CXCR4 Mutations Research Articles

Long-Term Follow-up of Previously Treated Patients Who Received Ibrutinib for Symptomatic Waldenstrom's Macroglobulinemia: Update of Pivotal Clinical Trial

MYD88 mutations are present in 95% of WM patients and trigger malignant cell growth through Bruton's Tyrosine Kinase and Hematopoietic Cell Kinase, both targets of ibrutinib. CXCR4 mutations are found in 30-40% of previously treated WM patients and confer in vitro resistance to ibrutinib. We therefore performed a prospective study that examined the activity of ibrutinib in previously treated WM patients (Treon et al, NEJM 2015). The findings showed that ibrutinib was highly active in previously treated WM patients and supported the first ever FDA and EMA drug approval for the treatment of WM. Herein, we provide a long-term follow-up of this study. Sixty-three symptomatic WM patients who received at least one prior therapy were enrolled. Their baseline characteristics were as follows: median age 63 (range 44-86 yrs); 48 (76%) were male, IPSSWM scores were low (n=14; 22%), intermediate (n=27; 43%), and high (n=22; 35%); median serum IgM was 3,520 (range 724-8,390 mg/dL); median hemoglobin level was 10.5 (range 8.2-13.8 g/dL); median serum B2M level was 3.9 (1.3-14.2 mg/L); adenopathy >1.5 cm (n=37; 59%); splenomegaly >15 cm (n=7; 11%), and median bone marrow disease involvement was 60% (range 3-95%). The median prior therapies was 2 (range 1-9), and 25 (40%) patients were refractory to their previous therapy. MYD88 and CXCR4 genotyping was performed for 63 and 62 patients, respectively and is shown in Table 1. Patients were initiated on 420 mg a day of ibrutinib, and dose de-escalation for toxicity was permitted. Patients received protocol administered ibrutinib for 40 months, and transitioned to commercial supply thereafter. Patients could consent to continue follow-up for response assessment after 40 months on commercial drug supply. Ibrutinib was administered until progression or intolerance. The median time on ibrutinib was 46.6 (range 0.5-60 months). Improvements in categorical responses occurred with prolonged treatment. The overall and major (>PR) response rates were 90.4% and 77.7%, respectively, and were not impacted by prior lines of therapy or relapsed or refractory status. Seventeen (27%) patients achieved a VGPR. No complete responses were observed. At best response, median serum IgM level declined from 3,520 to 821 mg/dL (p<0.0001). At baseline 46/63 (73%) patients had a serum IgM >3,000 mg/dL versus 4/63 (6%) patients at best response (p<0.0001). At best response, median bone marrow involvement declined from 60% to 20% (p<0.0001), and the median hemoglobin level increased from 10.5 to 14.2 g/dL (p<0.0001). The impact of MYD88 and CXCR4 mutation status on responses and time to at least a minor (overall) and PR or better (major) response are shown in Table 1.Figure 1 shows the Kaplan Meier curves for progression free survival (PFS) for all study participants (A), and those genotyped by MYD88 and CXCR4 mutation status (B). With a median study follow-up of 47.1 months, the median PFS for patients with MYD88MutCXCR4WT has not been reached. By comparison, the median PFS was 45 months for patients with MYD88MutCXCR4Mut, and 21 months for those with MYD88WTCXCR4WT (Log-rank p=0.0093 for 3-way comparison). Three patients died due to disease progression.Adverse events (Grade >2) that were at least possibly related to protocol therapy in >5% of patients during protocol therapy were as follows: anemia (n=4); atrial fibrillation (n=6); GERD (n=5); hypertension (n=5); neutropenia (n=14); pneumonia (n=6); skin infection (n=3); thrombocytopenia (n=9). Seven patients (11%) had atrial arrhythmia [Grade 1 (n=1); Grade 2 (n=5); Grade 3 (n=1)] on ibrutinib. Six of these patients continued ibrutinib with medical management for their atrial arrhythmia. Four patients came off protocol therapy for toxicity: atrial fibrillation (n=1); infection not related to drug therapy (n=1), procedure related hematoma (n=1), and thrombocytopenia (n=1). The findings from the long-term follow-up of this pivotal study confirm that ibrutinib is highly active in symptomatic patients with relapsed and refractory WM, and produces durable responses. Prolonged ibrutinib therapy is associated with improvements in categorical responses, including attainment of VGPR. Ibrutinib response activity and PFS are impacted by MYD88 and CXCR4 mutation status in this patient population. (ClinicalTrials.gov number, NCT01614821.) [Display omitted] DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding. Laubach:Novartis, Takeda, Celgene: Consultancy; Novartis, Takeda, Celgene, Onyx: Research Funding. Castillo:Pharmacyclics: Consultancy, Research Funding; Abbvie: Research Funding; Millennium: Research Funding; Janssen: Consultancy, Research Funding. Palomba:Merck: Consultancy. Advani:Pharmacyclics: Consultancy; Pharmacyclics: Research Funding; Janssen: Research Funding; Infinity: Research Funding; Juno Therapeutics: Consultancy; Agensys: Research Funding; Celgene: Research Funding; Nanostring: Consultancy; Cell Medica: Research Funding; Genentech: Research Funding; Merck: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Gilead: Consultancy; Seattle Genetics: Research Funding; Bayer Healthcare Pharmaceuticals: Research Funding; FortySeven: Research Funding; Spectrum: Consultancy; Sutro: Consultancy; Regeneron: Research Funding; Millennium: Research Funding; Kura: Research Funding.

TAS4464, a Novel, Highly Potent NEDD8-Activating Enzyme Inhibitor Shows Antitumor Activity in Ibrutinib-Insensitive Mantle Cell Lymphoma Via Inactivation of the NF-κB Pathway

