Double-hit Lymphoma Research Articles

Complete Remission with Devimistat (CPI-613) in Refractory Burkitt Lymphoma

Complete Remission with Devimistat (CPI-613) in Refractory Burkitt Lymphoma

VIP152, a Selective CDK9 Inhibitor, Induces Complete Regression in a High-Grade B-Cell Lymphoma (HGBL) Model and Depletion of Short-Lived Oncogenic Driver Transcripts, MYC and MCL1, with a Once Weekly Schedule

VIP152, a Selective CDK9 Inhibitor, Induces Complete Regression in a High-Grade B-Cell Lymphoma (HGBL) Model and Depletion of Short-Lived Oncogenic Driver Transcripts, MYC and MCL1, with a Once Weekly Schedule

Do MYC Alterations Matter in HIV-Associated Large B Cell Lymphomas? the “Euromyc” Study (a European retrospective study)

Do MYC Alterations Matter in HIV-Associated Large B Cell Lymphomas? the “Euromyc” Study (a European retrospective study)

VIP152 Is a Novel CDK9 Inhibitor with Efficacy in Chronic Lymphocytic Leukemia

VIP152 Is a Novel CDK9 Inhibitor with Efficacy in Chronic Lymphocytic Leukemia

Btm-3566, a Novel Activator of the Mitochondrial Stress Response Promotes Robust Therapeutic Responses in Vitro and In Vivo in Diffuse Large B-Cell Lymphoma

Relapsed/refractory diffuse large B-cell lymphomas (r/r-DLBCL) are a therapeutic challenge, especially in patients not suitable for high dose chemotherapy, stem cell transplantation or patients who fail CAR-T-cell therapy. r/r-DLBCLs are highly heterogeneous both clinically and molecularly, which imposes a pressing need to develop novel therapies to improve outcomes in patients independently of the molecular subtype. We describe here BTM-3566, a first-in-class compound with activity against a variety of B-cell malignancies but with greatest effect in DLBCL. BTM-3566 activates the mitochondrial integrated stress response (ISR) through a novel mechanism regulated by the mitochondrial protein FAM210B. BTM-3566 induces apoptosis in DLBCL lines in vitro and complete tumor regression in vivo in DLBCL PDX mouse models harboring genetic alterations associated with poor prognosis.BTM-3566 is an oral small molecule based on a pyrazolothiazol-backbone, developed for treatment of diffuse large B-cell lymphoma (DLBCL). BTM-3566 induces apoptosis and complete cell killing in DLBCL lines a with an IC 50 of ~200 - 500 nM. Responsive DLBCL cell lines include ABC, GCB, and double-hit and triple-hit lymphoma lines. Pharmacokinetic studies in mice showed suitability for once daily dosing, with > 50% of oral bioavailability and close to 6 hours of serum half-life. 14-day dosing in mice and dogs demonstrated excellent tolerability at therapeutic doses. BTM-3566 showed stability in human hepatocytes (IC < 5 ml/min*kg) as well and a favorable in vitro safety profile. In xenograft models using the double-hit DLBCL line SU-DHL-10, BTM-3566 treatment resulted in complete regression in all tumor-bearing animals. Most importantly, no subsequent tumor growth occurred for 2 weeks after cessation of therapy, indicating that treatment with BTM-3566 resulted in a durable complete remission in this model of double-hit DLBCL. Expansion studies into human DLBCL PDX models harboring a range of high-risk genomic alterations, including Myd88 mutations and MYC and BCL2 rearrangements, demonstrated response in 100% of the lines with complete tumor regression in 6 of 8 PDX models tested (Table 1).Transcriptome and proteome analyses revealed that BTM-3566 strongly activated the ATF4-integrated stress response (ISR), indicated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and subsequent upregulation of the transcription factor ATF4. Of the four eIF2a-kinases in the human genome we determined, using CRISPR-Cas 9 gene depletion, that HRI was uniquely required for BTM-3566 eIF2a phosphorylation, induction of ATF4 ISR and apoptosis. HRI is described as being activated by mitochondrial-related stress, including heme depletion, increased ROS generation or blockage of mitochondrial ATP synthesis which result in an increase in mitochondrial proteostasis including activation of mitochondrial protease OMA1. We determined that BTM-3566 activates OMA1 without acting as a classical mitochondrial toxin. Treatment with BTM-3566 induced OMA1-dependent OPA1 processing and mitochondrial fragmentation in as little as 30 minutes of treatment, in the absence of any reduction in mitochondrial oxygen consumption or membrane depolarization. This data indicates that BTM-3566 represents a new class of compounds that activate the mitochondrial protease OMA1.Gene expression-based profiling of BTM-3566 sensitivity in over 400 cancer cell lines showed that FAM210B, a mitochondrial membrane protein, negatively correlated with response to BTM-3566. Notably, overexpression of FAM210B completely prevents OMA1 activation and causes complete resistance to BTM-3566-induced apoptosis in DLBCL cell line BJAB and the Burkitt lymphoma cell line Ramos. Thus, FAM210B serves as a strong predictor of BTM-3566 sensitivity, as well as revealing a novel mechanism of regulation of OMA1 activation.In summary, we describe here a novel, highly potent activator of the mitochondrial ISR, which is well tolerated in mice and dogs, has favorable pharmacokinetics and induces robust DLBCL regression in-vivo. An IND application in B-cell malignancies will be completed by early Q1 2022 with initiation of first in human clinical trials the first half of 2022. [Display omitted] DisclosuresSchwarzer: Bantam Pharmaceutical: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Anantha: Bantam Pharmaceutical: Consultancy, Current Employment, Current holder of stock options in a privately-held company. Cooper: Bantam Pharmaceutical: Consultancy. Hannink: Bantam Pharmaceutical: Research Funding. Hembrough: Bantam Pharmaceutical: Consultancy. Levine: Bantam Pharmaceutical: Consultancy, Current holder of individual stocks in a privately-held company. Luther: Bantam Pharmaceutical: Current holder of individual stocks in a privately-held company, Ended employment in the past 24 months, Membership on an entity's Board of Directors or advisory committees. Stocum: Bantam Pharmaceutical: Current Employment, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Liesa-Roig: Bantam Pharmaceutical: Research Funding. Kostura: Bantam Pharmaceutical: Consultancy, Current holder of stock options in a privately-held company, Patents & Royalties: Named Inventor on patents with assignment to Bantam Pharmaceutical.

