non-GC Diffuse Large B-cell Lymphoma Research Articles

How to improve RCHOP as frontline therapy for diffuse large B-cell lymphoma: a systematic review and meta-analysis of 21 randomized controlled trials.

RCHOP is the standard of care for patients with diffuse large b-cell lymphoma (DLBCL) but failures occur in approximately 40% of them. We performed a meta-analysis of 21 randomized controlled trials (RCTs) comparing experimental regimens with RCHOP. We searched the database of PubMed with proper criteria, and data of efficacy (Progression Free Survival-PFS) in the ITT population were extracted and analyzed. Cross comparisons of RCTs were performed by using the CINEMA software. Odds ratio (OR) and 95% confidence intervals (95%, CI) are reported. The literature search yielded 21 RCTs including 5785 patients in the RCHOP arm and 5648 patients in the experimental arm. Odds ratio (OR) for PFS in the total cohort was OR (95%, CI): 0.87 (0.76-0.99), p=0.02. Among different strategies to improve RCHOP, addition of a novel agent on RCHOP improved PFS. In total 1740 patients in the RCHOP arm were compared with 1755 in the RCHOP plus a novel agent arm, and the OR (95% CI) for PFS was 0.84 (0.71-0.97), p=0.02. Indirect comparisons of nine studies adding a novel agent on RCHOP does not give prominence to any agent. Subgroup analysis according to cell of origin was performed for non-GC DLBCL patients. In this subgroup, 1546 patients treated with RCHOP were compared with 1538 patients treated with experimental regimens. The OR (95% CI) for PFS was 0.86 (0.73-1.02), p=0.34. Overall survival data extracted from 18 studies showed no superiority of experimental regimens over RCHOP. Efficacy of RCHOP backbone is marginally improved when adding a novel anti-lymphoma agent.

Final Results of a Phase I/II Trial of Carfilzomib + R-CHOP for Frontline Treatment of Patients with Non-Germinal Center Diffuse Large B-Cell Lymphoma (DLBCL)

INTRODUCTION: Recent data from the REMoDL-B trial show that addition of the proteasome inhibitor bortezomib to R-CHOP improves progression free survival (PFS) of patients (Pts) with activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL) compared to R-CHOP, but peripheral neuropathy limited dose intensity. Relative to bortezomib, carfilzomib (K) displays comparable clinical activity in plasma cell neoplasms but does not cause neurotoxicity. K has preferential clinical activity in relapsed non-germinal center (non-GC) DLBCL treated with chemotherapy (Torka, et al. Blood Adv. 2023). To explore the safety and efficacy of K in combination with R-CHOP (KR-CHOP), we performed a phase I trial to identify the recommended phase 2 dose (RP2D) in all DLBCL Pts followed by a phase II extension exclusively in non-GC DLBCL. METHODS: Between 2015-2020, Pts with untreated de novo or transformed DLBCL, adequate organ function and ECOG PS ≤ 3 were enrolled at 3 US academic medical centers. As previously reported [Hill, et al, Blood (2018), 132 (Supl.1):1692], during phase I dose escalation, the RP2D of K was 20 mg/m 2 on days (D) 1 & 2 of cycle 1 and 56 mg/m 2 on D1 & 2 of cycles 2-6. Rituximab was given on D2 and CHOP was given on D3. All Pts received pegfilgrastim and zoster prophylaxis. To assess the cardiac safety of combining K with anthracycline, we compared echocardiogram data at baseline and conclusion of therapy from study Pts and control NHL Pts treated with CHOP. To gauge for differential clinical activity of KR-CHOP vs R-CHOP, we compared PFS and overall survival (OS) rates for study Pts with non-GC DLBCL vs. Pts with non-GC DLBCL receiving standard of-care (SOC) R-CHOP at the Cleveland Clinic during the same period. The outcomes were also compared between propensity score (PS)-matched pairs, to account for differences in baseline demographics and clinical risk. RESULTS: 48 Pts were enrolled, and 47 treated; 18 in dose levels < RP2D and 29 at RP2D.Baseline demographics and treatment outcomes are shown in the Table. All-grade (G) hematologic adverse events (AEs) were: anemia, 35% (22% ≤ G2, 13% G3); thrombocytopenia, 24% (15% ≤ G2, 2% G3, 7% G4); febrile neutropenia, 9% (all G3/G4). Non-hematologic AEs included: nausea, 49% (47% ≤ G2, 2% G3); vomiting, 23% (21% ≤ G2, 2% G3), peripheral sensory neuropathy, 27% (all ≤ G2); dyspnea, 24% (all ≤G2). There was one case of G3 congestive heart failure. Echocardiograms from 42 study Pts who completed 6 cycles of KR-CHOP showed no statistically significant difference in change in ejection fraction from baseline compared to 39 control Pts [95% vs. 94%, respectively (P = 0.59)]. The overall response rate was 89% (70% CR, 19% PR). At a median follow-up of 31 months, 3-year Kaplan-Meier estimates of PFS and OS for the study population were 79% and 87%, respectively. There were 8 deaths in the study: 4 due to lymphoma; 2 due to infection; 2 due to other causes. Comparing outcomes of the 36 study Pts with non-GC DLBCL to 172 control Pts receiving R-CHOP as SOC, more control Pts had stage IV disease (54% vs 34%, p=0.03) and older age (median 67 vs 66 years old, p=0.03), but less frequently >1 extranodal site of disease (23% vs. 42%, P = 0.02). PS matching using logistic regression identified 25 case-control pairs of Pts with non-GC DLBCL according to gender, age, and other International Prognostic Index (IPI) elements (see Table). In analysis of PS-matched pairs of Pts with non-GC DLBCL, Pts treated with KR-CHOP had better 3-year PFS and OS relative to Pts treated with R-CHOP as SOC [96% vs. 50% (p=0.002) (see Figure) and 67% vs. 96% (p = 0.02), respectively]. In Cox univariate analysis, treatment with R-CHOP vs. KR-CHOP was associated with an increased risk of PFS and OS with hazard ratios (HR) of 6.15 [95% confidence interval (CI) 1.71 - 22.05, P = 0.002] and 3.19 [(95% CI, 1.01 -10.11), P = 0.02], respectively. CONCLUSION: KR-CHOP can be delivered safely, without excess cardiac toxicity or peripheral neuropathy. The superior PFS and OS rates for Pts with non-GC DLBCL vs. controls receiving R-CHOP are encouraging but should be interpreted with caution due to inherent selection bias in clinical trial populations. These data suggest that, like bortezomib, carfilzomib (K) may have preferential clinical activity in ABC DLBCL. Future randomized trials are justified using advanced molecular profiling to further investigate which Pts with DLBCL may benefit from addition of a proteasome inhibitor as part of frontline treatment.