Background: Although ibrutinib, a Bruton tyrosine kinase inhibitor, is becoming widely used to treat patients with mantle cell lymphoma (MCL), about one-third of patients do not respond, leaving them with limited therapeutic options. Effective therapy is, therefore, urgently needed for ibrutinib-resistant MCL patients. The mechanisms of ibrutinib intrinsic and acquired resistance in MCL include activation of the alternative nuclear factor-κB (NF-κB) pathway, CARD11 mutation, and BTK C481S mutation. PLCG2, CXCR4, and MYD88 mutations are resistant mechanisms in other cancers. A characteristic common to these resistance mechanisms is the activation of NF-κB signaling mediated by two signaling cascades, the canonical and non-canonical pathways. TAS4464, a highly selective, and currently the most potent, inhibitor of NEDD8-activating enzyme (NAE), inhibits both the canonical and non-canonical pathways of NF-κB via NAE inhibition. Here, we report the antitumor activity of TAS4464 in ibrutinib-insensitive MCL preclinical models via modulation of both NF-κB pathways.Materials and methods: The biological activity of TAS4464 was evaluated in 7 different human MCL cell lines. Intracellular ATP levels were measured to assess in vitro cell growth. Effects on the cell cycle were analyzed by using fluorescence-activated cell sorting. The effects of TAS4464 on NEDD8 conjugation and cullin-RING ubiquitin ligase (CRL) substrate levels were evaluated by western blot analysis. Activated p65 and RelB (NF-κB subunits) were quantified by using commercial DNA-binding ELISA kits. The levels of NF-κB-targeted gene transcripts were assessed by quantitative real-time-PCR. Antitumor activities and the survival benefit of TAS4464 were evaluated in subcutaneous and systemic human MCL xenograft models, respectively.Results: TAS4464 in the nanomolar range inhibited cell growth and at 100 nmol/L induced apoptosis within 24 h in all tested MCL cell lines, whereas ibrutinib was less active (GI50 values >1 µmol/L) in 4 of the 7 cell lines tested. In ibrutinib-resistant cells, non-canonical NF-κB signaling (MAVER-1 and Z-138), or both non-canonical and B-cell antigen receptor (BCR)-independent canonical NF-κB signaling (GRANTA-519 and JVM-2), is reportedly activated. Ibrutinib suppressed p65 activity in BCR dependent-ibrutinib sensitive (REC-1) and moderately sensitive (JEKO-1 and Mino) cell lines but did not suppress p65 in the ibrutinib-insensitive cell lines, suggesting that ibrutinib inhibits only the BCR signal-driven canonical NF-κB pathway. In both ibrutinib-sensitive and -insensitive cell lines, TAS4464 inhibited NEDD8 conjugation to CRLs, with subsequent accumulation of CRL substrate proteins, including phosphorylated-IκBα (p-IκBα) and phosphorylated-p100 (p-p100), which are components of the canonical and non-canonical NF-κB pathways, respectively. This in turn suppressed p65 (canonical) and RelB (non-canonical) activity, and NF-κB-targeted gene expression. Intravenous administration of TAS4464 once or twice a week induced strong antitumor effects and led to tumor regression in subcutaneous human MCL Mino and Z-138 xenograft models; antitumor effects included inhibiting cullin neddylation, inducing caspase cleavage, and downregulating NF-κB-targeted gene transcription in the tumors. The antitumor activity of TAS4464 exceeded those of ibrutinib and bortezomib. Furthermore, in a human MCL GRANTA-519 systemic xenograft model, intravenous administration of TAS4464 once or twice a week doubled the survival times of mice, whereas ibrutinib did not prolong survival.Conclusions: TAS4464 inhibits cell growth and induces apoptosis in MCL cell lines through inactivation of both the canonical and non-canonical NF-κB pathways, in contrast to the limited effects of ibrutinib in BCR signal-driven cell lines. Our results suggest that inhibition of NF-κB pathways by modulating the amounts of p-IκBα and p-p100 is a potential treatment approach for MCL. TAS4464 exerts marked antitumor activity in subcutaneous human MCL xenograft models, in which neither bortezomib nor ibrutinib is effective, and it has a survival benefit in a human MCL systemic xenograft model. Thus, TAS4464 may be a valuable addition to current treatment options for refractory/relapsed MCL. DisclosuresMuraoka:Taiho Pharmaceutical Co., Ltd.: Employment. Yoshimura:Taiho Pharmaceutical Co., Ltd.: Employment. Tsuji:Taiho Pharmaceutical Co., Ltd.: Employment. Hashimoto:Taiho Pharmaceutical Co., Ltd.: Employment. Nakagawa:Taiho Pharmaceutical Co., Ltd.: Employment. Mizutani:Taiho Pharmaceutical Co., Ltd.: Employment. Ohkubo:Taiho Pharmaceutical Co., Ltd.: Employment. Matsuo:Taiho Pharmaceutical Co., Ltd.: Employment. Utsugi:Taiho Pharmaceutical Co., Ltd.: Employment. Iwasawa:Taiho Pharmaceutical Co., Ltd.: Employment.

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.

Acquired Von Willebrand Disease in Patients with Waldenström Macroglobulinemia

Introduction: Acquired von Willebrand disease (VWD) is uncommonly observed in patients with Waldenström Macroglobulinemia (WM), and necessitates treatment intervention. The clinical characteristics for WM patients with acquired VWD, as well as outcomes following therapy have not been well characterized.Methods: We carried a retrospective study in patients with new diagnosed diagnosed WM who were evaluated at our institution, and tested for the presence of VWD. The presence of any VWD was evaluated by measuring levels of VW factor antigen (VWFAG), VW factor activity (VWFACT) and factor VIII (FVIII). Low-level VWD was diagnosed if VWFAG, VWFACT and FVIII levels were between 30% and 50%. VWD was diagnosed if VFWAG, VWFACT and FVIII were <30%. Univariate and multivariate logistic regression models were fitted to evaluate association of clinical factors and presence of any VWD. We also evaluated the effect of response to therapy on VWFAG, VWFACT and FVIII levels.Results: A total of 320 patients with WM were tested for VWD, of whom 271 (85%) showed no evidence of deficiency, and 49 (15%) showed any VWD. Patients were tested because they provided a history of easy bruising or bleeding. Thirty patients had low-level VWD (61%) and 19 were VWD (39%). The median IgM level of patients with any VWD was 5,660 mg/dl (range 1,800-10,300 mg/dl) and 2270 mg/dl (95% CI 25-11,000 mg/dl) in patients without VWD. Patients with any VWD were more likely to have IgM levels 3,000-5,999 mg/dl and 6,000+ mg/dl (47% vs. 31%, and 45% vs. 6%, respectively; p<0.001) and less likely to have white blood cell count (WBC) >6 K/uL (29% vs. 50%; p=0.006) than patients without VWD. There were no differences in age, sex, hemoglobin levels, platelet count and bone marrow involvement between WM patients with any and without VWD. In the multivariate regression analyses, patients with IgM levels 3,000-5,999 mg/dl and 6,000+ mg/dl had OR 11.1 (95% CI 3.7-33.3; p<0.001) and OR 48.7 (95% CI 14.7-160.8; p<0.001), respectively, of having any VWD. WBC was not associated with presence of any VWD (p=0.34). In the subset of patients who had genomic testing performed (n=125), patients with any VWD were more likely to carry CXCR4 mutations (77% vs. 37%, p<0.001) than patients without VWD. There were no clinical or genomic differences between patients with low level VWD and actual VWD. Of 18 patients with any VWD who received therapy for WM, 6 patients (33%) received proteasome inhibitors + rituximab, 5 (28%) received alkylators + rituximab, 5 (28%) received ibrutinib and 2 (11%) received rituximab alone. Three patients (17%) achieved very good partial response (VGPR), 6 (33%) partial response, 8 (44%) minor response and 1 (11%) stable disease. At the moment of best response, the median IgM level had decreased from 6241 mg/dl to 2274 mg/dl, the median VWFAG level had increased from 21% to 64%, the median level VWFACT had increased from 23% to 58% and the median FVIII had increased from 21% to 57% (p<0.001 for all comparisons). Before therapy, 6 patients (33%) had low-level VWD and 12 (67%) had VWD. After therapy, 14 patients (78%) did not have VWD, 4 (22%) had low-level VWD and no patients had VWD. The depth of response to therapy (change in IgM) correlated with the percentage of change in VFWAG, VWFACT and FVIII (see Figure).Conclusion: Patients with WM can present with acquired VWD, which seems to strongly correlate with high serum IgM levels. VWD improves and in most cases resolves with response to WM therapy. The depth of response to therapy correlates with the degree of improvement in VWD. [Display omitted] DisclosuresCastillo:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.

