ALBERT

Description: Fibrous sheath interacting protein 1 (FSIP1), a spermatogenesis-related testicular antigen, is expressed in abundance in breast cancers, particularly in those overexpressing human epidermal growth factor receptor 2 (HER2); however, little is known about its role in regulating the growth and metastasis of breast cancer cells. We and others have shown...

Description: Tissue transglutaminase (TG2) enables survival of human malignant pleural mesothelioma cells in hypoxia Cell Death and Disease 8, e2592 (February 2017). doi:10.1038/cddis.2017.30 Authors: Sara Zonca, Giulia Pinton, Zhuo Wang, Maria Felicia Soluri, Daniela Tavian, Martin Griffin, Daniele Sblattero & Laura Moro

Description: Background: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease of malignant B cells most often classified by tumor gene expression and/or mutations. DLBCL is also characterized by a tumor microenvironmental influence that has not been well described. Immuno-oncology-targeting agents such as immune checkpoint inhibitors have limited clinical activity in DLBCL, highlighting the need for a better understanding of the DLBCL tumor microenvironment for rational drug development, combinations, and disease stratification. Here, we systematically characterize the immune composition of more than 100 DLBCL tumors using 2 imaging technologies and provide insight into the complexity of DLBCL disease biology. Methods: A total of 110 cases of newly diagnosed DLBCL were analyzed by multiplex immunohistochemistry (IHC; n=70) and multiplexed ion beam imaging (MIBI) (n=40), each with 10 common markers (CD20, CD3, CD8, Foxp3, CD56, CD163, CD11c, CD56, PD-1, PD-L1) and a few platform-specific markers. Both IHC and MIBI images were digitalized to generate marker-positive cell counts (including single, double, or triple positivity), cell density (cell count/mm2), and population fraction (% of total nucleated cells). Marker density was analyzed for the major components of tumor-infiltrating cell types and correlation between any pair of markers. In addition, RNAseq data and fluorescence in situ hybridization (FISH) data on MYC, BCL-2, and BCL-6 translocation were generated. Results: In the IHC cohort, T cells (CD3+), dendritic cells (DCs; by CD11c+), and macrophages (CD163+)were the major immune components, with median population fractions of 22%, 16%, and 2.7%, respectively. Natural killer cells (CD56+CD20−) were a minor component at a median of 0.1%. A significant negative correlation was observed between tumor cells (CD20+) and CD4+ (Spearman ρ = −0.47; P = 1.3 × 10−04), and CD8+ T cells (Spearman ρ = −0.42; P = 1.4 × 10−03) cells, and an unexpectedly negative correlation between DCs (CD11c+) and macrophages (CD163+, r = −0.63; P = 9.8 × 10−06) was found. Similar to follicular lymphoma, 2 PD-1+ T-cell populations were identified: PD-1bright and PD-1dim. The PD-1bright cells co-stained with CXCR5, indicating T follicular helper cells (Tfh). The PD-1dim were expressed on exhausted effector cells that co-stained with Tim3 or Lag3. The median population fraction of Tim3+ or Lag3+ among T cells was 25%, whereas that of PD-1dim among T cells was 0.2%, indicating that PD-1 was not a useful marker for exhausted T cells. PD-L1 was predominantly found on DCs and macrophages, and the median population fraction of PD-L1+ among tumor cells was only 6.2%. By unsupervised hierarchical clustering on marker density, 3 major immune-infiltration patterns (P1, P2, and P3) were identified. The first 2 segments (P1 and P2) were 20% and 25% of the total cases, respectively. Both were characterized by high T-cell, macrophage, and DC infiltration. P1 was enriched for