Retrospective Review of the Safety and Efficacy of High-Dose Methotrexate for Prevention of CNS Relapse in Diffuse Large B-Cell Lymphoma at Kaiser Permanente- Northern California (Jan 2015 - June 2019)

Retrospective review of the safety and efficacy of high-dose methotrexate for prevention of CNS relapse in diffuse large B-cell lymphoma at Kaiser Permanente- Northern California (Jan 2015 - June 2019)Background: Central nervous system (CNS) relapse occurs in 10-12% of high-risk diffuse large B-cell lymphoma (DLBCL) patients. Prophylactic intravenous high-dose methotrexate (HD-MTX) is recommended by international guidelines to reduce this risk despite limited evidence to support such practice. Recent retrospective studies have cast doubt on the clinical benefit of such treatment. There is limited data on safety and efficacy of such treatment in a community oncology setting.Methods: We conducted a retrospective analysis of adults ≥ 18 years diagnosed with DLBCL treated with systemic therapy and HD-MTX as CNS prophylaxis at Kaiser Permanente Northern California from 1/2015 - 6/2019. We abstracted patient demographics, clinical information, treatment, toxicity, and health care utilization from the electronic health record. Descriptive statistics were used to evaluate patients' outcomes.Results: Of 33 patients (median age: 61; range: 23 - 81; age ≥ 60: 57.5%), most had stage IV disease (78.7%) and an ECOG performance status of 0 or 1 (66.5%). Patients with CNS-IPI score of 2-3 (intermediate-risk) was 30.3%, while higher CNS-IPI scores of 4-6 (high-risk) was 51.5%. Other patient characteristics include double hit lymphoma (12.1%), kidney/adrenal gland involvement (33%), and/or epidural involvement (24.2%). Most common therapies were R-CHOP (51.5%) and R-EPOCH (27.2%). The median number of HD-MTX doses was 3 (range 1-4). The median cumulative dose was 7 gm/m2 (range 3-10.5). With regards to the treatment schedule, 63.6% received HD-MTX intercalated with systemic chemotherapy and 36.4% received HD-MTX after completion of preplanned treatment. Overall, renal toxicity was the most common adverse side effect. The rates of grade 1, 2 and 3 renal toxicity were 12.1%, 9% and 6%, respectively. Other notable side effects experienced were neutropenic fever requiring hospitalization (27.2%) and grade 3 transaminitis (6%). No patients experienced grade 3 mucositis. The median duration of hospital stay was 12 days (range 4-37) and 12.1% required suspension of future HD-MTX. With a median follow up of 31.3 months (range: 0.79 - 58.4) 69.6% are alive and 15.1% had CNS relapse despite prophylactic HD-MTX.Conclusions:In this community oncology setting, patients with DLBCL who were deemed high risk for CNS relapse and received HD-MTX for prophylaxis experienced similar CNS relapse rate compared to those who did not in previous studies. Our findings are in line with recent retrospective reviews, which further support the lack of benefit of such prophylactic treatment. This study underscores the need for further research to prevent CNS disease and improved patient selection criteria for prophylactic treatment among high risk DLBCL patients. DisclosuresNo relevant conflicts of interest to declare.