Carfilzomib-Based Combination Regimens Are Highly Effective Frontline Therapies for Multiple Myeloma and Waldenström's Macroglobulinemia

Introduction:Multiple myeloma (MM) and Waldenström's macroglobulinemia (WM) are related plasma cell disorders that respond to treatment with proteasome inhibitors. Several early phase studies have shown that carfilzomib (Cz) is highly effective as the initial treatment of MM and results in deep remissions in most patients. A randomized phase III study is evaluating the efficacy of Cz or bortezomib with lenalidomide and dexamethasone as frontline therapy in MM and could lead to changes in the treatment of choice (NCT01863550). A clinical trial also showed good tolerance and efficacy of Cz in the frontline treatment of WM. In this study we aim to report the efficacy and safety of frontline Cz-based combinations in MM and WM in a standard of care setting.Methods:In this retrospective, single center study, we queried our ambulatory and inpatient (IPATC) database to identify subjects who were diagnosed with MM or WM and received Cz as initial therapy from 4/1/2014 to 11/30/2016. Investigational Cz was excluded. We identified 54 patients with MM (including one with concurrent lambda AL amyloidosis) and 6 patients with WM. Responses were assessed based on guidelines published by the International Myeloma Working Group (Kumar et al. Lancet 2016) and the VI international workshop on WM (Owen et al. BJH 2013). The Kaplan-Meier method was used to estimate time-to-event endpoints including progression free survival (PFS). Statistical software SAS 9.3 (SAS, Cary, NC) and S-Plus 8.2 (TIBCO Software Inc., Palo Alto, CA) were used for all the analyses.Results:All MM patients were treated with either KRd (Cz, lenalidomide, dexamethasone) (n=52, 96%) or KCd (Cz, cyclophosphamide and dexamethasone) (n=2, 4%). Median age at diagnosis was 66 years (range 60-70 years). Revised International Staging System (R-ISS) stage I, II or III was present in 20 (37%), 28 (52%) and 6 (11%) patients, respectively. Patients presented with either CRAB criteria (n=48, 89%) or biomarkers of malignancy (n=4, 7%) and 22% had extramedullary disease. Gene expression profiling-70 (GEP-70, Quest Diagnostics) was available for 25 patients (Table 1a). Patients with both high-risk GEP and FISH had early relapse (Table 1b). The maximum doses of Cz used were 36 mg/m2 (n=44), 27 mg/m2 (n=6) and not available (n=4). After a median of 3 cycles, eligible patients underwent autologous stem cell transplant (ASCT) (n= 36, 77%). Fifty-two patients were evaluable for response. After a median of 3 cycles of initial treatment, overall response (ORR) was 98% [complete remission (CR; n=9, 17%), very good partial response (VGPR; n=27, 52%) and partial response (PR; n=15, 29%)]. After ASCT, responses deepened to CR (n=18, 50%), VGPR (n=9, 25%) and PR (n=4, 11%). Eleven patients did not undergo ASCT (comorbidities: 6, insurance issues: 1, patient preference: 3, progression: 1). The median number of cycles received in this group was 4 and best responses were CR (n=3, 30%), VGPR (n=2, 20%), PR (n=5, 50%) while 1 not evaluable. At a median follow up of 7.4 months, overall survival (OS) is 100% while median PFS has not been reached (Figure 1). Five patients discontinued therapy due to toxicity (one of each with pericarditis, shortness of breath, pulmonary toxicity, neuropathy, retroperitoneal bleeding) and 3 due to progressive disease. There were no reported cases of heart failure.Patients with WM, n=6, [MYD88 mutation positive (n=5, 83%), CXCR4 mutation positive (n=0)] were treated with Cz, rituximab and dexamethasone induction followed by maintenance as described by Treon et al. (Blood 2014). Median age was 57 years (range 52-74 years). Patients presented with B symptoms (n=6, 100%) and anemia (n=5, 83%). After a median of 6 cycles of therapy, ORR was 66% [CR: n=1 (17%), PR: n=3 (50%)] while 2 (33%) had minimal response. All patients had resolution of B symptoms and anemia after treatment. One patient (MYD88 positive, CXCR4 negative) relapsed 27 months after starting initial treatment. Only one patient had severe toxicity (debilitating neuropathy attributed to rituximab) prompting therapy discontinuation after 3 cycles (Alfaraj et al, Ann Hematol. 2016). At a median follow up of 33.5 months, OS is 100%. Mutational status did not seem to impact response to therapy.Conclusion:Cz based regimens are generally well tolerated and offer a neuropathy sparing approach with excellent responses in newly diagnosed MM and WM making them a good choice for frontline treatment of these diseases. [Display omitted] DisclosuresLee:Celgene: Consultancy; Eutropics Pharmaceuticals: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees; Pimera Inc: Consultancy; Daiichi Sankyo: Research Funding; Takeda: Consultancy. Orlowski:BioTheryX: Consultancy, Membership on an entity's Board of Directors or advisory committees. Manasanch:quest diagnostics: Research Funding; merck: Research Funding; celgene: Consultancy; takeda: Consultancy; sanofi: Research Funding; adaptive biotechnologies: Consultancy.

Molecular and genetic biomarkers implemented from next-generation sequencing provide treatment insights in clinical practice for Waldenström macroglobulinemia

Waldenström macroglobulinemia (WM) is a distinct type of indolent lymphoplasmacytic lymphoma (LPL) with a high frequency of MYD88L265P mutation. Treatment for WM/LPL is highly variable in clinic and ibrutinib (a Bruton tyrosine kinase inhibitor, BTKi) has become a new treatment option for WM. To investigate the clinical impact of genetic alterations in WM, we assembled a large cohort of 219 WMs and 12 LPLs dividing into two subcohorts: a training cohort, patients sequenced by a same targeted 29-gene next-generation sequencing (NGS) panel, and a validation cohort, patients sequenced by allele specific-PCR or other targeted NGS panels. In both training and validation subcohorts, MYD88L265P and TP53 mutations showed favorable and adverse prognostic effects, respectively. CXCR4 nonsense/missense mutations (CXCR4NS/MS), cytogenetic complex karyotypes, and a family history of lymphoma/leukemia in first-degree relatives were associated with significantly worse clinical outcomes only or more in the validation subcohort. We further investigated the efficacy of various treatments and interaction with genetic factors in the entire cohort. Upfront dexamethasone usage was associated with poorer clinical outcomes in patients who received non-proteasome-containing chemotherapy as first-line treatment independent of genetic factors. Maintenance rituximab was associated with better survival. Ibrutinib/BTKi showed potential benefit in relapsed/refractory patients and patients without CXCR4NS/MS including those with TP53 mutations. In conclusion, genetic testing for MYD88L265P, TP53, and CXCR4 mutations and cytogenetic analysis provide important information for prognosis prediction and therapy selection. The findings in these study are valuable for improving treatment decisions on therapies available for WM/LPL patients with integration of NGS in clinic.