PD-1+ T cells, whereas P2 was void of any PD-1+ cells. The third segment (P3) that comprised 55% of the cases was predominantly tumor cells with low T-cell, macrophage, and DC infiltration. PD-L1+ cells were primarily found in segments P1 and P2 but rare in segment P3. Additional analysis on the associations between the 3 immune-infiltration patterns and prognostic DLBCL molecular features such as cell-of-origin, double-hit gene signature, and double-hit FISH will also be presented. Conclusions: These data show the complexity of DLBCL disease biology and show classification of DLBCL at the immune-infiltration level as 3 distinct patterns. The overall low expression of PD-1+ T cells and the restricted pattern of PD-L1+ tumor cells provide a possible explanation for the lack of clinical activity of PD-1/PD-L1 blockade in DLBCL. We also observed other exhausted T cells expressing Lag3 and Tim3, suggesting alternative therapeutic opportunities in stratified populations. These data also highlight the opportunity to develop rational immuno-oncology-targeted agents based on the immune infiltration pattern of DLBCL and selection of patients who may respond more favorably to particular agents. Disclosures Huang: Celgene Corporation: Employment, Equity Ownership. Nakayama:Celgene Corporation: Employment, Equity Ownership. Stokes:Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Lee:Celgene Corporation: Employment, Equity Ownership. Ren:Celgene Corporation: Employment, Equity Ownership. Marella:Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Newhall:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

Description: Background Treatment of relapsed/refractory multiple myeloma (RRMM) remains challenging as durable remissions are achieved in patient sub-groups only. Identifying patients that are likely to benefit prior to or early after starting relapse treatments remains an unmet need. MUKseven is a trial specifically designed to investigate and validate biomarkers for treatment optimization in a 'real-world' RRMM population. Design In the randomized multi-center phase 2 MUKseven trial, RRMM patients (≥2 prior lines of therapy, exposed to proteasome inhibitor and lenalidomide) were randomized 1:1 to cyclophosphamide (500 mg po d1, 8, 15), pomalidomide (4 mg days 1-21) and dexamethasone (40 mg; if ≥75 years 20 mg; d1, 8, 15, 21) (CPomD) or PomD and treated until progression. All patients were asked to undergo bone marrow (BM) and peripheral blood (PB) bio-sampling at baseline, cycle 1 day 14 (C1D14, on-treatment) and relapse. For biomarker discovery and validation, IGH translocations were profiled by qRT-PCR, copy number aberrations by digital MLPA (probemix D006; MRC Holland), GEP by U133plus2.0 array (Affymetrix), PD protein markers by IHC and PB T-cell subsets by flow cytometry for all patients with sufficient material. Primary endpoint was PFS, secondary endpoints included response, OS, safety/toxicity and biomarker validation. Original planned sample size was 250 patients but due to a change in UK standard of care during recruitment with pomalidomide becoming available, a decision was made to stop recruitment early. Results In total, 102 RRMM patients were randomized 1:1 between March 2016 and February 2018. Trial entry criteria were designed to include a real-world RRMM population, permitting transfusions and growth factor support. Median age at randomization was 69 years (range 42-88), 28% of patients had received ≥5 prior lines of therapy (median: 3). Median follow-up for this analysis was 13.4 months (95% CI: 12.0-17.5). 