ALBERT

Description: CD40 ligand (CD40L) is a potent inducer of normal and malignant B-cell proliferation through interaction with CD40. We and others have observed excess mast cells (MC) in bone marrow (BM) biopsies of WM patients, which are commonly found admixed with tumor aggregates. (Tournilhac et al, JCO 2004, 22:571S). We therefore sought to clarify the role of MC in WM. Co-culture of 0.5% paraformaldehyde fixed, or sublethally irradiated HMC-1, LAD, and KU mast or basophilic cell lines and sorted BM lymphoplasmacytic cells (LPC) from 10 WM patients resulted in MC dose-dependent tumor colony formation and/or proliferation as assessed by 3H-thymidine uptake studies. As demonstrated by immunohistochemical, multicolor flow cytometric (CD117+FceRI+) and/or RT-PCR analysis, CD40L was expressed on BM MC from 29 of 31 (94%), 11 of 13 (85%), and 7 of 9 (78%) of WM patients, respectively. In contrast, cell surface CD40L expression was not detected by immunohistochemistry (p=0.00005) and flow cytometry (p=0.003) in 5 normal donors, and only faint expression for 1 of 5 normal donors by RT-PCR (p=0.09). Moreover, by multicolor flow cytometry, CD40 was expressed on BM tumor cells from 14/17 (83%) patients. CD40 functionality was confirmed either by the G28.5 CD40 agonistic antibody which induced dose dependent proliferation or by the rh-CD40L which partly prevented serum starvation-induced-apoptosis of WM LPC from 4/4 and 3/4 patients respectively. Importantly, expansion of tumor cells from 3 of 4 patients in mixed cultures with paraformaldehyde fixed MC was blocked in a dose dependent manner by use of a CD40L blocking protein (CD40:Fc). These studies demonstrate that CD40L is constitutively expressed on the cell surface of BM MC in WM and support the growth of WM tumor cells, and therefore provide the framework for therapeutic targeting of MC and MC-WM cell interactions in WM.

Description: Background Elotuzumab is an approved monoclonal antibody targeting SLAMF7 on plasma and NK cells that enhances the activity of lenalidomide, pomalidomide, and bortezomib in multiple myeloma (MM). A recent study showed improved outcomes with the combination of pomalidomide, bortezomib, and dexamethasone vs. bortezomib and dexamethasone in relapsed or refractory MM (Richardson PG et al., Lancet Oncol 2019). We therefore studied elotuzumab with pomalidomide, bortezomib, and dexamethasone (elo-PVD) in relapsed and refractory MM. Methods The primary objective was to determine the overall response rate (ORR). Patients with relapsed and refractory disease and ≥1 prior lines of treatment (including lenalidomide and a proteasome inhibitor) were eligible to participate. Prior treatment with pomalidomide was permitted. Elotuzumab was weekly for the first 2 cycles and then every other week. Pomalidomide was given on days 1-21; bortezomib was on days 1, 8, 15; and dexamethasone was weekly. Each cycle was 28 days. Results The trial has completed accrual in September 2018 with 48 patients receiving treatment. The median age was 64 (range 40-80), and median number of prior regimens was 3 (range 1-9); 25% had high risk FISH. All patients had prior lenalidomide and proteasome inhibitor (bortezomib 96%, 29% carfilzomib) and were refractory to their last line of therapy. Other prior therapies included: autologous stem cell transplant (48%), pomalidomide (33%), daratumumab (25%), and isatuximab (4%). 46 patients were assessable for response (2 patients did not complete cycle 1 and were not evaluable for response: 1 due to rapid disease progression; 1 stroke. The median length of follow up was 18.8 months (range 0.5-23.4): 16 patients continue on study; 27 patients discontinued for progressive disease; 3 patients discontinued for adverse events (AEs) (sepsis, pneumonia, stroke); 1 patient underwent auto SCT; and 1 patient was lost to follow up. Best ORR was 61% (PR = 16, VGPR = 10, CR = 2). ORR for patients with prior anti-CD38 antibody, 46%; carfilzomib, 46%; pomalidomide, 43%. Median PFS was 9.8 months (95% CI 6.8-Inf). In patients with 1 prior line of therapy, ORR was 74% and median PFS was not reached (95% CI 12-Inf); 18 month PFS was 68%. Grade ≥ 3 hematologic AEs included anemia (10%), neutropenia (29%), and thrombocytopenia (15%). Additional common grade ≥ 3 AEs included lung infection (27%) and hypophosphatemia (15%). Common non-hematologic AEs all grades included fatigue (grade 1-2 only, 70%), upper respiratory infection (grade 1-2, 56%; grade 3, 2%), diarrhea (grade 1-2 only, 42%), constipation (grade 1-2 only, 35%), hyperglycemia (grade 1-2, 46%; grade 3, 4%), and sensory neuropathy (grade 1-2 only, 31%), with 2 possibly related deaths (sepsis, pneumonia). Conclusions Elo-PVD is one of the first trials of a quadruplet regimen in relapsed and refractory MM incorporating a monoclonal antibody. In patients with refractory disease, elo-PVD shows encouraging responses. With the limitations of cross trial comparisons and small patient numbers, for patients with 1 prior line of treatment and refractory disease, a PFS at 18 months of 68% with elo-PVD compares favorably with a median PFS of 17.8 months in a similar subgroup of PVD in the OPTIMISMM trial (Dimopoulos MA et al., ASH 2018). Patients who received prior pomalidomide, carfilzomib, and/or anti-CD38 monoclonal antibody also benefited. Treatment was well-tolerated with manageable toxicity and with attention to infectious AEs. Disclosures Yee: Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Karyopharm: Consultancy; Adaptive: Consultancy; Amgen: Consultancy, Honoraria. Lipe:Celgene: Consultancy; amgen: Research Funding; amgen: Consultancy. Nadeem:Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy. O'Donnell:Celgene: Consultancy; Amgen: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Takeda: Consultancy. Branagan:Pharmacyclics: Consultancy; Janssen: Consultancy; Surface Oncology: Consultancy. Lohr:Celgene: Research Funding; T2 Biosystems: Honoraria. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Richardson:Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Raje:Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene Corporation: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Merck: Consultancy. OffLabel Disclosure: The combination of elotuzumab, pomalidomide, bortezomib, and dexamethasone is an off-label use in relapsed and refractory multiple myeloma.