Bone marrow involvement and subclonal diversity impairs detection of mutated CXCR4 by diagnostic next-generation sequencing in Waldenström macroglobulinaemia.

CXCR4 mutations impact disease presentation and treatment outcomes in Waldenström macroglobulinaemia (WM). Non-uniform testing for CXCR4 mutations may account for discordant findings in WM clinical trials. We compared two approaches used in these trials for detection of the most common CXCR4 (S338X) variant: targeted next-generation sequencing (NGS) using unselected bone marrow (BM) samples, and combined allele-specific polymerase chain reaction (AS-PCR) and Sanger sequencing with unselected and CD19-selected BM samples. Our findings showed that targeted NGS frequently yielded false-negative results. Both CD19 selection and AS-PCR markedly improved detection of CXCR4S338X mutations. Sensitivity was adversely impacted by low BM involvement and CXCR4 mutation clonality.

Flow cytometry detection of CD138 expression continuum between monotypic B and plasma cells is associated with both high IgM peak levels and MYD88 mutation and contributes to diagnosis of Waldenström macroglobulinemia

Differential diagnosis of Waldenström macroglobulinemia (WM) with other indolent B-cell malignancies is still a challenge. Here, we propose an original and simple analysis of routine flow cytometry (FCM) unraveling the characteristic ongoing plasma cell (PC) differentiation of WM tumor B-cells. FCM analysis of both B-cells and PC was performed on a series of 77 patients with IgM peak. MYD88 and CXCR4 mutations were studied using an allele-specific PCR and by high throughput sequencing. Twenty seven (35%), 46 (58%) and 4 (5%) patients were classified as WM, IgM monoclonal gammopathy of undetermined significance (MGUS) or other B-NHL respectively. MYD88 mutation was found in 25/27 WM (93%) and in 29/46 MGUS (63%). Using FCM, monotypic B-cells were found in 27/27 WM (100%) and 34/46 MGUS (74%). Monotypic CD138pos/CD38pos PCs were detected in 23/27 WM (85%) and 25/46 MGUS (54%). Highlighting the ongoing PC differentiation of WM tumor B-cells by FCM, we evidenced a CD138 expression continuum between monotypic B-cells and PCs. This pattern remained absent in control samples and was significantly associated with higher IgM peaks (p=6.10-5 ) and MYD88 mutations (p=10-3 ) in both WM and MGUS cases. FCM exploration of both B-cells and PC led to identify a CD138 expression continuum as an objective marker of ongoing PC differentiation of WM tumor cells and was strongly associated with increased IgM peak levels and MYD88 mutations. This approach could contribute to place FCM at the forefront of WM diagnosis.

Targeting of CXCR4 by the Naturally Occurring CXCR4 Antagonist EPI-X4 in Waldenström's Macroglobulinemia.

CXCR4 expression and downstream signaling have been identified as key factors in malignant hematopoiesis. Thus, up to 40% of all patients with Waldenström's macroglobulinemia (WM) carry an activating mutation of CXCR4 that leads to a more aggressive clinical course and inferior outcome upon treatment with the Bruton's tyrosine kinase inhibitor ibrutinib. Nevertheless, little is known about physiological mechanisms counteracting CXCR4 signaling in hematopoietic neoplasms. Recently, the endogenous human peptide EPI-X4 was identified as a natural CXCR4 antagonist that effectively blocks CXCL12-mediated receptor internalization and suppresses the migration and invasion of cancer cells towards a CXCL12 gradient. Here, we demonstrate that EPI-X4 efficiently binds to CXCR4 of WM cells and decreases their migration towards CXCL12. The CXCR4 inhibitory activity of EPI-X4 is accompanied by reduced expression of genes involved in MAPK signaling and energy metabolism. Notably, the anti-WM activity of EPI-X4 could be further augmented by the rational design of EPI-X4 derivatives showing higher binding affinity to CXCR4. In summary, these data demonstrate that a naturally occurring anti-CXCR4 peptide is able to interfere with WM cell behaviour, and that optimized derivatives of EPI-X4 may represent a promising approach in suppressing growth promoting CXCR4 signaling in WM.

Hematologic disorder–associated Cxcr4 gain-of-function mutation leads to uncontrolled extrafollicular immune response

The extrafollicular immune response is essential to generate a rapid but transient wave of protective antibodies during infection. Despite its importance, the molecular mechanisms controlling this first response are poorly understood. Here, we demonstrate that enhanced Cxcr4 signaling caused by defective receptor desensitization leads to exacerbated extrafollicular B-cell response. Using a mouse model bearing a gain-of-function mutation of Cxcr4 described in 2 human hematologic disorders, warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome and Waldenström macroglobulinemia, we demonstrated that mutant B cells exhibited enhanced mechanistic target of rapamycin signaling, cycled more, and differentiated more potently into plasma cells than wild-type B cells after Toll-like receptor (TLR) stimulation. Moreover, Cxcr4 gain of function promoted enhanced homing and persistence of immature plasma cells in the bone marrow, a phenomenon recapitulated in WHIM syndrome patient samples. This translated in increased and more sustained production of antibodies after T-independent immunization in Cxcr4 mutant mice. Thus, our results establish that fine-tuning of Cxcr4 signaling is essential to limit the strength and length of the extrafollicular immune response.

TREC Screening for WHIM Syndrome.

T cell receptor excision circle (TREC) quantification is a recent addition to newborn screening (NBS) programs and is intended to identify infants with severe combined immunodeficiencies (SCID). However, other primary immunodeficiency diseases (PID) have also been identified as the result of TREC screening. We recently reported a newborn with a low TREC level on day 1 of life who was diagnosed with WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome, a non-SCID primary immunodeficiency caused by mutations in the chemokine receptor CXCR4. We have now retrospectively reviewed the birth and clinical histories of all known WHIM infants born after the implementation of NBS for SCID. We identified six infants with confirmed WHIM syndrome who also had TREC quantification on NBS.Three of the six WHIM infants had low TREC levels on NBS. All sixpatients were lymphopenic but only one infant had a T cell count below 1,500 cells/μL. The most commonclinical manifestation was viral bronchiolitis requiring hospitalization. One infant died of complications related toTetralogy of Fallot, a known WHIM phenotype. The results suggest that WHIM syndrome should be considered in the differential diagnosis of newborns with low NBS TREC levels. Not applicable.