A Phase II Trial of Anakinra for the Prevention of CAR-T Cell Mediated Neurotoxicity

A Phase II Trial of Anakinra for the Prevention of CAR-T Cell Mediated Neurotoxicity

Single-route CNS prophylaxis for aggressive non-Hodgkin lymphomas: real-world outcomes from 21 US academic institutions

AbstractProphylaxis is commonly used to prevent central nervous sy stem (CNS) relapse in diffuse large B-cell lymphoma (DLBCL), with no clear standard of care. We retrospectively evaluated 1162 adult patients across 21 US academic centers with DLBCL or similar histologies who received single-route CNS prophylaxis as part of frontline therapy between 2013 and 2019. Prophylaxis was administered intrathecally(IT) in 894 (77%) and using systemic high-dose methotrexate (HD-MTX) in 236 (20%); 32 patients (3%) switched route due to toxicity and were assessed separately. By CNS-International Prognostic Index (IPI), 18% were considered low-risk, 51% moderate, and 30% high. Double-hit lymphoma (DHL) was confirmed in 243 of 866 evaluable patients (21%). Sixty-four patients (5.7%) had CNS relapse after median 7.1 months from diagnosis, including 15 of 64 (23%) within the first 6 months. There was no significant difference in CNS relapse between IT and HD-MTX recipients (5.4% vs 6.8%, P = .4), including after propensity score matching to account for differences between respective recipient groups. Weighting by CNS-IPI, expected vs observed CNS relapse rates were nearly identical (5.8% vs 5.7%). Testicular involvement was associated with high risk of CNS relapse (11.3%) despite most having lower CNS-IPI scores. DHL did not significantly predict for CNS relapse after single-route prophylaxis, including with adjustment for treatment regimen and other factors. This large study of CNS prophylaxis recipients with DLBCL found no significant difference in CNS relapse rates between routes of administration. Relapse rates among high-risk subgroups remain elevated, and reconsideration of prophylaxis strategies in DLBCL is of critical need.

Primary breast double-hit lymphoma management and outcomes: a real-world multicentre experience

BackgroundPrimary breast double-hit lymphoma (PB-DHL) is a rare, highly aggressive malignancy that poses challenges regarding accurate diagnosis and selecting optimal treatment regimens.MethodsWe retrospectively reviewed 48 cases of patients diagnosed with PB-DHL in six academic centres between June 2014 and June 2020 in China. Study-specific data were recorded, including treatment options, therapeutic evaluation, prognostic factors and relapse patterns, and the overall survival (OS) and progression-free survival (PFS) were evaluated.ResultsIn total, 48 patients were enrolled, with 14 patients treated with DA-EPOCH-R/MA (rituximab, dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, alternating with high-dose methotrexate and cytarabine), 18 patients treated with DA-EPOCH-R (rituximab, dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin), and 16 patients treated with R-HyperCVAD (rituximab, hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, alternating with cytarabine plus methotrexate). The overall 5-year OS and PFS rates were 41.7% (95% confidence interval [CI], 27.6–56.8%) and 37.5% (95% CI, 24.0–52.6%), respectively. Of the three treatment regimens, the 5-year OS was higher in DA-EPOCH-R/MA group than in the DA-EPOCH-R or R-HyperCVAD subgroups (57.1% vs. 38.9% vs. 31.3%; P = 0.016), as was the 5-year PFS (50.0% vs. 38.9% vs. 25.0%; P = 0.035). Autologous stem cell transplantation (ASCT) prolonged the OS and PFS compared with non-ASCT patients (5-year OS: 72.2% vs. 23.3%; P < 0.001; 5-year PFS: 72.2% vs. 16.7 %, P < 0.001). Multivariate analysis identified tumour size, risk stratification, treatment with DA-EPOCH-R/MA, breast irradiation, and ASCT as significant prognostic factors.ConclusionsDA-EPOCH-R/MA is a promising regimen for PB-DHL, and breast irradiation yields complementary benefits for prognosis. ASCT significantly decreased disease relapse, providing a potential curative PB-DHL intervention and justifying ASCT as first-line therapy for young patients. More effective treatment strategies for PB-DHL patients remain encouraging.

ABCL-021: FRONT-MIND: A Phase III, Randomized, Double-Blind, Placebo-Controlled Study Comparing Efficacy and Safety of Tafasitamab + Lenalidomide + R-CHOP vs R-CHOP Alone for Newly-Diagnosed High-Intermediate and High-Risk Diffuse Large B-Cell Lymphoma (DLBCL)