Combination of Phosphodiesterase 4 (PDE4) Inhibitor Roflumilast and R-CHOP As First-Line Therapy for High-Risk Diffuse Large B Cell Lymphoma Patients

BACKGROUND PDE4 inhibition is a biologically well-informed strategy with activity in B-cell malignancies including diffuse large B-cell lymphoma (DLBCL) (PMID: 15331441, PMID: 26503641). Roflumilast (R) is a PDE4 inhibitor, currently FDA-approved for COPD (PMID: 26338438 ). Building on preclinical and clinical activity of R in B-cell lymphomas, both alone and in combination with other agents (PMID: 27756749); a phase 1 evaluation was conducted, adding R to rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in patients with untreated high risk DLBCL. METHODS: This is a single center, phase 1b, open label, non-randomized, single arm, investigator initiated trial (IIT) of R + R-CHOP (RR-CHOP) for treatment naïve high-risk DLBCL patients. Key eligibility criteria included: non-GCB DLBCL (Han's algorithm), NCCN-IPI risk score of ≥ 2, Ann Arbor stage II-IV; key exclusion criteria include active CNS involvement and documented history of severe depression (a specific concern related to the use of PDE4 inhibitors). Patients received R-CHOP every 21 days for 6 cycles and R PO daily (500 mcg), throughout the 18-week treatment period. Paired germline-tumoral exome sequence was completed for 70% of patients enrolled in the trial, and DLBCL genetic subtype defined by the probabilistic classification tool LymphGen (PMID: 32289277). Primary end point was assessment and grading of adverse events (AE) according to the CTCAE version 4.03 criteria. Eligible patients were included in the intent-to-treat analysis. Secondary end points were estimation of complete response (CR) as per Lugano criteria, progression free survival (PFS), overall survival (OS) and their relationship to DLBCL genetic subtypes. RESULTS: Ten patients met eligibility criteria for enrollment. Sixty percent were male, 50 % were Hispanics (HI), median age at diagnosis was 59 years old (y.o) (33 y.o-79 y.o), 50% were stage 2, and 50% were stage 3-4; 50% received CNS prophylaxis given high risk and 70% had International Prognostic Index (IPI) scores of 3 to 5. Standard doses of R-CHOP were able to be delivered safely, with addition of 500 mcg of R on daily basis. Regarding hematologic adverse events (AE); 1 patient (10%) experienced neutropenia grade 3, and two patients (20%) had neutropenia grade 2. Anemia occurred in 2 patients (20%), all grade 1-2. Regarding non-hematologic AE; anorexia occurred in 40% of the cohort, all grade1; diarrhea occurred in 50%, all grade 1; headache occurred in 60%, with 40% being grade 2, and 20% being grade 3, with the latter judged to be unrelated to RR-CHOP by the investigators. Forty percent had grade 1 increase of BNP; 40% of patients had nausea, 30% being grade 1-2, and 10% being grade 3; and 40% experienced weight loss, all grade 1-2. Of 10 enrolled patients, 9 were found evaluable for assessment of efficacy, with 66% achieving CR, 22% partial response and 11 % stable disease. At 50 months of follow up, PFS was 60% at 1 year (y) and 50% at 2 y. Median PFS was not reached. OS was 100% at 1 y and 86% at 2 y, with a median OS of 38.9 months. (Figure 1 and Figure 2). The recommended R dose for phase 2 is 500 mcg PO daily. LymphGen assigned the DLBCLs in this cohort to the A53, MCD, EZB and B2N subtypes. Notably, 3 of the 3 DLBCLs in our cohort classified as MCD or A53 DLBCLs, which normally predict poor outcome, reached CR and are long term responders. Examination of 461 primary DLBCLs (NCBI dbGaP Genomic Variation in DLBLC cohort) uncovered significantly higher PDE4B expression in in the MCD subtype, suggesting that addition of R may be particularly beneficial in this DLBCL subset. CONCLUSION: Phosphodiesterase Inhibition in DLBCL is a well biologically informed strategy with the potential to enhance and perhaps optimize the duration of responses to R-CHOP. On this difficult to treat subgroup of non-GC DLBCL, with known subpar outcomes, RR-CHOP demonstrated to be safe, with manageable AEs that did not differ from what we have historically seen with R-CHOP alone. Early efficacy data from this combination shows 10% improvement compared to our own institutional historical controls of treatment naïve non-GC DLBCL patients treated with R-CHOP. Based on this data, we are pursuing a phase 2 multisite IIT of RR-CHOP in patients with newly diagnosed high risk DLBCL. Despite the small size cohort, our data suggest that tailoring this strategy to the poor outcome MCD/A53 genetic subgroups may increase the likelihood of success. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Primary thyroid lymphoma (PTL): a rare case report and review of the literature

Primary thyroid lymphoma (PTL) is a rare malignancy of the thyroid, accounting for 5% of all thyroid malignancies and approximately 2% of extranodal lymphomas. PTL occurs mainly in elderly females and diffuse large B-cell lymphoma (DLBCL) is most common. Here we report a case of non-germinal center diffuse large B-cell lymphoma (non-GC DLBCL) occurring in a 62-year-old female who was admitted to the hospital because of multiple palpable left neck masses. The patient then underwent thyroid ultrasound, detecting multiple nodules in the two lobes and isthmus of the thyroid gland, in which the largest nodule was in the left lobe with TIRADS 5 measuring 47x33 mm, and many cervical lymph nodes. Microscopically, thyroid tissue showed proliferation of tumor cells with large nuclei, coarse chromatin, narrow rims of cytoplasm, and scattered mitoses. There was 01 destructured lymph node, proliferating cells with characteristics similar to those in the thyroid gland; the remaining lymph nodes were chronically inflamed. Immunohistochemically, the tumor cells were positive for CD20, CD79a, Ki67 (40%), and negative for CD10, BCL6, MUM1, CD5, BCL2, c-MYC, CD15, CD30, EBV. Primary thyroid lymphomas are rare with the prognosis of patients depending on the histological classification of the tumor and the stage of the disease. They need to be evaluated and treated individually because there is no unified treatment due to their rarity.

Programmed cell death-ligand 1 (PD-L1)+ tumour cells and low-reacting programmed cell death 1 (PD1)+ tumour-infiltrating lymphocytes predict poor prognosis in Epstein-Barr virus+ diffuse large B-cell lymphoma.