16 patients remained on trial at time of analysis (median number of cycles: 19.5; range 8-28). More patients achieved ≥PR with CPomD compared to PomD: 70.6% (95% CI: 56.2-82.5%) vs. 47.1% (CI: 32.9-61.5%) (P=0.006). Median PFS was 6.9 months (CI: 5.7-10.4) for CPomD vs. 4.6 months (CI: 3.5-7.4) for PomD, which was not significantly different as per pre-defined criteria. Follow-up for OS is ongoing and will be presented at the conference. High-risk genetic aberrations were found at following frequencies: t(4;14): 6%, t(14;16)/t(14;20): 2%, gain(1q): 45%, del(17p): 13%. Non-high risk lesions were present as follows: t(11;14): 22%, hyperdiploidy: 44%. Complete information on all high-risk genetic markers was available for 71/102 patients, of whom 12.7% had double-hit high-risk (≥2 adverse lesions), 46.5% single-hit high-risk (1 adverse lesion) and 40.8% no risk markers, as per our recent meta-analysis in NDMM (Shah V, et al., Leukemia 2018). Median PFS was significantly shorter for double-hit: 3.4 months (CI: 1.0-4.9) vs. single-hit: 5.8 months (CI: 3.7-9.0) or no hit: 14.1 months (CI: 6.9-17.3) (P=0.005) (Figure 1A). GEP was available for 48 patients and the EMC92 high-risk signature, present in 19% of tumors, was associated with significantly shorter PFS: 3.4 months (CI: 2.0-5.7) vs. 7.4 (CI: 3.9-15.1) for EMC92 standard risk (P=0.037). Pharmacodynamic (PD) profiling of cereblon and CRL4CRBN ubiquitination targets (including Aiolos, ZFP91) in BM clots collected at baseline and C1D14 is currently ongoing. Preliminary results for the first 10 patients demonstrate differential change of nuclear Aiolos (Figure 1C), with a major decrease in Aiolos H-scores in 7/10 patients from baseline to C1D14 and reconstitution at relapse. T-cell PB sub-sets were profiled at baseline and C1D14 by flow cytometry. Specific sub-sets increased with therapy from baseline to C1D14, e.g. activated (HLA-DR+) CD4+ T-cells, as reported at last ASH. CD4+ T-cell % at baseline was associated with shorter PFS in these analyses in a multi-variable Cox regression model (P=0.005). PD and T-cell biomarker results will be updated and integrated with molecular tumor characteristics and outcome. Discussion Our results demonstrate that molecular markers validated for NDMM predict treatment outcomes in RRMM, opening the potential for stratified delivery of novel treatment approaches for patients with a particularly high unmet need. Additional immunologic and PD biomarkers are currently being explored. Disclosures Croft: Celgene: Other: Travel expenses. Hall:Celgene, Amgen, Janssen, Karyopharm: Other: Research funding to Institution. Walker:Janssen, Celgene: Other: Research funding to Institution. Pawlyn:Amgen, Janssen, Celgene, Takeda: Other: Travel expenses; Amgen, Celgene, Janssen, Oncopeptides: Honoraria; Amgen, Celgene, Takeda: Consultancy. Flanagan:Amgen, Celgene, Janssen, Karyopharm: Other: Research funding to Institution. Garg:Janssen, Takeda, Novartis: Other: Travel expenses; Novartis, Janssen: Research Funding; Janssen: Honoraria. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Wang:Celgene Corporation: Employment, Equity Ownership. Boyd:Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Pierceall:Celgene: Employment. Thakurta:Celgene: Employment, Equity Ownership. Cook:Celgene, Janssen-Cilag, Takeda: Honoraria, Research Funding; Janssen, Takeda, Sanofi, Karyopharm, Celgene: Consultancy, Honoraria, Speakers Bureau; Amgen, Bristol-Myers Squib, GlycoMimetics, Seattle Genetics, Sanofi: Honoraria. Brown:Amgen, Celgene, Janssen, Karyopharm: Other: Research funding to Institution. Kaiser:Takeda, Janssen, Celgene, Amgen: Honoraria, Other: Travel Expenses; Celgene, Janssen: Research Funding; Abbvie, Celgene, Takeda, Janssen, Amgen, Abbvie, Karyopharm: Consultancy.