<h3>Context</h3> Tafasitamab is an Fc-modified, humanized anti-CD19 monoclonal antibody, which induces enhanced <i>in vitro</i> antibody-dependent cell-mediated cytotoxicity and phagocytosis. Preclinical studies data suggest that tafasitamab+lenalidomide may synergistically enhance cytotoxicity to malignant B-cells. Tafasitamab+lenalidomide is FDA-approved in adult patients with relapsed/refractory DLBCL who are ineligible for autologous stem cell transplantation. R-CHOP is the current standard of care (SOC) for patients with newly-diagnosed DLBCL. Data from the Phase Ib FIRST-MIND (NCT04134936) study in newly-diagnosed DLBCL patients suggest that tafasitamab+lenalidomide when added to R-CHOP is tolerable in patients with treatment-naïve DLBCL. <h3>Objective</h3> To assess the efficacy and safety of tafasitamab+lenalidomide+R-CHOP <i>vs</i> R-CHOP alone in previously untreated, high-intermediate and high-risk patients with DLBCL. <h3>Design</h3> FRONT-MIND is a Phase III, multicenter, randomized, double-blind, placebo-controlled study. Patients will be followed-up for up to 60 months after end of treatment. <h3>Setting</h3> Patients will be enrolled from approximately 350 centers in America, Europe, and Asia-Pacific. <h3>Patients</h3> Eligible patients (n=880) will be aged 18–80 years with previously untreated local biopsy-proven, CD20-positive DLBCL (IPI ≥3 if >60 years/age-adjusted IPI 2–3 if ≤60 years), and ECOG PS 0–2. Patients with transformed lymphoma (except double or triple hit lymphoma) are excluded. <h3>Interventions</h3> Patients will be randomized to receive six 21-days (D) cycles of tafasitamab (12 mg/kg intravenous [IV], D 1, 8, and 15)+lenalidomide (25 mg orally, D1–10)+R-CHOP or six 21-D cycles of tafasitamab placebo (D1, 8, and 15)+lenalidomide placebo (orally, D1–10)+R-CHOP. <h3>Main Outcome Measures</h3> The primary endpoint is investigator-assessed progression-free survival. Secondary endpoints include investigator-assessed event-free survival, overall survival, and safety. <h3>Results</h3> Not yet available. <h3>Conclusions</h3> As 30–50% of patients overall relapse or are refractory to first-line therapy, there remains a high unmet need to improve treatment options for newly diagnosed patients, particularly those with high-risk DLBCL. The combination of tafasitamab, lenalidomide, and R-CHOP may have synergistic potential. Preliminary data from the FIRST-MIND study suggest that tafasitamab±lenalidomide+R-CHOP is tolerable in patients with treatment-naïve DLBCL. The present study will provide further evaluation of clinical benefits and safety when adding tafasitamab+lenalidomide to current SOC in newly diagnosed patients with high-intermediate and high-risk DLBCL treated with R-CHOP.

Screening Strategy for Detecting Double-Hit Lymphoma in a Resource-Limited Setting

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements [double-hit lymphomas (DHL)] are aggressive lymphomas. Current literature recommends fluorescent in-situ hybridization analysis (FISH) in all cases of diffuse large B-cell lymphoma (DLBCL) to identify cases of DHL. However, this approach is not feasible in a resource-limited setting. We analyzed cases of de novo high-grade B-cell non-Hodgkin lymphoma using histomorphology, immunohistochemistry and FISH to identify which cases need to undergo FISH testing in a resource-limited setting. Cases of de novo high-grade B-cell non-Hodgkin lymphoma that included DLBCL, not otherwise specified and B-cell lymphoma unclassifiable (BCLU) with features intermediate between DLBCL and Burkitt lymphoma diagnosed over a period of 5 years were analyzed by Hans algorithm, MYC, BCL2, and Ki67. MYC, BCL2, and BCL6 break apart FISH was tested in selected cases. One hundred and nine cases were obtained, of which 102 had DLBCL morphology and 7 had BCLU/blastoid morphology. BCL2 expression was noted in 48 cases (44%), MYC in 33 cases (30.3%) and MYC/BCL2 co-expression in 24 cases (22%). FISH testing could be done in 42 consecutive cases, of which 5 cases had MYC and BCL2 co-rearrangement (11.9%) (double-hit) and 2 cases showed rearrangement for only MYC (4.7%) (single-hit). Single-hit lymphoma/DHL showed significant independent positive correlation with BCLU/blastoid morphology, CD10 expression, germinal center B-cell phenotype, and MYC/BCL2 co-expression. The sensitivity and specificity of each parameters include BCLU/blastoid morphology (42% vs. 94%), CD10 positive (50% vs. 88%), germinal center B-cell phenotype (57% vs. 82%), MYC/BCL2 co-expression (85% vs. 80%). Selected candidates for FISH (any one of the above parameters) using this strategy showed a sensitivity and specificity of 100% and 68%, respectively (P=0.001). We propose a highly sensitive screening strategy for detection of MYC/BCL2 rearrangement in high-grade B-cell lymphoma in a resource-limited setting (pending validation in a larger cohort).

Finding the True Hits in Double-Hit Lymphomas

Finding the True Hits in Double-Hit Lymphomas

Dual targeting of the DNA damage response pathway and BCL-2 in diffuse large B-cell lymphoma