Epstein-Barr virus (EBV)+ diffuse large B-cell lymphoma (DLBCL) has specific tumour cell characteristics, and these patients have worse outcomes than EBV-negative DLBCL patients. We compared 38 EBV+ DLBCL patients with 43 methotrexate-associated EBV+ B-cell lymphoproliferative disorders (MTX+/EBV+ BLPDs) and 30 non-germinal centre (GC) subtype DLBCL. Lymphoma cells of the EBV+ DLBCL group were positive for BCL2 in 17 patients (44.7%), CMYC in 23 patients (60.5%), and p53 in 33 patients (86.8%), which was significantly higher than in the MTX+/EBV+ BLPD group (P < 0.05), and were positive for CD30 in 29 patients (76.3%), compared with two in non-GC subtype DLBCL (6.7%) (P < 0.0001). Significantly more EBV+ DLBCL patients (n = 16, 42.1%) had programmed cell death-ligand 1 (PD-L1)+ tumour cells than patients with non-GC subtype DLBCL (n = 5, 16.7%; P = 0.024), and PD-L1+ tumour cells were more common in advanced stages than in early stages (P = 0.048). Twenty-five EBV+ DLBCL patients (69.4%) had few reactive PD1+ tumour-infiltrating lymphocytes (TILs), compared with 12 patients with MTX+/EBV+ BLPDs (37.5%) (P = 0.008). In the EBV+ DLBCL group, CD30, BCL2, CMYC, and p53 expression was not related to patient prognosis. Poor outcomes were associated with PD-L1+ tumour cells (P = 0.001) and low-reacting PD1+ TILs (P = 0.02), while their combination conferred a worse outcome (P < 0.0001). Immune evasion by PD-L1+ tumour cells and exhaustion of PD1+ TILs may occur in EBV+ DLBCL patients, and PD-L1/PD1 interactions may influence tumour progression and poor prognosis.

The Humanized EBV-LPD Recaptilates Human Non GC DLBCL

Several mouse models have been developed for diffuse large B cell lymphoma (DLBCL) such as Emu-cmyc Transgenic mice, BCL6 knock in mice, λ-myc mice and so on.However, the histopathology of these mice does not fully recapitulate that of the human DLBCL. For non- GC type in particular, it is not an exaggeration to say that none of these mice show the similarity to it.The xenograft model in which human tumor cells are transplanted into immunodeficient mice which is frequently used for the pre-clinical study for new drug, is also problematic in that the tumor cells derive form human while the cancer microenvironment from the mouse.We established the new mouse model which well recaptilates human non GC DLBCL. Human umbilical cord blood was transplanted into NOD/Shi-scid-IL2Rγnull mice(NOG mice), which is called as humanized NOG mice. The humanized NOG mice are infected with low dose EBV that infects only the primates. These mice develop EBV+ lymphoproliferative disease (LPD). We analyzed the histopathology of the LPD and compared with EBV+ DLBCL.Both EBV positive tumor cells are Mum 1 positive Bcl 6 negative CD 10 negative corresponding to non-GC DLBCL. Both tumor microenvironments showed massive infiltrate of CD 163 positive macrophages. (Figure) The localization of the cells which express cytokines such as IFN beta, TNF and IL-10 are overlapped between them.Particularly, the LPD which EBV infected humanized mice develops consists of not only EBV infected human CD20 positive cells but also human stromal cells such as human CD163 positive macrophages.In conclusion, the LPD developed in EBV infected humanized mice well recapitulate human non GC DLBCL and can be a useful model mouse for drug discovery targeting tumor microenvironment including immunocheck point drugs. [Display omitted] DisclosuresKotani:Janssen Pharma: Research Funding.

Cell of Origin and Treatment Impact on the Outcome of Monomorphic Post-Transplant Lymphoproliferative Disorder-Diffuse Large B-Cell Lymphoma Subtype

Introduction : Cell of origin (COO) determination of diffuse large B-cell lymphoma (DLBCL) is important, as it has prognostic and therapeutic relevance. Patients with germinal center (GC) like DLBCL have more favorable outcomes than those with non-GC DLBCL, when treated with standard immunochemotherapy. In monomorphic post-transplant lymphoproliferative disorder (PTLD), DLBCL subtype, the biologic significance of COO is not well established. The aims of this study were to evaluate the impact of the COO on clinical presentation, outcome and response to different treatment regimens. Methods : We conducted a retrospective review of all monomorphic PTLD of the DLBCL subtype diagnosed and treated at Columbia University from 2000-2018. COO classification into GC and non-GC subtype was determined by immunohistochemistry using the Hans algorithm. In situ hybridization for Epstein-Barr encoded small RNAs (EBER) was performed to evaluate for EBV infection of the neoplastic lymphocytes. Outcomes according to therapeutic regimens were assessed using the revised response criteria for malignant lymphoma (2007). Overall survival (OS) and progression free survival (PFS) were estimated by the Kaplan-Meier method and compared by the log rank test. Results : Cell of origin: Biopsy material for COO subtyping was available for 49 monomorphic PTLD-DLBCL diagnosed during the study period. By immunohistochemistry 20/49 cases (40.8%) were GC and 29/49 cases (59.2%) were non-GC. Median age of presentation was 52 years (range 3-72) and 55 years (range 3-75), for the GC and non-GC respectively. Median time from transplant to PTLD onset was 3 years, range 7 months-25 years vs. 6 years, range 2 months-24 years for GC vs non-GC respectively. There was no significant difference in the incidence of EBV positivity, immunosuppressive therapy, organ transplanted, stage, LDH, ECOG and IPI between the 2 groups. Of note, extranodal involvement, specifically of the gastrointestinal tract was extremely common in both subtypes (16/20 [80%] in GC and 25/29 [86%] in non-GC). While acknowledging the heterogeneity of therapies administered, a trend suggesting better prognosis for the GC type was noted, although not statistically significant. The median PFS was 151.0 months for GC vs 17.0 months in non-GC (p= 0.1095), while the median OS was 151.0 months for GC vs 36.7 months for non-GC (p=0.1006) (Figure 1A, 1B). Impact of Treatment: Of the 49 cases, 44 were adults (age≥18). The two most common first line therapies administered were R-EPOCH (25) and R-CHOP (9). Ten patients received 6 other therapies including Rituximab monotherapy (3), CHOP (2), palliative care (2), RCEOP (1), EPOCH (1), and radiation (1). When focusing on patients who received R-CHOP or R-EPOCH as initial therapy, no significant differences in age, stage, LDH, extranodal disease, ECOG, IPI, immunosuppressive therapy and COO was identified between the 2 groups. The complete response (CR) rate for R-CHOP was 6/9 (66.6%) vs. 21/25 (84%) for R-EPOCH (p= 0.2701). Primary refractory disease was present in a third of the patients receiving R-CHOP vs. (4/25) 16% of the patients treated with R-EPOCH (p=0.2701). Treatment related mortality was low in both groups (1/9 (0.1%) vs 1/25 (0.04%) patients died during first line therapy with RCHOP vs REPOCH respectively). No difference was seen in terms of PFS and OS in the 2 groups. Median PFS and OS for RCHOP was 80.0 and 83.9 months respectively, the medians for R EPOCH were not reached (log-rank test p= 0.5430, 0.2855 for PFS and OS, respectively) (Figure 1C-1D). When the OS and PFS curves for RCHOP and REPOCH were analyzed based on COO, again no difference was noted. Conclusions : Non-GC subtype is more common than GC in monomorphic PTLD DLBCL. Clinical characteristics, EBV infection and time of onset post transplant is not different in the 2 subtypes. There is a trend suggesting better PFS and OS of PTLD DLBCL GC subtype, although not statistically significant, mirroring the outcome of GC vs non-GC COO in DLBCL of immunocompetent patients. With regard to therapy, R-EPOCH did not improve OS or PFS when compared to R-CHOP, but did not result in increased toxicity or treatment related mortality, which in our series was extraordinarily low for both therapies (&lt;=0.1%). Given the retrospective nature of our analysis, further studies of a larger cohort of patients is ongoing to validate these observations. Disclosures Sawas: Seattle Genetics, Gilead, Daiichi Sanko: Consultancy; Affimed: Research Funding. Lue:Kymera Therapeutics: Honoraria; Astex Pharmaceuticals: Honoraria. Marchi:Spectrum Pharmaceuticals, Verastem Oncology: Research Funding. Radeski:Corvus Pharmaceuticals: Research Funding. O'Connor:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Other: Travel Support, Research Funding; ADCT Therapeutics, Affimed, Agensys, Merck, Seattle Genetics, Spectrum, Trillium, and Verastem Oncology.: Research Funding.