Description: Fludarabine and rituximab are commonly used in combination in the treatment of Waldenstrom’s macroglobulinemia (WM), though long term outcome of this regimen remains to be defined. We therefore examined the outcome of 43 WM patients treated on a clinical trial whose eligibility included 〈 2 prior therapies, and no previous nucleoside analogue or rituximab treatment. Therapy consisted of 6 cycles (25 mg/m2/day for 5 days) of fludarabine and 8 infusions (375 mg/m2/week) of rituximab over 31 weeks. 43 patients were enrolled with a median age of 61, and median prior therapies of 0. Responses were: CR (n=2); VGPR (n=14); PR (n=21); MR (n=4); for an overall and major response rate of 95.3% and 86.0%, respectively. At best response, median bone marrow disease involvement declined from 55% to 5% (p

Description: Recently, a consensus panel of experts recommended alkylator therapy, nucleoside analogues and rituximab as appropriate first line therapies for WM (Semin Oncol2003; 30:121). It was also recommended that candidates for future autologous transplantation should have limited alkylator or nucleoside analogue exposure due to potential stem cell harm. CHOP-R (cyclophosphamide, adriamycin, vincristine, prednisone, rituximab) is a highly active, stem cell sparing regimen that has been extensively evaluated in other low-grade Non-Hodgkin’s lymphoma patients. No published experience in WM exists. As such, we analyzed the outcome of 13 patients with WM who received CHOP-R at our Institution. Patients had a median age of 54 (range 44–72) yrs, and a median of 1 (range 0–5) prior therapies. 8 of 13 (62%) patients had relapsed (n=2) or were refractory (n=6) to fludarabine. Five patients had received rituximab previously. Patients received a median of 6 cycles (range 2–6) of standard dose CHOP, and 6 infusions (range 2–6) of Rituximab (375 mg/m2). Three patients received additional rituximab (4 weekly infusions every 6 months) as maintenance therapy. Median IgM for all patients declined from 4,975 (range 1,960–12,400) to 1,575 (range 62–8,230) mg/dL (p=7 x 10−5); median S.V. 2.8 (range 1.5–12) declined to 1.6 (range 1.3–4.8) CP (p=0.04). Importantly, the median hematocrit rose from 30.7 (range 21.9–35.6) to 39 (range 24.3–43.2) p=0.005. Clinical responses were as follows: 3 CR/CRu; 7 PR; 2 MR. One patient is in stable disease after 3 cycles of CHOP-R. With a median follow-up of 8 (range 3–28) months, no patient has progressed. Therapy was well tolerated for most patients. Two patients had febrile neutropenia with documented bacteremia and recovered without any complication. Circulating effector cell levels pre- and post-CHOP-R were also evaluated in 6 patients since rituximab activity is mediated in part by ADCC activity. No significant change in CD3+, CD4+, CD8+, and CD16+/CD56+ effector cell levels occurred following CHOP-R as assessed by multicolor flow cytometry.