Standard chemotherapies for diffuse large B-cell lymphoma (DLBCL), based on the induction of exogenous DNA damage and oxidative stress, are often less effective in the presence of increased MYC and BCL-2 levels, especially in the case of double hit (DH) lymphomas harboring rearrangements of the MYC and BCL-2 oncogenes, which enrich for a patient’s population characterized by refractoriness to anthracycline-based chemotherapy. Here we hypothesized that adaptive mechanisms to MYC-induced replicative and oxidative stress, consisting in DNA damage response (DDR) activation and BCL-2 overexpression, could represent the biologic basis of the poor prognosis and chemoresistance observed in MYC/BCL-2-positive lymphoma. We first integrated targeted gene expression profiling (T-GEP), fluorescence in situ hybridization (FISH) analysis, and characterization of replicative and oxidative stress biomarkers in two independent DLBCL cohorts. The presence of oxidative DNA damage biomarkers identified a poor prognosis double expresser (DE)-DLBCL subset, characterized by relatively higher BCL-2 gene expression levels and enrichment for DH lymphomas. Based on these findings, we tested therapeutic strategies based on combined DDR and BCL-2 inhibition, confirming efficacy and synergistic interactions in in vitro and in vivo DH-DLBCL models. These data provide the rationale for precision-therapy strategies based on combined DDR and BCL-2 inhibition in DH or DE-DLBCL.

Dose-adjusted EPOCH and rituximab for the treatment of double expressor and double-hit diffuse large B-cell lymphoma: impact of TP53 mutations on clinical outcome

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, including one-third of cases overexpressing MYC and BCL2 proteins (double expressor lymphoma, DEL) and 5-10% of patients with chromosomal rearrangements of MYC, BCL2 and/or BCL-6 (double/triple-hit lymphomas, DH/TH). TP53 mutations are detected in 20-25% of DEL. We report the efficacy of dose-adjusted EPOCH and rituximab (DA-EPOCH-R) in a series of 122 consecutive patients, including DEL (n=81, 66%), DEL-MYC (n=9, 7%), DEL-BCL2 (n=13, 11%), or high-grade lymphomas (DH/TH) (n=19, 16%). Central nervous system (CNS) prophylaxis included intravenous methotrexate (n=66), intrathecal chemotherapy (IT) (n=40) or no prophylaxis (n=16). Sixty-seven patients (55%) had highintermediate or high International Prognostic Index (IPI) and 30 (25%) had high CNS-IPI. The 2-year progression-free survival (PFS) and overall survival (OS) for the entire study population were 74% and 84%, respectively. There was a trend for inferior OS for DH/TH (2-year OS: 66%, P=0.058) as compared to all the others. The outcome was significantly better for the IPI 0-2 versus IPI 3-5 (OS: 98% vs. 72%, P=0.002). DA-EPOCH-R did not overcome the negative prognostic value of TP53 mutations: 2-year OS of 62% versus 88% (P=0.036) were observed for mutated as compared to wild-type cases, respectively. Systemic CNS prophylaxis conferred a better 2-year OS (94%) as compared to IT or no prophylaxis (76% and 65%, respectively; P=0.008). DA-EPOCH-R treatment resulted in a favorable outcome in patients with DEL and DEL with single rearrangement, whereas those with multiple genetic alterations such as DEL-DH/TH and TP53 mutated cases still have an inferior outcome.

Real-World Experience of Axicabtagene Ciloleucel and Tisagenlecleucel for Relapsed or Refractory Aggressive B-cell Lymphomas: A Single-Institution Experience

BackgroundCD19-directed chimeric antigen T-cell receptor (CAR-T) therapies have revolutionized the treatment of patients with relapsed/refractory (R/R) aggressive B-cell lymphomas (aBCL). The results of the landmark ZUMA-1 and JULIET trials have been reproducible in real-world settings across multiple institutions, and patients with double (DHL) or triple (THL) hit lymphomas have demonstrated non-inferior outcomes compared to non-DHL/THL counterparts. Materials and MethodsThis retrospective cohort study included 53 patients with R/R aBCL who received CAR-T from October 2017 to June 2020 at the University of California, Los Angeles. Patient characteristics, lymphoma-related variables and outcomes of interest were summarized using descriptive statistics and compared between groups by Fisher's exact test. Kaplan-Meier methods were used for analysis of OS, progression free survival (PFS), and duration of response (DOR). Univariate and multivariate cox regression analysis were performed to evaluate for significant prognostic variables. ResultsWith a median follow-up of 15.2 months, this cohort demonstrated overall response rate and complete response rate of 72% and 64% (n = 34), respectively. The median DOR, PFS and OS were not reached, 7.9 and 17.7 months, respectively. By univariate analysis, DHL/THL status was the only clinical feature significantly associated with relapse post-CAR-T (OR 5.9, P = .015). ConclusionsOur single-institution, real-world cohort of R/R aBCL patients demonstrated similar efficacy outcomes to those of the ZUMA-1 and JULIET trials and published real-world studies. Our findings suggest DHL/THL patients may benefit from novel CAR-T constructs, maintenance strategies with immunomodulatory agents or allogeneic-HCT.