Description: Abstract 3287 Background: Durable responses with lenalidomide monotherapy have been reported in patients with non-Hodgkin lymphoma. In relapsed/refractory diffuse large B-cell lymphoma (DLBCL), higher responses were observed in the activated B-cell-like (ABC) subtype than in the germinal centre B-cell (GCB)-like subtype (Czuczman, et al. British Journal of Haematology, 2011, 154, 477–481). Herein, the molecular mechanisms involved in the differential efficacy of lenalidomide in DLBCL subtypes were investigated. Methods: A panel of DLBCL cell lines, with 5 of ABC-subtype and 11 of non-ABC subtype, was collected and cell of origin subtype was confirmed based on literature, molecular and genetic analysis. The direct antiproliferative effect of lenalidomide on DLBCL cells was assessed using the 3H-thymidine incorporation assay and apoptosis analysis. The molecular mechanisms involved in the antiproliferative efficacy of lenalidomide in DLBCL subtypes were investigated by western blot, immunohistochemistry (IHC) and qRT-PCR analysis of key signaling events during B-cell receptor (BCR)-dependent NF-κB activation. The critical roles of interferon regulatory factor 4 (IRF4), and cereblon (CRBN) in lenalidomide efficacy were established by knock-in or knock-down of these proteins in sensitive ABC cells. Finally, a mouse xenograft model was used to confirm the antitumor effect of lenalidomide and the relevance of the molecular mechanism involved. Results: Using DLBCL cell lines, lenalidomide treatment was found to preferentially suppress proliferation of ABC-DLBCL cells in vitro at a concentration range of 0.01–100 μM (the median plasma concentration at Cmax for patients receiving 25 mg lenalidomide is 2.2 μM) and delay tumor growth in a human tumor xenograft model of OCI-Ly10 cells (lenalidomide 3–30 mg/kg, p.o. qdX28), with minimal effect on non-ABC-DLBCL cells. This tumoricidal effect of lenalidomide was associated with downregulation of IRF4, a survival factor in ABC-DLBCL cells. Treatment with lenalidomide for 1–3 days, similar to the inhibitors of PKCb and MALT1 (LY-333,531 and z-VRPR-fmk, respectively), was found to significantly (p

Description: Key Points Pomalidomide leads to rapid immune activation in vivo correlating with clinical outcome in relapsed myeloma. Baseline expression of ikaros/aiolos protein in tumor cells is not predictive of outcome.

Description: Background: The zinc finger transcription factors, Aiolos (IKZF3) and Ikaros (IKZF1) were identified as lenalidomide (LEN) and pomalidomide (POM)-induced substrates of the cereblon (CRBN)-dependent Culin4 E3-ligase complex. While recent studies suggest that the anti-proliferative activity of LEN and POM in multiple myeloma (MM) cell lines in vitro is due in part to the targeted ubiquitination and subsequent proteasomal degradation of Aiolos and Ikaros, the downstream molecular mechanisms remain unknown. Using inducible shRNA-mediated knockdown combined with kinetic analyses, we systematically investigated the biological mechanisms associated with the degradation of Ikaros and Aiolos in MM cell lines that are sensitive to or have acquired resistance to LEN and POM. Results: In MM1.S and U266 MM cell lines stably engineered with doxycycline (DOXY)-inducible shRNAs, knockdown of either Ikaros or Aiolos showed a reduction in cell proliferation (80%-90%) as measured by 3H-thymidine incorporation after a 4 day treatment with DOXY. We demonstrated that this anti-proliferative effect is inherently tied to and precedes the induction of apoptosis, which was maximized (60%-80% AnnV+/ToPro3+) 5 days following Aiolos or Ikaros knockdown compared with a control shRNA. shRNA-mediated knockdown of Aiolos or Ikaros was furthermore associated with decreases in both c-Myc and IRF4 protein expression levels (70%-90% and 60%-80%, respectively) that were maximized by day 4. In turn, shRNA knockdown of either c-Myc or IRF4 elicited anti-proliferative (〉 80% inhibition) and pro-apoptotic (50%-80%) responses as early as 48hrs after shRNA induction. These data suggest that the reduction of c-Myc and IRF4 protein levels downstream of Aiolos and Ikaros degradation account for the apoptotic effect and marks the onset of the cytotoxic response induced by LEN and POM in MM cells. To define the temporal