Genetic characterization of B-cell prolymphocytic leukemia: a prognostic model involving MYC and TP53

AbstractB-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessments of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex (≥3 abnormalities) in 73% of the patients and highly complex (≥5 abnormalities) in 45%. The most frequent chromosomal aberrations were translocations involving MYC [t(MYC)] (62%), deletion (del)17p (38%), trisomy (tri)18 (30%), del13q (29%), tri3 (24%), tri12 (24%), and del8p (23%). Twenty-six (76%) of the 34 patients exhibited an MYC aberration, resulting from mutually exclusive translocations or gains. Whole-exome sequencing revealed frequent mutations in TP53, MYD88, BCOR, MYC, SF3B1, SETD2, CHD2, CXCR4, and BCLAF1. The majority of B-PLL used the IGHV3 or IGHV4 subgroups (89%) and displayed significantly mutated IGHV genes (79%). We identified 3 distinct cytogenetic risk groups: low risk (no MYC aberration), intermediate risk (MYC aberration but no del17p), and high risk (MYC aberration and del17p) (P = .0006). In vitro drug response profiling revealed that the combination of a B-cell receptor or BCL2 inhibitor with OTX015 (a bromodomain and extra-terminal motif inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We concluded that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease.

Identification of 2 DNA methylation subtypes of Waldenström macroglobulinemia with plasma and memory B-cell features

AbstractEpigenetic changes during B-cell differentiation generate distinct DNA methylation signatures specific for B-cell subsets, including memory B cells (MBCs) and plasma cells (PCs). Waldenström macroglobulinemia (WM) is a B-cell malignancy uniquely comprising a mixture of lymphocytic and plasmacytic phenotypes. Here, we integrated genome-wide DNA methylation, transcriptome, mutation, and phenotypic features of tumor cells from 35 MYD88-mutated WM patients in relation to normal plasma and B-cell subsets. Patients naturally segregate into 2 groups according to DNA methylation patterns, related to normal MBC and PC profiles, and reminiscent of other memory and PC-derived malignancies. Concurrent analysis of DNA methylation changes in normal and WM development captured tumor-specific events, highlighting a selective reprogramming of enhancer regions in MBC-like WM and repressed and heterochromatic regions in PC-like WM. MBC-like WM hypomethylation was enriched in motifs belonging to PU.1, TCF3, and OCT2 transcription factors and involved elevated MYD88/TLR pathway activity. PC-like WM displayed marked global hypomethylation and selective overexpression of histone genes. Finally, WM subtypes exhibited differential genetic, phenotypic, and clinical features. MBC-like WM harbored significantly more clonal CXCR4 mutations (P = .015), deletion 13q (P = .006), splenomegaly (P = .02), and thrombocytopenia (P = .004), whereas PC-like WM harbored more deletion 6q (P = .012), gain 6p (P = .033), had increased frequencies of IGHV3 genes (P = .002), CD38 expression (P = 4.1e-5), and plasmacytic differentiation features (P = .008). Together, our findings illustrate a novel approach to subclassify WM patients using DNA methylation and reveal divergent molecular signatures among WM patients.

Variable expression of CXCR4 in molecular subtypes of infiltrating gliomas.

Glioblastomas (GBMs) and diffuse intrinsic pontine gliomas (DIPGs) are infiltrating gliomas with poor prognosis. CXCR4 has been linked to glioma cell invasion, survival, proliferation, and angiogenesis. This study aimed to evaluate the expression of CXCR4 in molecular subtypes of adult and pediatric infiltrating gliomas. We evaluated the expression of CXCR4 in 21 DIPGs and 44 adult infiltrating gliomas (25 GBM, 8 astrocytomas, and 11 oligodendrogliomas) by immunohistochemistry. Mutations in 315 cancer genes and rearrangements in 28 genes were evaluated by next-generation sequencing. CXCR4 was expressed in -DIPGs and adult infiltrating gliomas in tumor cells (28.6% and 5.6%, respectively) and endothelial cells (14.3% and 19.4%?, respectively). In adult gliomas, there was a correlation between CXCR4 expression and mutations in EGFR, PIK3CA, TERT promoter, and CDKN2A/B loss. In contrast, CXCR4 expression was not detected in IDH1/IDH2 mutant gliomas. These associations were confirmed using The Cancer Genome Atlas (TCGA) database. CXCR4 is expressed in a subset of DIPGs and GBMs, but it is not expressed in astrocytomas or oligodendrogliomas. CXCR4 expression is variable and it is influenced by tumor genomic alterations. It is important to consider CXCR4 expression in clinical trials that evaluate the efficacy of CXCR4 inhibitors in the treatment of gliomas.

Congenital Neutropenia Is Also Associated with a High Cancer Risk: A Study from the French Severe Chronic Neutropenia Registry