Changing for the Better: Discovery of the Highly Potent and Selective CDK9 Inhibitor VIP152 Suitable for Once Weekly Intravenous Dosing for the Treatment of Cancer

Selective inhibition of exclusively transcription-regulating positive transcription elongation factor b/CDK9 is a promising new approach in cancer therapy. Starting from atuveciclib, the first selective CDK9 inhibitor to enter clinical development, lead optimization efforts aimed at identifying intravenously (iv) applicable CDK9 inhibitors with an improved therapeutic index led to the discovery of the highly potent and selective clinical candidate VIP152. The evaluation of various scaffold hops was instrumental in the identification of VIP152, which is characterized by the underexplored benzyl sulfoximine group. VIP152 exhibited the best preclinical overall profile in vitro and in vivo, including high efficacy and good tolerability in xenograft models in mice and rats upon once weekly iv administration. VIP152 has entered clinical trials for the treatment of cancer with promising longterm, durable monotherapy activity in double-hit diffuse large B-cell lymphoma patients.

Ofatumumab Plus Chemotherapy Followed By G-CSF For Mobilization Of Peripheral Blood Stem Cells (PBSC) In Patients With B-Cell Non-Hodgkin's Lymphomas (NHL)

![Graphic][1] Background The in-vivo use of anti-CD20 mAbs during PBSC mobilization has the potential to improve the cure rates following autologous stem cell transplantation in patients with NHL (Khouri et al. J Clin Oncol 2005). Ofatumumab, a type I human antibody, binds to a different epitope of CD20 than rituximab. We studied the feasibility of PBSC mobilization following ofatumumab with ifosfamide/etoposide (a standard chemo-regimen at our center). In addition, as the very late antigens (VLA)-4 and VLA-6 are expressed on hematopoietic stem cells (HSC) and both play a role in homing and engraftment of HSC, we studied these markers on HSC progenitor (CD34+) cells. Patients and Methods Eligibility criteria included patients with chemosensitive NHL with 100,000/mm3 and an ANC > 1500/mm3. Ofatumumab was given at 1000 mg on day 1 and 2000 mg of day 8. Chemotherapy (days 2-4) and G-CSF were used as per standard protocols at our center. Each patient provided a PB sample prior to mobilization and an aliquot of leukapheresis product (LP) after mobilization for detection of leukocyte subsets including T-, B-, natural killer (NK)- and dendritic cell (DC)-subsets, and VLA-4 and VLA-6 molecules on CD34+ PBSC by multicolor FACS analyses. Differences between median values of leukocyte subsets between LP and PB were determined using the Wilcoxon Signed Rank test. Results Seventeen patients were studied; Median age was 52 years (range, 25-68). Histologies included: Diffuse large cell (n=9), Follicular (n=4), Mantle cell (n=3) and double-hit lymphoma (n=1). Median number of prior chemotherapies received was 2 (range, 1-4). Fourteen (82.3%) patients mobilized PBSC after a median of 3 (range, 1-6) apheresis. WBC count at day one of collection was 19.6 (range, 4.8-31) k/μL. Three (17.6%) patients failed collection of at least 2x106 CD34+cells/kg but then mobilized successfully after receiving Plerixafor. All patients were able to receive an autologous transplantation with BEAM. Median number of CD34 infused was 5.56 x10^6/kg(range, 3.79-8.85). Median times to recovery of ANC > 500/mm3 and a platelet count of >20,000/mm3 were 11 days (range, 10-12) and 10 days (range, 9-15), respectively, following transplantation. With a median follow-up time of 9 months (range, 2-11 months) post-transplant, 16 (94%) patients remain alive in remission and 1 patient died of progression. Three patients experienced grade 2 toxicity with ofatumumab (hypertension, rash, and bone pain). None had > grade 2. Correlative studies were undertaken in 14 patients. LP had a significantly higher median WBC than paired PB samples (31.1 vs. 4.3 k/uL; p=0.001) but a significantly lower median percentage of lymphocytes (3.0% vs. 18.5%; p=0.001). Among the lymphocytes, LP had significantly higher percentages of CD3+ CD4+ T cells (56.9% vs. 46.7%; p=0.048) and CD3-CD16-CD56+ NK cells (13.7% vs. 7.24%; p=0.022), respectively than paired PB samples. There were no significant differences in total T (CD3+), T suppressor/cytotoxic (CD3+ CD8+) B (CD19+), and CD16+ NK cells. In addition, total dendritic cells (DC: Lin-HLA-DR+), and DC subsets comprised of myeloid-derived DC (mDC: CD11c+CD123-) and plasmacytoid-derived DC (pDC: CD11c-CD123+) were assessed in LP and PB samples. Compared with PB, LP had a significantly higher percentage of total DC (0.40 vs. 0.32; p=0.022) but significantly lower percentages of mDC (17.25 vs. 61.9; p=0.001) and pDC (13.35 vs. 21.85; p=0.035), respectively. The median percentage of HSC progenitors (CD34+ CD38dim) in PB was significantly lower in PB than in LP (12.3% vs. 46.4%, p=0.001). Moreover, LP specimens were assesssed for VLA-4/CD49d and VLA-6/CD49f expression. Median percentage of VLA-4 expression was significantly higher in LP than in PB samples (99.05% vs. 55.65%, p= 0.001). In contrast, the median percentage of VLA-6 expression was significantly lower in LP than in PB (22.15% vs. 60.3%, p=0.003). Conclusions PBSC mobilization with ofatumumab is feasible with minimal toxicity. This resulted in an increase in the recruitment of T-helper, NK, and DC subsets in the leukopheresis product, compared with levels in PB. Although others have reported a positive association between the expression of VLA-4 on CD34+ CD38dim cells and time to engraftment, we were unable to confirm this in our study. Since this is most likely due to the small sample size, additional studies are warranted. Disclosures: Khouri: GlaxoSmithKline: Research Funding. Off Label Use: Ofatumumab in stem cell mobilization. [1]: /embed/inline-graphic-2.gif