Description: INTRODUCTION: In previous studies, we observed that CC-5013 enhanced rituximab mediated ADCC killing of lymphoplasmacytic cells (BJH2005; 128:192). As such, we conducted a phase II study of CC-5013 with rituximab in WM patients naïve to either agent. Intended therapy was as follows: Weeks 1-48 CC-5013 (25 mg po qD for 3 weeks, then 1 week off); Weeks 2–5, 13–16 Rituximab (375 mg/m2/week). METHODS: Twelve patients were enrolled, 10 of whom were previously untreated with a median age of 65 (range 53–76 yrs), along with baseline BM involvement of 50 (range 5–90%), serum IgM of 4175 (range 1180–7130 mg/dL), hematocrit of 31.9% (range 24–36.6%), and B2M of 3.5 (range 1.8–6.0 mg/L). RESULTS: Four patients were taken off study due to intolerance to therapy (3 to CC-5013, 1 to CC-5013 and Rituximab) and were non-evaluable. Unexpected acute decreases in hematocrit occurred in 10/12 (85%) patients within the first 2 weeks of therapy, resulting in hospitalization for 4 patients. The median decrease in hematocrit, which coincided with CC-5013 administration was 4.2% (range 1.7-7.5%), and was not associated with blood loss, hemolysis, or generalized myelosuppression. Other grade 3/4 toxicities attributed to CC-5013 therapy were expected and included neutropenia (n=4); tinnitus (n=2); thrombocytopenia (1); myositis (n=1); and rash (n=1). Five patients had a spike in serum IgM levels following rituximab, accompanied with epistaxis (n=1), headaches and blurry vision (n=2) requiring plasmapheresis. Toxicities led to the discontinuation and dose reduction of CC-5013 for 8 and 4 patients, respectively, and discontinuation of rituximab in 1 patient due to recurring allergic reactions. Discontinuation of CC-5013 within the first 28 days of therapy occurred in 6 out of 8 patients. Responses among the 8 evaluable patients: PR (n=3); MR (n=4); SD (n=1); ORR 88% among evaluable patients. In two patients with bulky adenopathy and splenomegaly, a significant reduction in the node/spleen size was noted by week 12. Both of these pts have subsequently achieved a PR. With a median follow-up of 7+ months, no patient who received at least 2 months of CC-5013 have progressed. CONCLUSION: The response data achieved with CC-5013 and rituximab in this study is encouraging, though the dose and schedule of CC-5013 administration, and mechanism for the unexpected acute drops in hematocrit observed in WM patients warrants further investigation. Such studies are in progress in our laboratory.

Description: We report the long-term outcome of a multicenter, prospective study examining fludarabine and rituximab in Waldenström macroglobulinemia (WM). WM patients with less than 2 prior therapies were eligible. Intended therapy consisted of 6 cycles (25 mg/m2 per day for 5 days) of fludarabine and 8 infusions (375 mg/m2 per week) of rituximab. A total of 43 patients were enrolled. Responses were: complete response (n = 2), very good partial response (n = 14), partial response (n = 21), and minor response (n = 4), for overall and major response rates of 95.3% and 86.0%, respectively. Median time to progression for all patients was 51.2 months and was longer for untreated patients (P = .017) and those achieving at least a very good partial response (P = .049). Grade 3 or higher toxicities included neutropenia (n = 27), thrombocytopenia (n = 7), and pneumonia (n = 6), including 2 patients who died of non–Pneumocystis carinii pneumonia. With a median follow-up of 40.3 months, we observed 3 cases of transformation to aggressive lymphoma and 3 cases of myelodysplastic syndrome/acute myeloid leukemia. The results of this study demonstrate that fludarabine and rituximab are highly active in WM, although short- and long-term toxicities need to be carefully weighed against other available treatment options. This study is registered at clinicaltrials.gov as NCT00020800.