Introduction: Congenital neutropenia (CN) is characterized by chronic neutropenia due to a constitutional genetic defect.1 To date, these diseases have not been considered to be frequently associated with malignant solid tumors, unlike the risk of secondary myelodysplastic syndrome leukemia, which is well-known in CN. Methods: The French Severe Chronic Neutropenia Registry (FSCNR) has prospectively enrolled CN patients since 1993. Solid tumors, identified during routine patient follow-up, were classified according to WHO criteria. We included localized lymphoma in the spectrum of malignant solid tumors. We calculated the incidence of malignant solid tumors in a cohort of CN patients. Results: Among 868 patients with various CN subtypes followed for a total of 16617 person-years, 24 patients who developed a malignant solid tumor were identified. Those cancers are described in Table 1, including the CN genetic anomaly. Cancers were almost always diagnosed in adulthood, with median age at diagnosis of 38.1 (range 10-72) years; only 3 cancers were diagnosed before age of 20 years. The cancer rate was 1.2% at 30 years of age, 7% at 40 years and 24% at 50 years (Fig. 1A). The risk-of-cancer percentages depended mainly on the associated genetic deficiency. Solid tumors were roughly distributed as follows: 33% among WHIM (CXCR4) patients, 5.3% among GATA2 patients, 2.7% among ELANE patients, 1.9 % among SBDS patients and 0.8% among for all other subtypes combined (Fig. 1B). Human papillomavirus (HPV) was the cause of cancer for 2/5 in WHIM patients and 2/6 in GATA2 patients. Three Lymphoma were identified, one in GATA2 patient and 2 in WHIM patients. Notably, our cohort's follow-up is skewed to the right, with less efficient monitoring of adults, with still limited long-term follow-up beyond 40 years. Therefore, we probably underestimated the solid-tumor risk in CN patients, as many patients, if alive, are no longer followed in hematology centers. Among 103 patients who underwent hematopoietic stem-cell transplantation (HSCT), 76 were long-term survivors. None of them developed solid tumors, which differs strikingly from the high malignancy risk associated with Fanconi anemia post-HSCT. Lastly, the FSCNR also includes and follows patients with idiopathic neutropenia. Among the 232 idiopathic neutropenia patients, followed for a total of 2866 person-years, no malignancy has been observed so far. Conclusion: Our data lead us to advance that CN patients should be considered at risk of developing solid cancers, especially after the age of 30 years. This risk, at first glance, depended on the CN-associated genetic anomaly, with CXCR4 mutation, GATA2, SBDS and ELANE being the most frequent. HSCT was not associated with a higher risk and may, in contrast, be protective. These findings warrant confirmation but represent a compelling reason to prolong follow-up into adulthood of CN patients diagnosed during childhood. No indication was found of a specific high solid-tumor risk associated with idiopathic neutropenia. Reference Donadieu J, Beaupain B, Fenneteau O, Bellanne-Chantelot C. Congenital neutropenia in the era of genomics: classification, diagnosis, and natural history. Br.J.Haematol. 2017; 179(4): 557-574. Acknowledgments: The French SCN registry is supported by grants from Amgen, Chugai, Prolong Pharma, X4 Pharma, Inserm, the Association 111 les Arts, the Association RMHE, the Association Sportive de Saint Quentin Fallavier. The authors thank the association IRIS and Mrs Grosjean and Mr Gonnot(ASSQF), the association Barth France for their support. Disclosures Hermine: Roche: Consultancy; Celgene BMS: Consultancy, Research Funding; AB Science: Consultancy, Current equity holder in publicly-traded company, Honoraria, Patents &amp; Royalties, Research Funding; Alexion: Research Funding; Novartis: Research Funding. Blaise:Jazz Pharmaceuticals: Honoraria. Sicre de Fontbrune:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. cohen Beaussant:X4 Pharmaceuticals, Inc.: Current Employment.

Mutational Patterns and Correlation to Chip-Related Mutations in Hematological Malignancies - a Study on Mutation Frequencies of 122 Genes in 28 Entities Including 3096 Cases

Background: Acquired somatic mutations are crucial for the development of the majority of cancers. In hematological malignancies, some molecular mutations are very specific for certain entities (e.g. BRAF in HCL, MYD88 in LPL), while others were detected in a variety of malignancies (e.g. mutations in TP53, TET2, DNMT3A, RUNX1). Moreover, mutations in genes related to CHIP (clonal haematopoiesis of indeterminate potential; ASXL1, TET2,DNMT3A) were detected in an age-related manner. Aim: (1) Analysis/comparison of mutation frequencies of 122 selected genes in 3096 cases with 28 different hematological malignancies for identification of "mutation-driven" entities. (2) Correlation of CHIP-related mutations with mutational landscapes. Methods: Whole-genome sequencing (WGS) was performed for all 3096 patients. For this, 151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). The Illumina tumor/unmatched normal workflow was used for variant calling. All reported p-values are two-sided and were considered significant at p&amp;lt;0.05. Results: Entities with the highest numbers of mutations (median n=4) and thus potentially with the largest impact of mutations on pathogenesis comprised aCML (range: 1-7), CMML (1-6), MDS/MPN-U (2-5) and s-AML (2-8), whereas the lowest numbers (median n=0) were observed for CML (0-3), MGUS (0-2), MLN_eo (0-3), NK cell neoplasm (0-3) and PPBL (0-2). In the total cohort of 3096 cases, the most frequently mutated genes were TET2 (14%), ASXL1 (13%), TP53 (10%), SF3B1 (9%), DNMT3A (9%) and SRSF2 (9%). Entities with high frequencies of specific mutations (&amp;gt; 50%) comprised: aCML (ASXL1, 86%), BPDCN (TET2, 67%), BL (TP53, 60%), CMML (TET2, 67%; ASXL1, 58%), FL (KMT2D, 87% and CREBBP, 73%), HCL (BRAF, 100%), LPL (MYD88, 98%; CXCR4, 51%), MDS/MPN-U (ASXL1, 60%), MPN (JAK2, 68%), B-NHL (TP53, 50%) and T-NHL (STAT3, 52%). Mutations enriched in distinct entities included SETBP1 (26% in MDS/MPN overlaps), CSF3R (30% in MDS/MPN-U), STAT3 (only in T-NHL and NK cell neoplasm, 52% and 23%), NOTCH1 and PHF6 (T-ALL, 38% and 30%) and MYC and ID3 (almost exclusively in BL, 30% each). Genes predominantly mutated in myeloid neoplasms comprised e.g. SF3B1 (with the exception of CLL), JAK2, NPM1, RUNX1, IDH2, CEBPA, STAG2, NF1 and GATA2. By contrast, mutations in KMT2D, MYD88, ARID1A, ATM, CXCR4, BIRC3 and CD79B were detected almost exclusively in lymphoid malignancies. A broad distribution across entities was observed for mutations in TET2, ASXL1,DNMT3A, TP53, BCOR and ETV6. Thus, the first three, i.e. CHIP-related genes were also mutated with a high frequency in lymphoid neoplasms. In line with this, gene mutations found in the largest number of entities comprise DNMT3A (n=23 entities), TET2 (n=21), ASXL1, TP53, NRAS (n=19, respectively), KRAS and BCOR (n=17, respectively). Further, we compared the mutational patterns of cases with at least one CHIP-associated mutation (n=920 cases in the total cohort, "CHIP+") with cases without such mutations (n=2176, "CHIP-") to decipher CHIP-correlated mutation patterns. Significant differences with respect to accompanying mutations were mainly detected for myeloid neoplasms (MDS, mutations in n=12 genes significantly different in CHIP+ vs. CHIP- without CHIP genes themselves; AML, n=7; MPN, n=4; aCML, n=2; CMML, n=1) but also for MPAL (n=3), T-ALL (n=2), B-ALL, FL and LPL (n=1, respectively). Mutations in TP53 were found significantly enriched in CHIP- cases in 4 different entities, moreover mutations in KRAS, WT1 and SF3B1 were more abundant in CHIP- cases (in CMML, AML and aCML, respectively). By contrast, CHIP+ cases were characterized by high frequencies of mutations in RUNX1 (in n=4 entities), SRSF2, IDH2, NRAS (n=3) and EZH2 (n=2). Conclusions: (1) Certain mutations showed a broad distribution within or even across the myeloid/lymphoid lineage, including CHIP-related mutations frequently detected also in lymphoid malignancies. (2) The median numbers of mutations were low in entities that are defined by chromosomal fusions (CML, MLN_eo) or in entities that are regarded as "pre-malignant" (MGUS, PPBL), while especially MDS/MPN overlap cases seem to be mutation-driven (high number of mutations). (3) We deciphered different mutation patterns in CHIP+ (RUNX1, SRSF2, IDH2, NRAS, EZH2) and CHIP- (TP53, KRAS, WT1, SF3B1) cases across all entities, suggesting differences in pathophysiology. Disclosures No relevant conflicts of interest to declare.