Disseminated Double-Hit Lymphoma in a Young Adult

High-grade B-cell lymphoma with MYC and BCL2 and-or BCL6 rearrangements, also called double-hit lymphoma, is an aggressive mature B-cell lymphoma that carries both MYC and BCL2 and/or BCL6 translocations. This can present as a diagnostic challenge since clinical characteristics, morphology and immunophenotype are not accurate indicators of underlying genetic aberrations in this type of lymphoma. We report a case of a 25-year-old male who presented with a one-year history of cough, gradually increasing abdominal girth and jaundice. Definitive diagnosis was made post-mortem with additional ancillary studies using immunohistochemistry staining and fluorescent in-situ hybridization studies.

Targeting Bromodomain and External Domain Epigenetic Reader Protein As Effective Strategy for Double-Hit and Triple-Hit B-Cell Lymphoma

Approximately 12% of diffuse large B cell lymphoma (DLBCL) are double-hit lymphomas (DHL) with translocations in both MYC (MYC/8q24) and BCL2 (14;18) (q32; q21) and/or BCL6 (3q27) and prognosis is generally poor. Triple hit lymphomas (THL) are a subset of DHL exhibiting simultaneous gene translocations of MYC, BCL2 and BCL6, and demonstrate aggressive behavior, worse than that of DHL. Currently, there are no guidelines for the treatment of this group of patients, and disease remains challenging to treat. Bromodomain and external domain (BET) proteins are known as “epigenetic readers” and are enriched at the regions regulating transcription of oncogenes and many BET inhibitors (BETi) are now being clinically evaluated in several cancers.The overall goal of this study was to investigate the effect of BET inhibition through pharmacological inhibitors on single hit, DHL and THL lymphomas. We have performed extensive cytogenetic analysis on 14 human B cell lymphoma cell lines to identify MYC, BCL2 and BCL6 translocations. We identified DOHH2, OCILy1, OCILy10 as DHL, while only VAL represents THL. Raji was identified as single MYC translocation (SHL) and U2932 as WT MYC. Proliferation rate for each cell line was assessed by thymidine incorporation assays in the presence or absence of BET inhibition through pharmacological inhibitors JQ1, IBET762 or OTX015 in WT, SHL, DHL and THL B cells. BET inhibition through each BET inhibitors used individually led to significant (p=0.001) inhibition of cell proliferation in DHL and THL cells or WT- and SH- MYC expressing cells. As expected, BET inhibition led to almost complete inhibition of MYC mRNA and MYC protein in multiple lymphoma cell lines regardless of cytogenetic features. Overexpression of MYC partially rescued the effects of BET inhibition in these cells. Chromatin immunoprecipitation assay was performed to further examine the MYC regulatory mechanisms by BET inhibition. JQ1 treatment reduced BRD4 occupancy on MYC promoter but had no effect on BRD4 occupancy at the promoter region of BCL6. Overall these findings suggest that BET inhibition may suppress proliferation of DHL and THL cells via disruption of transcriptional network led by oncogenic MYC. Interestingly, the effect of BET inhibition on cell survival was modest in all MYC expressing cell lines regardless of their cytogenetic characteristics. Histone acetylation marks are major binding sites for BET proteins. We sought to assess the effect of combining BET inhibitors with histone deacetylase inhibitor vorinostat on proliferation and survival in WT, DHL and THL cells. Combination of sub-lethal doses of IBET and vorinostat decreased proliferation of WT, DHL and THL cells to a greater extent as compared to either drug alone (p<0.0001). IBET/vorinostat combination had no greater effect than IBET or SAHA alone on cell survival or signaling proteins (MCl-1, BCL2 and BIM) in any of the MYC expressing cell lines. These results suggest that combination of BET and/or HDAC pathway can greatly influence cell proliferation but had very limited impact on survival of MYC expressing WT, DHL or THL cells in-vitro. We observed that BET inhibition attenuated BCL6 expression but had no effect on BCL2 expression in DHL and THL expressing B cells. Next we tested the combination of JQ1 and IBET with a BCL2 inhibitor ABT199 (ABT) and assessed survival in THL and DHL cells. ABT alone decreased cell survival in a dose dependent manner, while co-treatment of IBET or JQ1 with ABT significantly suppressed survival as compared to ABT alone (p<0.001). We also evaluated the immunomodulatory activity of the BET inhibitors on the immune checkpoint PD-L1 and CD47, an anti-phagocytic ligand expressed on tumor cells. CD47 surface expression was observed in both DHL and THL cells, however constitutive PD-L1 was only expressed on THL cells; Both CD47 and PD-L1 expression decreased with JQ1 and IBET treatment (p<0.03). These data suggest that BET inhibitors may play an immunomodulatory role in suppressing MYC in the DHL and THL lymphomas.This study shows for the first time the role of BET epigenetic reader protein in DHL and THL aggressive B-cell lymphoma. Using several pharmacological inhibitors, we provide comprehensive mechanistic studies of its action to provide a rationale for the use of BET inhibitor therapy alone or in combination with BCL2 or checkpoint inhibitors for patients with double-hit and triple-hit lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Dynamic BH3 Profiling Reveals Novel Therapeutic Strategies for the Treatment of Double-Hit Lymphoma