Phase I Trial of Carfilzomib + R-CHOP (CarR-CHOP) for Frontline Treatment of Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

Phase I Trial of Carfilzomib + R-CHOP (CarR-CHOP) for Frontline Treatment of Patients with Diffuse Large B-Cell Lymphoma (DLBCL)

A phase 1 study of ibrutinib in combination with R-ICE in patients with relapsed or primary refractory DLBCL

AbstractIn the postrituximab era, approximately half of the patients with relapsed or refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) fail to achieve a chemosensitive response to standard salvage therapy, and are thus ineligible to proceed to autologous stem cell transplantation with curative intent. The Bruton tyrosine kinase inhibitor ibrutinib demonstrates single-agent activity in rel/ref DLBCL, particularly of non–germinal center (non-GC) cell of origin. We conducted a single-center phase 1 study evaluating dose-escalated ibrutinib, in a 3-by-3 design, in combination with rituximab, ifosfamide, carboplatin, and etoposide (R-ICE) in physiologically transplant-eligible rel/ref DLBCL patients. Twenty-one patients have been treated and are evaluable for toxicity with no dose-limiting toxicities observed through expansion with ibrutinib at 840 mg daily at dose level 3. Of the 20 patients evaluable for response, per modern International Conference on Malignant Lymphoma criteria, 11 patients achieved complete remission (CR) and 7 patients achieved partial remission for an overall response rate of 90%. All evaluable patients with non-GC DLBCL achieved a metabolic CR. Ibrutinib in combination with R-ICE demonstrates tolerability and efficacy in rel/ref DLBCL, particularly of non-GC phenotype. This treatment program warrants further investigation in later-phase studies. This trial was registered at www.clinicaltrials.gov as #NCT02219737.

Proteomic Profiling of Diffuse Large B-Cell Lymphomas

Objective: The aim of this study was to identify differences in proteome profiles of diffuse large B-cell lymphoma (DLBCL) of nongerminal center (non-GC) versus GC type in the search for new markers and drug targets. Methods: Six DLBCL, with 3 repeats for each, were used for the initial study by proteomics: 3 non-GC and 3 GC DLBCL cases. For immunohistochemistry, tissue microarrays were made from 31 DLBCL samples: 16 non-GC de novo lymphomas and 15 GC cases (11 transformed from follicular lymphomas and 4 de novo GC lymphomas). Proteome profiling was performed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Results: Ninety-one proteins were found differentially expressed in non-GC compared to GC type. The Cytoscape tool was used for systemic analysis of proteomics data, revealing 19 subnetworks representing functions affected in non-GC versus GC types of DLBCL. Conclusion: A validation study of 3 selected proteins (BiP/Grp78, Hsp90, and cyclin B2) showed the enhanced expression in non-GC DLBCL, supporting the proteomics data.

Lenalidomide Maintenance after Autologous Stem Cell Transplant in Patients with High-Risk Relapsed/Refractory Lymphomas Is Feasible and Compares Favorably to Historical Controls: Results of a Phase I/II Trial