Targeted Next Generation Sequencing Improves Diagnosis in Unclassifiable Leukemic Indolent B-Cell Non-Hodgkin Lymphoma and Identifies a Subset with Recurrent MYD88 Mutations in a Prospective Multicentre Study

Introduction: Patients with leukemic indolent B-cell Non-Hodgkin Lymphomas (LI B-NHL) that do not easily fall into a distinct pathological category with standard diagnostics can pose a challenge as their optimal management is uncertain. Lack of a clear diagnostic label can deny patients access to novel therapies as regulatory approval of drugs is largely granted within histological categories. Moreover, these patients may be excluded from participation in clinical trials. We report findings from a prospective study evaluating targeted next generation sequencing (NGS) of circulating malignant B-cells in this setting. Methods: Over a period of 4 years, 108 patients with LI B-NHL, deemed unclassifiable on standard peripheral blood (PB) diagnostics, were prospectively enrolled from 14 centres around the UK including ours. Patients with non-clonal lymphocytosis, confirmed CLL-type MBL, CLL (Matutes score 4 or 5), MCL, HCL, high grade lymphoma on standard PB diagnostics were excluded. Clinical and morphological characteristics were analysed. PB immunophenotyping was used for characterisation including ROR1 expression. CD15+ cells were positively selected with magnetic beads for germline DNA. Tumor-germline pairs were analysed with theEuroclonality-NGS DNA capture panel, and ARResT/Interrogate and complementary pipelines, to characterise clonal immunoglobulin rearrangements, translocations and somatic mutations (Stewart et al. ASH annual meeting 2019). Germline variants were subtracted to improve somatic mutation detection. Results: Median age was 72yrs with M:F ratio of 2:1. Incidental lymphocytosis on a routine blood count was the commonest mode of presentation with only a third of patients symptomatic at presentation. Median lymphocyte count was 12.4 x 109/L at the time of sampling. Just under 50% of cases had partial or complete CD5 expression, CD19 and 20 expression was moderate to strong in most cases while ROR1 was negative in most cases. NGS was able to assign a diagnostic category in 11/90 (12%) cases based on detection of IGH;CCND1 (6), IGH;BCL2 (2), IGH;BCL3 (2) translocations and in one case a BRAF mutation with confirmation of hairy cell leukaemia on immuno-morphology. These tests were not triggered by standard diagnostic pathways at initial presentation. Two novel translocations of uncertain significance were detected - RAD51B:BIRC3 and IGHMswitch-MICALL2/INTS1. Clonal IGHV-IGHD-IGHJ rearrangements were detected by NGS and/or Sanger sequencing with predominantly IGHV3 and IGHV4 gene usage, IGHV4-34 being the most common with 15 cases. The majority of rearrangements showed somatic hypermutations above 2% and none could be assigned to the 19 major CLL-associated stereotyped subsets (using ARResT/AssignSubsets). Somatic mutations were detected in 74/108 cases. The most frequently mutated genes were TP53 in 16/108 (15%) and the hotspot MYD88 L265P mutation in 14/108 (13%) with VAF of 20-46%. Other variants detected were distributed among genes associated with NFkB signalling and chromatin remodelling (Figure 1). The MYD88 L265P mutation was present in a fifth of cases with mutations detected (14/74) and was the sole change seen in 7 cases. Accompanying mutations in the remaining cases included CD79B (2), POT1 (1), NFKBIE (1). No concomitant CXCR4 mutations were detected. Median age of the MYD88 L265P cohort was 68 years with male predominance (M:F=10:4). 9/14 presented with an incidental lymphocytosis and median count of 11.18 x 109/L. Lymphadenopathy was present in 3/14 (21%) while 7/14 (50%) had splenomegaly. A paraprotein was detected in 6/14 cases (4 IgM, 1 IgG, 1 IgA). CD5 was expressed in 6/14 cases by flow cytometry. CD19 and 20 were uniformly positive, CD79b was variable and 12/14 cases tested did not express ROR1. Conclusion: This prospective study outlines the value of a targeted NGS panel in enhancing the diagnosis of unclassifiable LI B-NHL thereby improving patient access to novel therapies and clinical trials. It highlights recurrent MYD88 mutations in a subset of patients that have splenomegaly as a frequent feature. 5 year clinical follow up data, currently being gathered in both the MYD88 mutated and overall cohort, will be valuable in further characterisation and risk stratification to inform management of these patients. Disclosures Furtado: Abbvie: Other: Conference Support. El-Sharkawi:Abbvie: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Innate: Consultancy. Iyengar:Abbvie: Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Janssen: Honoraria; Beigene: Consultancy.

Abstract IA39: Investigating malignant transformation in Waldenstrom's macroglobulinemia