Introduction: Double-hit lymphomas (DHL), a subset of diffuse large B-cell lymphoma (DLBCL) harboring both a MYC and BCL-2 or BCL-6 gene rearrangement, are associated with rapidly progressive disease that is often refractory to aggressive therapy. Given these poor outcomes, novel therapeutic strategies and drug combinations are desperately needed.BCL-2 family proteins, consisting of both pro- and anti-apoptotic members, are the key mediators of the intrinsic pathway of apoptosis. BH3 profiling is a functional assay that uses synthetic peptides that mimic the BH3 domains of pro-apoptotic BCL-2 family members to measure mitochondrial “priming”, or how close a cell is to its apoptotic threshold. This tool is also extremely powerful in that it can identify which anti-apoptotic proteins (i.e. BCL-2, BCL-XL, MCL-1) the cell relies on for survival. Dynamic BH3 profiling (DPB) further demonstrates whether ex vivo drug treatment enhances priming, which is an effective predictor of in vivo response, or anti-apoptotic dependencies, which can identify synergistic partners to use with BH3 mimetics such as venetoclax.Methods: We utilized five DHL (Dogkit, DHL-4, DHL-6, OCI-Ly8, OCI-Ly18) and three non-DHL (OCI-Ly7, Toledo, and Pfeiffer) cell lines as well as two DHL patient-derived xenograft (PDX) models (available at http://www.PRoXe.org). We performed BH3 profiling in order to functionally measure overall priming as well as dependence on specific anti-apoptotic proteins. DBP was performed by treating cells with various concentrations of drugs for short intervals (16-24 hours) and subsequently performing BH3 profiling. We screened nine drugs, including conventional chemotherapy and targeted agents with suspected activity in lymphoma. Delta priming represents the change in cyctochrome c release following exposure to BH3 peptides in treated cells as compared to untreated cells. We assessed cell viability using annexin/PI staining and measured protein expression levels with immunoblotting.Results: Baseline BH3 profiling of DHL cell lines and cell lines derived from PDX models revealed that cells rely on multiple simultaneous anti-apoptotic proteins for survival. There was a universal dependency on BCL-2, as well as in some cases, MCL-1 and BCL-XL.Conventional chemotherapy, including doxorubicin, vincristine, mafosfamide and etoposide, had a variable effect on priming and BCL-2 dependency in DHL cell lines. Doxorubicin for example, significantly enhanced priming and BCL-2 dependency in the cell line Dogkit, but not in DHL-4 or DHL-6. In vivo treatment of two DHL PDX models with chemotherapy, including doxorubicin and cyclophosphamide, enhanced priming and dependency on the anti-apoptotic proteins, BCL-2, BCL-XL and MCL-1.DBP was also performed using a variety of targeted agents, including lenalidomide, ibrutinib, azacitidine, bortezomib and EPZ-6438 (an EZH2 inhibitor). Interestingly, treatment with the hypomethylating agent, azacitidine, consistently enhanced priming and BCL-2 dependency across DHL cell lines and cell lines derived from PDX mice. Azacitidine treatment resulted in enhanced cell death when combined with venetoclax in cell viability assays and was associated with increased protein expression of Bim and Bad. Treatment with bortezomib also enhanced priming, though non-specifically enhanced all anti-apoptotic dependencies.Drug treatment in non-DHL cell lines less reliably increased dependency on BCL-2 for survival, but also enhanced BCL-XL, MCL-1 and BFL-1 dependency, depending on the cell line.Conclusions: BH3 profiling can be used to identify targetable dependencies on BCL-2, BCL-XL and MCL-1 in DHL cells lines and cells derived from DHL PDX mice. DBP additionally provides a novel approach to rationally identify drug combinations with venetoclax and other emerging inhibitors of apoptotic proteins. This strategy can identify which chemotherapy would be most efficacious for a specific cell type, especially in combination with BH3 mimetics. Furthermore, we have identified azacitidine as a promising combination therapy to use with venetoclax for the treatment of DHL. Further exploration of the role of azacitidine in combination with venetoclax for the treatment of DHL is warranted. [Display omitted] DisclosuresDavids:Celgene: Consultancy; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Merck: Consultancy; InCyte: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees. Letai:AbbVie, AstraZeneca, Novartis: Consultancy, Research Funding.