Background: Lymphoma patients with residual hypermetabolic lesions on FDG-PET imaging after salvage chemotherapy have poor outcomes following autologous stem cell transplant (ASCT). We have previously shown progression free survival (PFS) of only 5 months (range: 1-19) in this population with only 7% of patients without progression at 12 months (Svoboda et al, BMT 2006). We hypothesized that these high-risk patients may benefit from continued therapy after ASCT. Lenalidomide is an immunomodulatory agent which has been used as maintenance in other hematologic malignancies, but its toxicity and efficacy have not been well described in lymphoma patients following ASCT.Methods: We are conducting a phase I/II prospective, open-label trial of lenalidomide maintenance after ASCT in lymphoma patients at high risk for relapse defined by residual FDG-PET positive lesions (SUV ≥ 2.5) immediately prior to ASCT. The primary objective of phase I was to determine the safety and dose-limiting toxicity (DLT) of lenalidomide maintenance. A 3+3 de-escalation design was used with a starting dose of lenalidomide at 10 mg on days 1 through 28 of each 28-day cycle. Lenalidomide was initiated 28-100 days post-ASCT and planned for up to 24 cycles. DLT was defined as non-hematologic toxicity ≥ grade 3 or hematologic toxicity ≥ grade 4 during the first 28 days of lenalidomide. The primary objectives of phase II were PFS and overall survival (OS). Survival outcomes were calculated from the date of ASCT. Enrollment began in 5/2012; we report data through 7/2016.Results: Fourteen patients were enrolled and 11 were evaluable (one patient withdrew consent and two progressed prior to initiation of lenalidomide). Eight (73%) evaluable patients had diffuse large B-cell lymphoma (DLBCL): 4 with germinal center (GC) origin and 4 non-GC by Hans algorithm. Three (27%) patients had Hodgkin lymphoma. Median age was 44 years (29-61), ECOG PS 0 (0- 1), prior therapies 2 (2-5). Median follow-up was 24 months (range 8-44), and median time on lenalidomide was 13 cycles (1-24). No DLTs were observed in phase I, and the dose of 10 mg daily was determined to be appropriate for phase II. Six (55%) patients discontinued lenalidomide: 3 due to disease progression, 2 at investigator’s discretion (1 subsequently progressed), and 1 due to grade 3 rash possibly related to lenalidomide. Of 3 patients who discontinued lenalidomide due to progression, 1 (non-GC DLBCL) died of disease progression, 1 (GC DLBCL) achieved complete remission (CR) with allotransplant, and 1 (non-GC DLBCL) remains on another active therapy. Overall, 8 (73%) patients remain in CR following ASCT, including 3 patients who discontinued lenalidomide. Of note, 1 patient developed adenocarcinoma of the colon 1 year after completion of lenalidomide, and 1 patient developed therapy-related acute myeloid leukemia at 10 months after discontinuing lenalidomide. At a median follow-up of 24 months, PFS of the complete cohort was 62.3% (95% CI: 0.28-0.84; Figure 1) and median PFS was not reached. OS was 75% (95% CI: 0.30-0.93; Figure 2) and median OS was not reached. When compared to the reported PFS of 7% at 12 months in the historical controls with identical high risk pre-transplant characteristics, the PFS of 62.3% (95% CI: 0.28-0.84) at 12 months was significantly improved (Z-test, p<0.05).Conclusion: We established feasibility of lenalidomide maintenance at 10 mg daily after ASCT in patients with relapsed/refractory lymphomas. Preliminary clinical outcomes observed in this phase I/II trial are very encouraging when compared to historical controls. To better understand the toxicity profile and validate the promising clinical benefit, the strategy of utilizing immunomodulatory agents as post-transplant maintenance should be studied in a larger cohort of high-risk lymphoma patients. [Display omitted] [Display omitted] DisclosuresSvoboda:Pharmacyclics: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding. Nasta:Millennium Pharmaceuticals: Research Funding. Mato:Abbvie: Research Funding; Acerta Pharma: Research Funding; Gilead Sciences: Research Funding; ProNAi: Research Funding; Pharmacyclics: Consultancy; TG Therapeutics: Consultancy; Theradex: Research Funding; TG Therapeutics: Research Funding; Gilead Sciences: Consultancy; Abbvie: Consultancy. Hwang:Novartis: Research Funding. Schuster:Janssen Research & Development: Research Funding; Gilead: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Research Funding; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria; Hoffman-LaRoche: Research Funding; Merck: Research Funding.

Preclinical Evaluation of Ibrutinib for Central Nervous System Lymphoma

IntroductionPrimary CNS lymphoma (PCNSL) is a diffuse large B-cell lymphoma (DLBCL), predominantly of non-germinal center (non-GC) subtype. Currently available treatments remain disappointed with a non-optimal complete remission rate and a high relapse rate. As such, novel therapeutic agents are urgently needed. Constitutive activation of the NF-kB pathway via mutations in B cell receptor (BCR) (CD79B) and mutation of MYD 88 and TBL1XR1 pathways plays an important role in PCNSL. Ibrutinib, an inhibitor of BCR signaling, has been found to have significant therapeutic activity in relapsed or refractory non-CNS non-GC DLBCL that display similar NFKB mutational patterns. We have performed a CNS pharmacokinetic (PK) study of Ibrutinib and evaluated its therapeutic activity against CNS lymphoma (CNSL) in murine models.MethodsCNS PK study in mice - The first study was done in a CNSL model created by intracerebral implantation of luciferase-transfected lymphoma cells (MC116) in nude mice in comparison to healthy nude mice without lymphoma cell implantation (N=5 per group). Once the tumor activity was confirmed by bioluminescence imaging in CNSL group, all the mice were treated with ibrutinib 12 or 30 mg/kg/day by oral gavage (OG) for 5 days. Blood and brain were collected 2 hours after the last dose. Ibrutinib was measured by ultra performance liquid chromatography with tandem mass spectrometry.Cerebro-spinal fluid (CSF) PK study in Sprague-Dawley (SD) rats - Ibrutinib 50 mg/kg single dose was given by OG (N=6). Microdialysates of CSF were collected from a ventricle of the brain and a carotid artery via microdialysis catheters every hour for 13 hours post-treatment. Drug concentrations were determined by capillary electrophoresis with laser detection.Preclinical therapeutic evaluation - A murine CNS lymphoma model was created by intracerebral injection of the OCI-Ly10 non-GC DLBCL cells, which were confirmed to have CD79 A and MYD 88 mutations and show high sensitivity to Ibrutinib in vitro. Animals were sacrificed if they showed severe clinical signs or symptoms or >20% weight loss. Ibrutinib 30 mg/kg was administered by OG daily starting at day 7 post-implantation and until the last sacrifice in the control group treated with vehicle. Therapeutic efficacy was determined by Kaplan-Meier survival analysis using date of sacrifice as endpoint.ResultsCNS PK study in mice - 1) Ibrutinib levels in plasma, normal brain tissue and brain-tumor were analyzed. Ibrutinib was not detected in healthy control mice treated with vehicle. In healthy mice treated with ibrutinib (12 mg/kg and 30 mg/kg), Ibrutinib mean plasmatic levels were 9.31 (±3.56) ng/mL and 42.2 (±20.9) ng/mL respectively. At 12 mg/kg dose level, ibrutinib was detectable but not quantifiable in brain tissue. After treatment with 30 mg/kg,iIbrutinib mean levels were 0.0077 (±0.0053) and 0.0071 (±0.00483) ng/g tissue in right and left cerebral hemispheres respectively. In CNSL mice treated with ibrutinib (12 mg/kg and 30 mg/kg), Ibrutinib mean plasmatic levels were 12.03 (±9.04) ng/mL and 42.64 (±42.04) ng/mL respectively, brain ibrutinib levels were 0.008 (±0.005) and 0.007 (±0.003) ng/g tissue for tumor-bearing right hemisphere and non-tumor bearing left hemisphere respectively for 30 mg/kg, while brain ibrutinib level was detectable but not quantifiable at 12mg/kg.CSF PK study in SD rats - CSF penetration of ibrutinib was determined to be ~5.2 % as calculated by area under the curve (AUC) ratio of CSF and blood samples. Cmax in CSF is ~0.35 uM, which is significantly higher than published EC50 concentrations for DLBCL cell lines.Preclinical therapeutic evaluation -Ibrutinib showed an excellent therapeutic activity with significant prolongation of survival (p=0.0137) (fig 1). At a median follow-up of 112 days, the median survival in treated CNSL group is 56 days compared to 33 days in the control group. At the end of the follow-up 33% of animals in treated CNSL group are still alive without treatment.ConclusionIbrutinib achieves high concentrations in brain and CSF in murine CNS PK models with promising single-agent preclinical therapeutic activity against CNS lymphoma in a murine model. Further studies to develop combination regimen are ongoing. [Display omitted] DisclosuresSoussain:Roche: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding.

