.gov under ID NCT02400437 Clin Cancer Res; 24(14); 3247-52. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Acquired ibrutinib resistance due to BTK Cys481 mutations occurs in B-cell malignancies, including those with MYD88 mutations. BTK Cys481 mutations are usually subclonal, and their relevance to clinical progression remains unclear. Moreover, the signaling pathways that promote ibrutinib resistance remain to be clarified. We therefore engineered BTK Cys481Ser and BTK WT expressing MYD88-mutated Waldenström macroglobulinemia (WM) and activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) cells and observed reactivation of BTK-PLCγ2-ERK1/2 signaling in the presence of ibrutinib in only the former. Use of ERK1/2 inhibitors triggered apoptosis in BTK Cys481Ser -expressing cells and showed synergistic cytotoxicity with ibrutinib. ERK1/2 reactivation in ibrutinib-treated BTK Cys481Ser cells was accompanied by release of many prosurvival and inflammatory cytokines, including interleukin-6 (IL-6) and IL-10 that were also blocked by ERK1/2 inhibition. To clarify if cytokine release by ibrutinib-treated BTK Cys481Ser cells could protect BTK WT MYD88-mutated malignant cells, we used a Transwell coculture system and showed that nontransduced BTK WT MYD88-mutated WM or ABC DLBCL cells were rescued from ibrutinib-induced killing when cocultured with BTK Cys481Ser but not their BTK WT -expressing counterparts. Use of IL-6 and/or IL-10 blocking antibodies abolished the protective effect conferred on nontransduced BTK WT by coculture with BTK Cys481Ser expressing WM or ABC DLBCL cell counterparts. Rebound of IL-6 and IL-10 serum levels also accompanied disease progression in WM patients with acquired BTK Cys481 mutations. Our findings show that the BTK Cys481Ser mutation drives ibrutinib resistance in MYD88-mutated WM and ABC DLBCL cells through reactivation of ERK1/2 and can confer a protective effect on BTK WT cells through a paracrine mechanism., (© 2018 by The American Society of Hematology.)

MYD88 mutations are present in 95% of Waldenstrom Macroglobulinaemia (WM) patients, and support diagnostic discrimination from other IgM-secreting B-cell malignancies. Diagnostic discrimination can be difficult among suspected wild-type MYD88 (MYD88 WT ) WM cases. We systematically reviewed the clinical, pathological and laboratory studies for 64 suspected MYD88 WT WM patients. World Health Organization and WM consensus guidelines were used to establish clinicopathological diagnosis. Up to 30% of suspected MYD88 WT WM cases had an alternative clinicopathological diagnosis, including IgM multiple myeloma. The estimated 10-year survival was 73% (95% confidence interval [CI] 52-86%) for MYD88 WT versus 90% (95% CI 82-95%) for mutated (MYD88 MUT ) WM patients (Log-rank P < 0·001). Multivariate analysis only showed MYD88 mutation status (P < 0·001) as a significant determinant for overall survival. Diffuse large B-cell lymphoma (DLBCL) was diagnosed in 7 (15·2%) and 2 (0·76%) of MYD88 WT and MYD88 MUT patients, respectively (Odds ratio 23·3; 95% CI 4·2-233·8; P < 0·001). Overall survival was shorter among MYD88 WT patients with an associated DLBCL event (Log-rank P = 0·08). The findings show that among suspected MYD88 WT WM cases, an alternative clinicopathological diagnosis is common and can impact clinical care. WM patients with MYD88 WT disease have a high incidence of associated DLBCL events and significantly shorter survival versus those with MYD88 MUT disease., (© 2017 John Wiley & Sons Ltd.)

Symptomatic hyperviscosity is a common clinical manifestation in patients with Waldenström macroglobulinaemia (WM) and high serum IgM levels. Prompt intervention is required to prevent catastrophic events, such as retinal or central nervous system bleeding. Identifying patients at high risk of symptomatic hyperviscosity might support the decision to treat asymptomatic patients before irreversible damage occurs. We carried out a large retrospective study in 825 newly diagnosed WM patients, of who 113 (14%) developed symptomatic hyperviscosity. The median serum IgM level at the time of symptomatic hyperviscosity was 61·8 g/l (range 31-124 g/l). Forty-four patients (36%) had symptomatic hyperviscosity at the time of WM diagnosis. A serum IgM level >60 g/l at diagnosis was associated with a median time to symptomatic hyperviscosity of 3 months, whereas the median time for patients with serum IgM level of 50·01-60 g/l was approximately 3 years. Adjusting for other clinical factors, the odds of developing symptomatic hyperviscosity were 370-fold higher with serum IgM levels >60 g/l, and showed an association with CXCR4 mutational status. Symptomatic hyperviscosity did not impact overall survival (P = 0·12). The findings support the use of serum IgM level >60 g/l as a criterion for initiation of therapy in an otherwise asymptomatic WM patient., (© 2017 John Wiley & Sons Ltd.)