order of events involving Aiolos, Ikaros, c-Myc and IRF4 in more detail, kinetic experiments following shRNA-mediated knockdown in parallel with drug treatments were performed. Data from these experiments showed that there is a distinct kinetic order of both LEN- and POM-mediated effects, initiated by immediate targeted degradation of Aiolos and Ikaros (within 90 min), followed by a decrease in c-Myc levels (24-48 hrs) with subsequent IRF4 downregulation (48-72 hrs), and finally, resulting in programmed cell death (3-5 days). Importantly, DOXY washout experiments, resulting in re-accumulation of Aiolos or Ikaros at early time points (24 hrs) partially overcame the antiproliferative effects of the shRNA-mediated knockdown of either target. Interestingly, upon the onset of c-Myc downregulation (24-48 hrs), the commitment to cell death could no longer be reversed in our experiments. Further, we generated MM1.S and U266 cells with acquired resistance to POM (10 µM; also cross-resistant to LEN) (MM1.S/PomR and U266/PomR , respectively), in which CRBN protein expression is substantially decreased (〉 90%). Consequently, in these resistant cell lines, neither Aiolos nor Ikaros are degraded in the presence of LEN or POM. However, bypass of CRBN-dependent Aiolos degradation by DOXY-induced knockdown rescued c-Myc and IRF4 downregulation and concomitant inhibition of growth (90% and 60%, respectively), suggesting that resistant MM cells with acquired CRBN loss remain dependent on Aiolos and Ikaros. Conclusions: For the first time, our studies showed that degradation of Aiolos and Ikaros sets up a molecular sequence of events culminating in programmed cell death in MM cells. Our mechanistic studies showed that c-Myc is a key intermediate factor whose downregulation is a rate-limiting step for the transcriptional downregulation of IRF4 as well as for the commitment to cell death. Taken together, our results demonstrate a molecular sequence of events underlying the mechanism of action of cytotoxicity of LEN or POM in MM cells. Quantitative measurements of Aiolos and Ikaros degradation, and c-Myc and IRF4 downregulation in clinical samples would help validate these findings. Disclosures Bjorklund: Celgene Corp: Employment, Equity Ownership. Havens:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Amatangelo:Celgene Corp: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Wang:Celgene Corp: Consultancy. Breider:Celgene Corp: Employment. Ren:Celgene Corp: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

Description: Treatment options for relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL) are limited with no standard of care; prognosis is poor, with 4- to 6-month median survival. Avadomide (CC-122) is a cereblon-modulating agent with immunomodulatory and direct antitumor activities. This phase 1 dose expansion study assessed safety and clinical activity of avadomide monotherapy in patients with R/R de novo DLBCL and transformed lymphoma. Additionally, a novel gene expression classifier, which identifies tumors with a high immune cell infiltration, was shown to enrich for response to avadomide in R/R DLBCL. Ninety-seven patients with R/R DLBCL, including 12 transformed lymphoma, received 3 to 5 mg of avadomide administered on continuous or intermittent schedules until unacceptable toxicity, disease progression, or withdrawal. Eighty-two patients (85%) experienced ≥1 grade 3/4 treatment-emergent adverse events (AEs), most commonly neutropenia (51%), infections (24%), anemia (12%), and febrile neutropenia (10%). Discontinuations because of AEs occurred in 10% of patients. Introduction of an intermittent 5/7‑day schedule improved tolerability and reduced frequency and severity of neutropenia, febrile neutropenia, and infections. Among 84 patients with de novo R/R DLBCL, overall response rate (ORR) was 29%, including 11% complete response (CR). Responses were cell-of-origin-independent. Classifier-positive DLBCL patients (de novo) had an ORR of 44%, median progression-free survival (mPFS) of 6 months, and 16% CR versus an ORR of 19%, mPFS of 1.5 months, and 5% CR in classifier-negative patients (P = .0096). Avadomide is being evaluated in combination with other antilymphoma agents. This trial was registered at www.clinicaltrials.gov as #NCT01421524.