Rituximab-Lenalidomide (REVRI) in Relapse or Refractory Primary Central Nervous System (PCNSL) or Vitreo Retinal Lymphoma (PVRL): Results of a "Proof of Concept" Phase II Study of the French LOC Network

BackgroundPrimary CNS lymphoma (PCNSL) is a diffuse large B-cell lymphoma (DLBCL), predominantly of non-germinal center (non-GC) subtype. Despite improvement of therapeutic results since the introduction of high-dose methotrexate (MTX) in first-line treatment, improvements of therapeutic results are needed in PCNSL, both in first-line treatment and at relapse. The roles of consolidation and maintenance therapy need to be addressed as well. The association of Rituximab and Lenalidomide has shown activity in non-CNS non-GC DLBCL, but its activity in PCNSL was unknown.MethodsIn this prospective, multicenter open-label phase II study, we enrolled patients over 18 with a refractory or relapse PCNSL or primary vitreo-retinal lymphoma (PVRL) of DLBCL type. The treatment consisted in an induction phase with 8 cycles of 28 days with R2 (Rituximab 375/m2 IV D1; Lenalidomide 20 mg/D D1-D21 for the first cycle then 25 mg/D D1-D21 for the subsequent cycle in the absence of hematologic toxicity), followed, in responder patients by a maintenance phase with 12 cycles of 28 days with Lenalidomide alone (10 mg/D, D1-D21). Corticosteroids were allowed during the first induction cycle in case of a threatening or symptomatic edema. Deep vein thrombosis prophylaxis was mandatory. Patient who received a complete month of treatment were evaluable for response. The primary end-point was the objective response rate (ORR) at the end of the induction phase, according to the international primary CNS lymphoma collaborative group (IPCG) criteria. The analysis was based of a Fleming's two-step design (P0 = 10 %; P1= 30%). A pilot exploration of circulating NK and T cells before and after treatment and correlation with therapeutic response was conducted. This study is registered with ClinicalTrials.gov, number NCT01956695ResultsBetween September 17, 2013 and September 29, 2015, fifty patients (median age: 69, range 46-86) were recruited from 10 centers. All the patients had previously received high-dose (HD) MTX. Median number of previous lines of chemotherapy was 2 (range, 1-4). Nine patients had previous received an HD chemotherapy followed by hematopoetic stem cell rescue. Initial diagnoses were PCNSL (n = 42) and PVRL (n = 8). At time of inclusion in the study, diagnoses were PCNSL (n = 41) and PVRL or isolated intra-ocular (IO) relapse of PCNSL (n=9). Seven patients had concomitant involvement of the cerebrospinal fluid (CSF). Patients were included either for a relapse after last treatment (80 %; median time to relapse = 5.5 months), or for a refractory disease (20 %). Thirty-four patients received concomitant corticosteroids during the first month of treatment. Forty-five patients were evaluable for response after the first cycle of treatment. Median number of induction cycles was 7 (range, 1-8). Grade 3 or 4 adverse events were reported in 11 patients (infection: n = 10, cutaneous rash: n =1). A second cancer (melanoma) occurred in one patient. Two patients withdrew their consent. During the induction phase, best observed responses were CR (n = 16), PR (n = 11), stable disease (n = 5) and progressive disease (n = 11) for an ORR of 63% (27/43). . At the end of the induction phase, 43 patients were evaluable for the primary objective. ORR was 39 % (17/43) including 13 CR (30%). A response has been observed in patients included for a PCNSL (n = 13, 32%) and for an IO relapse or PVRL (n = 4, 44%). Seventeen patients started the maintenance phase. With a median follow-up of 9 months (range, 1.1-15.4), median overall and progression-free survivals of the whole population were 15.3 months (95 % CI, 9.6 - non reached) and 8.1 months (95 % CI, 4.2 - non reached) respectively. Median duration of response in the responder patients was 8.9 months (95 % CI, 7.6- non reached). The results of the maintenance phase are pending. Results will be further updated.ConclusionThis phase II study demonstrates a significant activity of the rituximab-lenalidomide regimen in relapse or refractory PCNSL or PVRL. Updated results with a longer follow-up are awaited to better evaluate the PFS and the median duration of response. This regimen warrants to be added in the armamentarium drugs for PCNSL and further explored in combination with other chemotherapies in first-line treatment, as maintenance therapy, or as a chemo-free regimen for patients unfit for high-dose methotrexate.Funding: Celgne, Roche [Display omitted] DisclosuresGhesquieres:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche France: Research Funding; Mundipharma: Consultancy. Choquet:Celgene: Consultancy; Janssen: Consultancy. Morschhauser:Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria; Janssen: Honoraria; Servier: Consultancy, Honoraria. Soussain:Roche: Research Funding; Pharmacyclics: Research Funding; Celgene: Research Funding.

Enlargement of deep medullary veins during the early clinical course of Sturge-Weber syndrome

Therapeutic improvements are required for primary CNS lymphoma (PCNSL) and secondary CNS lymphoma. PCNSLs are predominantly diffuse large B-cell lymphoma (DLBCL) classified in the non-germinal center (non-GC) subgroup.1 The role of B-cell receptor (BCR) signaling to continuously activate the NF-κB pathway is well-established in non-GC DLBCL.2 Mutations of MYD88 , CD79B , and TBL1XR1 , genes involved in the NF-κB pathway, are frequently encountered in PCNSL.3 Ibrutinib, an inhibitor of BCR signaling, led to an objective response rate of 50% in patients with relapsed or refractory systemic non-GC DLBCL.4 As a small molecule (MW = 440), with promising CNS distribution,5 ibrutinib represents a potential treatment for PCNSL. We report a retrospective case series of patients with relapsed and refractory CNS lymphoma treated with ibrutinib. Acknowledgment: The authors thank Cathie Germain, MA, and Cynthia Burnett, BA, for assistance with patient scheduling; Jane Cornett, RN, and Anne Deboard, RN, for performing sedation for imaging; Yang Xuan, BS, for MRI acquisition and preprocessing; and Michael Behen, PhD, for cognitive assessment of the patients .