Description: The tumor necrosis factor (TNF) receptor family member, CD27, is a transmembrane co-stimulatory molecule present on primed T and B lymphocytes that also secrete a soluble form (sCD27). Recent evidence has suggested that interactions between CD27 and its TNF-like ligand, CD70, play a critical role in regulating B-cell activation and survival, though the detailed mechanism(s) by which this occurs remain unclear. Waldenstrom’s Macroglobulinemia (WM) represents a lymphoplasmacytic lymphoma characterized by a monoclonal IgM gammopathy and possesses a mast cell component that may contribute to its pathogenesis (Blood 104; 646a). Using ELISA assays, we observed that WM patients displayed significantly higher levels of sCD27 in their sera (median 7.45, range 0–19.42 U/ml) versus healthy donors (median 0, range 0–2.78 U/ml; p=2.5 x 10−7). CD27 was expressed in 7/7 patients using RT-PCR analysis, but was expressed on the cell surface of tumor cells in 5/12 patients using flow cytometric analysis. Conversely, CD70 expression was widely expressed on both tumor cells (6/6 patients) and mast cells (10/11 patients) using flow cytometric analysis. In order to define the functional role of sCD27 in WM, we cultured BCWM.1 (CD27−CD70+) WM cells, and LAD1 (CD27−CD70+) mast cells with sCD27 (0.1–50 ug/mL), and observed no effect on proliferation or induction of apoptosis. Culture of LAD1 cells with sCD27 did, however, result in marked upregulation of the TNF family ligands CD40L (CD154) and a proliferation induction ligand (APRIL), which previous work in our laboratory had implicated as mast cell proliferation and survival factors in WM. Taken together, these studies suggest a novel functional role for sCD27, and imply a pivotal role in the pathogenesis of WM.

Description: INTRODUCTION: Xbp-1 is a ubiquitously expressed basic leucine zipper protein which serves as key transcription factor for plasma cell differentiation and immunoglobulin production. XBP-1 mRNA undergoes splicing at an IRE-1a specific cleavage site to produce a spliced form (Xbp-1s) which due to a frame shift results in a more stable and potent transcription factor than the unspliced form (Xbp-1us). Previous observations demonstrated that Xbp-1 was highly expressed in multiple myeloma (MM), though its expression in other B-cell disorders including Waldenstrom’s macroglobulinemia (WM) remains to be clarified. METHODOLOGY: In this study, we investigated the different forms of Xbp-1 in CD19+ selected bone marrow (BM) cells from 61 patients with the consensus panel definition of WM using semi-quantitative PCR analysis. CD19+ selected BM cells from 6 healthy donors, and CD138+ selected BM cells from 4 MM patients were used as controls. All RT-PCR results were normalized to b-actin levels. Sequencing of Xbp-1 in CD19+ selected LPC was also performed for 20 WM patients. RESULTS: In 9/61 (15%) WM patients, Xbp-1 transcripts were undetectable. Among WM patients expressing Xbp-1, as well as all MM patients evaluated, higher levels of XBP-1us were observed in comparison to normal healthy donors (p=0.001). Decreased XBP-1s transcript levels were also observed among WM and MM patients versus healthy donors, but did not reach statistical significance. Sequence analysis of Xbp1 in CD19+ selected BM LPC demonstrated variants 10/20 WM patients in exon 1 that are currently under investigation. CONCLUSIONS: Abnormal expression of Xbp-1 is common in patients with WM, and includes loss of expression, and aberrations in splice patterns. The genetic basis for these findings is under investigation.

Description: Waldenström macroglobulinemia (WM) is a B-cell malignancy characterized by an IgM monoclonal gammopathy and bone marrow (BM) infiltration with lymphoplasmacytic cells (LPCs). Excess mast cells (MCs) are commonly present in WM, and provide growth and survival signals to LPCs through several TNF family ligands (CD40L, a proliferation-inducing ligand [APRIL], and B-lymphocyte stimulator factor [BLYS]). As part of these studies, we demonstrated that WM LPCs secrete soluble CD27 (sCD27), which is elevated in patients with WM (P 〈 .001 vs healthy donors), and serves as a faithful marker of disease. Importantly, sCD27 stimulated expression of CD40L on 10 of 10 BM MC samples and APRIL on 4 of 10 BM MC samples obtained from patients with WM as well as on LAD2 MCs. Moreover, the SGN-70 humanized monoclonal antibody, which binds to CD70 (the receptor-ligand partner of CD27), abrogated sCD27 mediated up-regulation of CD40L and APRIL on WM MCs. Last, treatment of severe combined immunodeficiency–human (SCID-hu) mice with established WM using the SGN-70 antibody blocked disease progression in 12 of 12 mice, whereas disease progressed in all 5 untreated mice. The results of these studies demonstrate a functional role for sCD27 in WM pathogenesis, along with its utility as a surrogate marker of disease and a target in the treatment of WM.