Ibrutinib produces high response rates and durable remissions in Waldenström macroglobulinemia (WM) that are impacted by MYD88 and CXCR4 WHIM mutations. Disease progression can develop on ibrutinib, although the molecular basis remains to be clarified. We sequenced sorted CD19 + lymphoplasmacytic cells from 6 WM patients who progressed after achieving major responses on ibrutinib using Sanger, TA cloning and sequencing, and highly sensitive and allele-specific polymerase chain reaction (AS-PCR) assays that we developed for Bruton tyrosine kinase ( BTK ) mutations. AS-PCR assays were used to screen patients with and without progressive disease on ibrutinib, and ibrutinib-naïve disease. Targeted next-generation sequencing was used to validate AS-PCR findings, assess for other BTK mutations, and other targets in B-cell receptor and MYD88 signaling. Among the 6 progressing patients, 3 had BTK Cys481 variants that included BTK Cys481Ser(c.1635G>C and c.1634T>A) and BTK Cys481Arg(c.1634T>C) Two of these patients had multiple BTK mutations. Screening of 38 additional patients on ibrutinib without clinical progression identified BTK Cys481 mutations in 2 (5.1%) individuals, both of whom subsequently progressed. BTK Cys481 mutations were not detected in baseline samples or in 100 ibrutinib-naive WM patients. Using mutated MYD88 as a tumor marker, BTK Cys481 mutations were subclonal, with a highly variable clonal distribution. Targeted deep-sequencing confirmed AS-PCR findings, and identified an additional BTK Cys481Tyr(c.1634G>A) mutation in the 2 patients with multiple other BTK Cys481 mutations, as well as CARD11 Leu878Phe(c.2632C>T) and PLCγ2 Tyr495His(c.1483T>C) mutations. Four of the 5 patients with BTK C481 variants were CXCR4 mutated. BTK Cys481 mutations are common in WM patients with clinical progression on ibrutinib, and are associated with mutated CXCR4 ., (© 2017 by The American Society of Hematology.)

Histological transformation to diffuse large B-cell lymphoma (DLBCL) rarely occurs in patients with Waldenström Macroglobulinemia (WM). We identified 20 patients out of a cohort of 1,466 WM patients who experienced histologic transformation. The 5, 10, and 15-year cumulative incidence rates were 1, 2.4, and 3.8%, respectively. Approximately half of the patients were naive to nucleoside analogues, and a quarter were previously untreated for WM at the time of transformation. More than 80% of patients presented with extranodal involvement, 65% with high IPI scores. DLBCL cells did not express CD10 but expressed BCL6 and BCL2. All patients were treated with chemoimmunotherapy. The median survival from histological transformation was 2.7 years. The median overall survival was shorter for transformed patients versus those who did not transform (estimated 9 vs. 16 years; P = 0.09). Histological transformation to DLBCL is rare, and is associated with inferior survival in WM. Am. J. Hematol. 91:1032-1035, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Whole-genome sequencing has identified highly prevalent somatic mutations including MYD88, CXCR4, and ARID1A in Waldenström macroglobulinemia (WM). The impact of these and other somatic mutations on transcriptional regulation in WM remains to be clarified. We performed next-generation transcriptional profiling in 57 WM patients and compared findings to healthy donor B cells. Compared with healthy donors, WM patient samples showed greatly enhanced expression of the VDJ recombination genes DNTT, RAG1, and RAG2, but not AICDA Genes related to CXCR4 signaling were also upregulated and included CXCR4, CXCL12, and VCAM1 regardless of CXCR4 mutation status, indicating a potential role for CXCR4 signaling in all WM patients. The WM transcriptional profile was equally dissimilar to healthy memory B cells and circulating B cells likely due increased differentiation rather than cellular origin. The profile for CXCR4 mutations corresponded to diminished B-cell differentiation and suppression of tumor suppressors upregulated by MYD88 mutations in a manner associated with the suppression of TLR4 signaling relative to those mutated for MYD88 alone. Promoter methylation studies of top findings failed to explain this suppressive effect but identified aberrant methylation patterns in MYD88 wild-type patients. CXCR4 and MYD88 transcription were negatively correlated, demonstrated allele-specific transcription bias, and, along with CXCL13, were associated with bone marrow disease involvement. Distinct gene expression profiles for patients with wild-type MYD88, mutated ARID1A, familial predisposition to WM, chr6q deletions, chr3q amplifications, and trisomy 4 are also described. The findings provide novel insights into the molecular pathogenesis and opportunities for targeted therapeutic strategies for WM.