Ibrutinib monotherapy in relapsed/refractory CNS lymphoma: A retrospective case series.

Therapeutic improvements are required for primary CNS lymphoma (PCNSL) and secondary CNS lymphoma. PCNSLs are predominantly diffuse large B-cell lymphoma (DLBCL) classified in the non-germinal center (non-GC) subgroup.1 The role of B-cell receptor (BCR) signaling to continuously activate the NF-κB pathway is well-established in non-GC DLBCL.2 Mutations of MYD88 , CD79B , and TBL1XR1 , genes involved in the NF-κB pathway, are frequently encountered in PCNSL.3 Ibrutinib, an inhibitor of BCR signaling, led to an objective response rate of 50% in patients with relapsed or refractory systemic non-GC DLBCL.4 As a small molecule (MW = 440), with promising CNS distribution,5 ibrutinib represents a potential treatment for PCNSL. We report a retrospective case series of patients with relapsed and refractory CNS lymphoma treated with ibrutinib.

MYC Associated and Double Protein Lymphoma: Subset Analysis of SWOG S9704

Background: Outside of Burkitt lymphoma (BL), MYC rearrangements confer negative prognostic significance, particularly when co-associated with BCL2 rearrangements. While “double hit lymphomas (DHL)”, occurring in 5-10% of diffuse large B cell lymphoma (DLBCL) patients (pts), have known inferior outcomes with R-CHOP (Savage Blood 2009; Barrans J Clin Oncol 2010), emerging reports suggest that “double protein lymphomas (DPL)” with immunohistochemical (IHC) overexpression of MYC and BCL2 also fare poorly with standard therapy. The frequency of DPL is reportedly higher than DHL and may account for 20-30% of DLBCL cases (Johnson J Clin Oncol 2012; Hu Blood 2013). The optimal management strategy for DHL and DPL is undefined, and there is limited data on the potential role of consolidative transplant. SWOG S9704 was a phase III randomized study of aggressive NHL treated with CHOP +/-R for 5 cycles and then either 3 additional cycles of CHOP +/-R for autologous stem cell transplant (ASCT) in first remission (Stiff N Engl J Med 2013), which gives a unique opportunity to evaluate DPL outcomes in a prospective dataset. Methods: Among 370 eligible pts, 260 had DLBCL or B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCLU); cases underwent morphologic review, and IHC analysis for MYC protein (>40% positive cells) was performed in 198 cases. BCL2 IHC (>30% positive cells), cell of origin (COO) classification (GC vs non-GC per Hans algorithm), and FISH studies for MYC were performed in all MYC IHC positive (+) cases with sufficient tissue. BCL2 FISH was also performed in cases positive for a MYC translocation via FISH. Clinical annotations were obtained through SWOG statistical center, and review of S9704 database. Results: There were 27 pts with MYC IHC+ among 198 available samples (13.6%), 19 of whom had concurrent BCL2 IHC+. Three of 6 evaluated pts had DHL via FISH (DHL group), and all were also DPL via IHC; thus, 16 pts had DPL without DHL. 8 patients with MYC IHC + had neither BCL2 positivity via FISH nor IHC (non-DHL, non-DPL group). For the 16 DPL pts (excluding 3 DHL pts), median (med) age was 59.9 y (range, 45-65y), 10 were male, 6 had bulky disease, all had elevated LDH (including 11 with LDH >2X ULN), and med AA IPI was 2. Among 11 evaluated pts for COO, 5 had GC whereas 6 had non-GC DLBCL phenotypes. All 3 DHL pts were female, and all had GC phenotype via Hans criteria. Among non-DHL/non-DPL pts (n=8), med age was 54.5y (range, 49-65y), 5 were male, 3 had bulky disease and all had elevated LDH. In terms of treatment, only 12 of 16 DPL pts were randomized (5 HCT, 7 no HCT), all 3 DHL pts were randomized (2 HCT) and 4 of 8 non-DHL/non-DPL pts were randomized. Reasons for non-randomization (n=8) were progression, lack of response or death. The med f/u for the entire cohort of 27 MYC IHC+ pts is 127 m (range, 94-147m); 21 pts have died (17 no HCT; 4 HCT groups). Med PFS for no HCT vs. HCT for all 27 MYC IHC + pts is 9m (95% CI: 5.0, 13.9) vs. not reached and 2-yr PFS is 16% (95% CI: 3.9%, 34.9%) vs. 63% (95% CI: 22.9%, 86.1%), respectively (Figure 1, intent-to-treat; Figure 2, randomized pts only). For the 16 DPL pts, 9 pts in the no HCT and 3 pts in the HCT group have progressed or died; med PFS for no HCT vs. HCT is 11m (95% CI: 6.7, 18.1) vs. 41m (95% CI: 7.1, NR), respectively, and 2-yr PFS is no HCT vs. HCT is 18% (95% CI: 2.9%, 44.2%) vs. 60% (95% CI: 12.6%, 88.2%), respectively Among 12 DPL pts actually randomized, 2-yr PFS was 29% for no HCT and 60% for HCT, but confidence intervals overlap substantially. All 3 DHL pts have progressed and died at a med of 6.5m. Conclusions: This is a subset analysis of the prospective S9704 trial; one of the largest data sets to address the question of upfront ASCT. True DHL were rare even in this high-risk cohort and had a dismal prognosis. The incidence of DPL was also less than reported in other series, and may reflect that adverse biologic features are more common in non-transplant, and thus older, populations. In an intent–to-treat analysis, there is a trend that MYC IHC+ and DPL patients consolidated with ASCT have improved outcomes; however, nearly one-third of MYC IHC+ pts were unable to be randomized due to early progression or death, suggesting that better induction regimens are needed. While these data are limited by small numbers, there seems to be a role for upfront ASCT in DPL which should be further explored. Support: CA32102, CA38926, CA21115 and in part by Bristol-Myers Squibb. Disclosures No relevant conflicts of interest to declare.