Abstract Waldenström's macroglobulinemia (WM) is an uncommon B-cell lymphoma that corresponds to the histopathologic classification of IgM-secreting lymphomplasmacytic lymphoma. It is also a disease characterized by a series of highly recurrent mutations. Whole-genome sequencing of CD19+ bone marrow cells from patients with WM led to the discovery of a somatic heterozygous c.978T&amp;gt;C mutation in Toll-like receptor adaptor protein MYD88, resulting in a leucine-to-proline substitution p.Leu265Pro (L265P) in over 90% of WM patients. Somatic activating mutations in MYD88 induce constitutive homodimerization and downstream signaling through the nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) pathway independent of receptor activation. Other somatic events characteristic of WM include deletions in chromosome 6q21-23 and activating nonsense and/or frameshift mutations in the carboxyl-terminal tail of CXCR4, found in 40-60% and over 30% of WM patients, respectively. Both of these events are found predominantly in MYD88 mutant WM and are frequently restricted to a subclonal population. Similar to related lymphomas, WM is thought to evolve from IgM-secreting monoclonal gammopathy of undermined significance (MGUS). The MYD88 mutations are easily detectible IgM MGUS stage, suggesting that it is a very early event in clonal development but may predate the malignant transformation into WM. This theory is support by several transgenic mouse models of mutant MYD88 that have demonstrated that p.Leu265Pro MYD88 mutation in B cells alone is not sufficient for lymphomagenesis (Knittel et al., Blood 2016; Sewastianik et al., Blood Adv 2019). Mutations in CXCR4 have been detected in IgM MGUS, though at lower rates than are typical of WM. Given that this and the transcriptional profile of CXCR4 activating mutations in WM are consistent with a pattern of suppression of tumor suppressors upregulated by mutant MYD88, it has been proposed as a possible transformation event from IgM MGUS to asymptomatic WM. Likewise, a number of careful genetic studies of IgM MGUS have suggested the emergence of somatic copy number alterations, particularly the deletions in 6q, to be related to the malignant transformation of WM (Schop et al., Cancer Genet Cytogenet 2006; Paiva et al., Blood 2015). We therefore reanalyzed our whole-genome sequencing data of WM patients and performed allele-specific PCR and 6q copy number assays to study the rates and relationship of CXCR4 mutations and chromosome 6q deletions in untreated MYD88 mutated WM. Both our 30-patient whole-genome and our 25-patient PCR cohort found no relationship between symptomatic and asymptomatic WM and the somatic events, indicating that they both were occurring at an earlier stage of WM development. Both studies also found that the large clonal deletions in chromosome 6q were not observed in the presence of CXCR4 mutations (p&amp;lt;0.01 for both). Using our previous RNASeq data of 55 WM patients, we compared the transcriptional signatures for CXCR4-mutated and 6q-deleted WM relative to WM patients with the MYD88 mutation alone, revealing an overlapping signature of 19 genes. Notably, the magnitude and relative direction of the changes were similar for CXCR4-mutated and 6q-deleted WM. Impacted genes included biologically relevant targets such as FAM110C, WNK2, CDK14, FOXO3, IGF2R, and HRK. Preliminary validation studies using qPCR and immunohistochemistry have confirmed these findings. We therefore propose that CXCR4 mutations and 6q deletions are not only appearing during the transformation from MGUS to WM but target a related set of genes that may play a critical role in the pathogenesis of MYD88 mutant WM. Functional studies to further characterize these genes and evaluate their potential as therapeutic targets for WM are ongoing. Citation Format: Zachary R. Hunter, Maria Luisa Guerrera, Guang Yang, Nicholas Tsakmaklis, Xia Lu, Steven P. Treon. Investigating malignant transformation in Waldenstrom's macroglobulinemia [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA39.

Long-Term Follow-Up of Ibrutinib Monotherapy in Symptomatic, Previously Treated Patients With Waldenström Macroglobulinemia.

PURPOSEWe report the long-term findings and final analysis of a pivotal multicenter trial of ibrutinib monotherapy in previously treated patients with Waldenström macroglobulinemia (WM).PATIENTS AND METHODSSixty-three symptomatic patients with median prior therapies of two (range, one to nine therapies), of whom 40% were refractory to their previous therapy, received ibrutinib at 420 mg/d. Dose reduction was permitted for toxicity.RESULTSThe median follow-up was 59 months, and overall and major response rates were 90.5% and 79.4%, respectively. At best response, median serum immunoglobulin M declined from 3,520 to 821 mg/dL, bone marrow disease involvement declined from 60% to 20%, and hemoglobin rose from 10.3 to 14.2 g/dL (P < .001 for all comparisons). Responses were impacted by mutated (Mut) MYD88 and CXCR4 status. Patients with MYD88Mut, wild-type (WT) CXCR4 showed higher major (97.2% v 68.2%; P < .0001) and very good partial (47.2% v 9.1%; P < .01) response rates and a shorter time to major response (1.8 v 4.7 months; P = .02) versus patients with MYD88MutCXCR4Mut. Conversely, four patients who had MYD88WT disease showed no major responses. The median 5-year progression-free survival (PFS) rate for all patients was not reached, and was 70% and 38% for those with MYD88MutCXCR4WT and MYD88MutCXCR4Mut WM, respectively (P = .02). In patients with MYD88WT, the median PFS was 0.4 years (P < .01 for three-way comparisons). The 5-year overall survival rate for all patients was 87%. Grade ≥ 3 adverse events in more than one patient at least possibly related included neutropenia (15.9%), thrombocytopenia (11.1%), and pneumonia (3.2%). Eight patients (12.7%) experienced atrial arrhythmia, and seven of the eight continued therapy with medical management.CONCLUSIONIbrutinib is highly active and produces long-term disease control in previously treated patients with WM. Treatment is tolerable. Response depth, time to major response, and PFS are impacted by MYD88 and CXCR4 mutation status.

Abstract 2119: Gene mapping in high-risk chronic lymphocytic leukemia pedigree identifies risk locus at chromosome 2q22.1

Abstract Chronic lymphocytic Leukemia (CLL) is an age-related heterogeneous B-cell leukemia. In familial studies CLL has been consistently shown to be one of the most heritable cancers, indicating that genetics play a factor in the risk of developing CLL. However, only a few genes so far have been found to confer disease-risk of CLL. We used shared genomic segment (SGS) analysis in one exceptionally large high-risk CLL pedigree of European ancestry and identified a genome-wide significant 0.93 Mb region on chromosome 2q22.1. This region was inherited through 26 meiosis to seven cases in the pedigree (p=2.0 × 10−7). This region overlaps a locus identified in a previous CLL linkage study, which adds evidence to this as a candidate region harboring disease risk alleles. There are two genes within our SGS region, CXCR4 and THSD7B. In particular, CXCR4, a B-cell chemokine receptor, is a strong functional candidate as it is overexpressed in many cancers, including CLL, and is current drug target for treatment of CLL. Rare germline mutations in CXCR4 have been previously identified in CLL cases and at least one variant (rs56400844) was found to segregate within a CLL pedigree. Interrogation of the germline coding space of the SGS region in five of the seven sharers did not identify any predicted deleterious shared alleles. Many non-coding alleles were shared, but further investigation will be necessary to prioritize those as potentially regulatory or if a haplotype of multiple alleles modulates the transcriptome. We next explored the germline coding space in the SGS region in two other existing data sources: 48 WGS from early onset, clinical high-risk or familial CLL cases and 161 WES from population-level CLL cases as part of the Oncology Research Information Exchange Network (ORIEN) Avatar Program. We identified a coding missense CXCR4 variant (rs377287446, AF= 0.0000559, CADD=26.2) in one case and an extremely rare 3' UTR variant (*290C&amp;gt;T, AF= 0.000007, singleton in gnomAD v3.0) present in two other cases. In summary, in the single largest high-risk pedigree studied to date, we have identified a genome-wide significant small region (&amp;lt;1 Mb) at 2q22.1 which overlaps a previously suggested susceptibility locus for CLL. Exome sequencing of the region in additional local CLL cohorts and previous studies have identified at least three candidate deleterious mutations in CXCR4, a strong functional candidate gene in the locus. Future work will concentrate on deciphering regulatory variants in the inherited SGS region to establish CXCR4 as a susceptibility risk gene for CLL. Citation Format: Julie Ellen Feusier, Michael J. Madsen, Karl Voelkerding, Martha Glenn, Nicola Camp. Gene mapping in high-risk chronic lymphocytic leukemia pedigree identifies risk locus at chromosome 2q22.1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2119.