Low lymphocyte-to-monocyte ratio predicts unfavorable prognosis in non-germinal center type diffuse large B-cell lymphoma

The peripheral blood lymphocyte to monocyte ratio (LMR) at diagnosis has been used to predict survival in diffuse large B-cell lymphoma (DLBCL) patients, but its prognostic significance with respect to different cell-of-origin (COO) subtypes remains unknown. We retrospectively analyzed 168 de novo DLBCL patients in this study and found that a low LMR (≤2.6) correlates with B symptoms, elevated LDH, advanced Ann Arbor stage and higher international prognostic index (IPI) score (p<0.05). The low LMR is a negative prognostic parameter for overall survival (OS) and event-free survival (EFS) in non-germinal center (GC) type DLBCL patients, as compared with the high LMR, especially in those treated with R-CHOP. However, the LMR has less correlation with the OS and EFS in GC type DLBCL patients (p=0.545 and 0.547, respectively). Multivariate analysis adjusting for IPI revealed that the low LMR indicates a shorter survival retain both OS and EFS in non-GC subtypes (p=0.023 and 0.005, respectively). In the non-GC DLBCL patients treated with R-CHOP a low LMR still showed a trend to predict poor EFS (p=0.052). In conclusion, these data suggest that a low LMR at diagnosis may imply a poor prognosis in non-GC subtype DLBCL patients, especially in those treated with R-CHOP, but not in those GC subtype DLBCL patients.

Way to go to exploit NK cells' versatile talents for cancer immunotherapy.

Way to go to exploit NK cells' versatile talents for cancer immunotherapy.

Influence of NK cell count on the survival of patients with diffuse large B-cell lymphoma treated with R-CHOP.

BackgroundAlthough adding rituximab to the chemotherapy regimen of cyclophosphamide, vincristine, doxorubicin, and prednisone (R-CHOP) has improved clinical outcomes of patients with diffuse large B-cell lymphoma (DLBCL), several recent studies have shown that the effect of rituximab is dominantly in the non-germinal center (non-GC) subtype compared to the germinal center (GC) subtype. Natural killer (NK) cell count, a surrogate marker of immune status, is associated with clinical outcomes in DLBCL patients in the rituximab era. We investigated whether the impact of NK cells on clinical outcomes differed according to the immunophenotype of DLBCL.MethodsThis study analyzed 72 DLBCL patients treated with R-CHOP between January 2010 and January 2014.ResultsLow NK cell counts (<100/µL) were associated with poor progression-free survival (PFS) and overall survival (OS) compared to high NK cell counts. In multivariate analysis, low NK cell count was an independent prognostic factor for PFS and OS. However, survival did not significantly differ between the GC and non-GC subtypes. We examined the clinical influence of NK cells according to the immunophenotype and found that low NK cell counts were significantly associated with poor PFS and OS in non-GC cases, but not in GC cases.ConclusionLow NK cell counts at diagnosis are associated with poor clinical outcomes in DLBCL patients treated with R-CHOP therapy. However, the impact is significant only in non-GC subtype DLBCL, not in the GC subtype.

Targeting The Interactions Of Fatty Acid Metabolism With PI3K/mTOR and MAPK As a Novel Therapeutic Strategy In Diffuse Large B-Cell Lymphoma (DLBCL)

BackgroundFatty acid metabolism is altered in several cancers, which includes de novo synthesis by fatty acid synthase (FASN) and increased utilization of fatty acids via β-oxidation to meet cellular energy requirements. PI3K and MEK/ERK signal transduction are primary biological pathways that activate tumor-related fatty acid metabolism. Notably, these concepts have emerged primarily from solid tumor studies; there is a comparative paucity of data in lymphoma. We examined the functional roles of fatty acid metabolism in DLBCL cells and their interaction with oncogenic signal transduction pathways including PI3K, MEK/ERK and hypoxia inducible factor-1 alpha (HIF-1a). We also investigated potential therapeutic implications of targeting fatty acid metabolism for the treatment of DLBCL. MethodsDLBCL cell lines OCI-LY3, OCI-LY19, SUDHL4, and SUDHL10 were examined in this study. Cerulenin (FASN inhibitor), Orlistat (FASN and LPL inhibitor), BEZ-235 (Dual PI3K/mTOR inhibitor), AZD6244 (MEK inhibitor) were used to study the effects of enzyme inhibition on lipid metabolism, cell signaling and cell death. We assessed cell viability with the MTT assay and apoptosis by flow cytometric analysis of Annexin-V/propidium iodide (PI). FASN or LPL mRNAs were quantified in DLBCL cell lines by quantitative RT-PCR as well as through gene expression profiling (GEP) analysis (by cell of origin) using the CaBIG dataset. Further, FASN and associated signaling pathways (MEK, ERK, and HIF-1a) were analyzed by Western blot. Finally, for investigation of potential interactions between FASN and HIF-1a or MAPK signaling, we utilized short hairpin RNA interference (shRNA) to knock out (KO) desired pathways. ResultsGEP analysis showed that genes related to fatty acid synthesis and mTOR were more prominently expressed in germinal center (GC) DLBCL (p=0.0006 vs GC control and p=0.0001 vs non-GC DLBCL), whereas genes involved in utilization of fatty acid for energy, PI3K and MAPK were preferentially expressed in non-GC DLBCL (p<0.0001 vs non-GC control and GC DLBCL). Protein level expression of FASN and CPT1 (relevant to β-oxidation) was readily detectable in all DLBCL cell lines, while the expression of LPL was low, except in SUDHL10 cells. We next examined FASN expression following KO of MEK, ERK, and/or HIF-1a using shRNA in OCI-LY3 and SUDHL10 cells. HIF-1a KO in OCI-LY3 cells and MEK, ERK KO in SUDHL10 significantly decreased FASN expression. Similarly, pharmacological inhibition of PI3K/mTOR or MEK using novel small molecule agents BEZ-235 or AZD6244 (selumetinib), we observed down-regulation of both HIF-1a and FASN expression. Treatment with BEZ-235 under fatty acid deprived conditions resulted in synergistic cell death 60% in OCI-LY3, compared to 25% with BEZ-235 alone. Additionally inhibition of MEK/ERK using AZD6244 or U0126 also resulted in down-regulation of CPT1 relevant to β-oxidation. Pharmacologic treatment with cerulenin, however, resulted in inhibition of MEK and ERK phosphorylation and apoptosis in SUDHL4 and OCI-LY3 cells. Furthermore, treatment with AZD6244 or cerulenin both resulted in time- and dose-dependent cell death (Annexin-V/PI) and decreased proliferation (by MTT) in GC and non-GC DLBCL cell lines (P<0.001). ConclusionsAltogether, we demonstrated that fatty acid metabolism is highly upregulated in DLBCL and that there was preferential gene expression in DLBCL cells in part according to cell of origin. Further, PI3K/mTOR, MAPK and HIF-1a constitutively activated oncogenic pathways in DLBCL appeared here to directly regulate fatty acid metabolism. In addition, we showed that targeting fatty acid metabolism may be harnessed as a potential therapeutic strategy. Continued investigation is warranted to delineate the mechanisms through which MAPK and HIF-1a regulate FASN expression and to determine the in vivo implications of FASN inhibition on DLBCL tumor growth. Disclosures:No relevant conflicts of interest to declare.