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BACKGROUND The immunologic effects of maintenance therapy in patients (pts) with AML in remission are not well-characterized but of high clinical interest, as rapid recovery of bone marrow (BM) after intensive chemotherapy (IC) may help delay relapse. Post IC, immunological interactions in the BM microenvironment present several immunosuppressive mechanisms. PD-L1 is commonly overexpressed on AML blasts, which is associated with worse prognosis. Oral azacitidine (Oral-AZA [CC-486]) is a hypomethylating agent recently approved in the US for pts with AML in complete remission (CR) or CR with incomplete hematologic recovery (CRi). To better understand the effects of Oral-AZA on immune cells, checkpoint expression of PD-L1/2 on AML blasts and normal myeloid progenitors (MPs), and the kinetics of T cell recovery and activation/exhaustion (eg, PD1, TIM3) were assessed. METHODS Biomarker-evaluable pts aged ≥ 55 years with AML were randomized 1:1 to Oral-AZA 300 mg (n=56) or placebo (PBO, n=52) post IC within 4 months of achieving CR. Flow cytometry evaluations of BM aspirates were performed at screening (ie, baseline [BL]), every 3 cycles until cycle 24 and every 6 cycles thereafter to cycle 36, or as clinically indicated. Correlative analyses of baseline immune parameters with median (med) relapse-free survival (RFS) were computed using Kaplan-Meier methods. RESULTS In the biomarker-evaluable pts, PD-L1 and PD-L2 expression at BL were higher on AML blasts (med intensity 7.1 and 2.9) than normal MPs (0.7 and 1.6). Most AML blasts were PD-L2+ (79%), whereas only 1.9% were PD-L1+. When stratified by the med, higher BL CD3 T cell numbers (as a % of total BM ) were associated with favorable RFS in both Tx arms (Oral-AZA: ≥ med, 562 days[d] and < med, 235d [P = .0308]; PBO: ≥ med, 325d and < med, 155d [P = .0391]). At cycle 3, pts in the Oral-AZA arm had a 1.7-fold increase in CD3 T cells from BL (PBO, 1.1; P = .0450), suggesting Oral-AZA can promote immunologic recovery during early Tx cycles. There was an inverse correlation between T cell exhaustion marker phenotypes (PD1/TIM3+) with CD4 (r = -.5967; P < .0001) and CD8 (r = -.2484; P = .0095) T cell numbers. An increase in RFS was seen in the PBO arm with lower PD1/TIM3+ CD4 numbers (< med, 429d; ≥ med, 155d; P = .0037), with a nominal increase observed in the Oral-AZA arm (< med, 428d; ≥ med, 303d; P = .6764). In a subset of pts, Oral-AZA appeared to suppress CD4 T cell exhaustion (PD1/TIM3+) compared with PBO. CONCLUSIONS Pts in CR/CRi post-IC have a unique immune profile defined by high expression of PD-L1 on a subset of blasts and a high % of PD-L2+ blasts. A higher BL CD3 T cell count after IC in BM was prognostic. Additionally, Oral-AZA appears to contribute to an increase in T cells while also suppressing exhaustion, potentially promoting T cell signaling that could activate functional immune-mediated responses against residual leukemic cells. Citation Format: Daniel L. Menezes, Wendy L. See, Alberto Risueno, Jianglin Ma, Ignazia La Torre, Barry Skikne, CL Beach, Keshava Kumar, Anjan Thakurta. Oral azacitidine modulates the immune microenvironment in acute myeloid leukemia (AML) patients in remission: A subanalysis from the QUAZAR AML-001 Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 505.

Multiple myeloma (MM) is a genetically heterogeneous disease where risk stratification and outcomes are associated with translocations of the immunoglobulin (Ig) and MYC loci, copy number abnormalities and mutations. Therefore, we designed a comprehensive MM targeted sequencing panel (MM panel) to investigate the common genomic abnormalities and validated it against known standards. The MM panel includes targeted mutation and translocation panels encompassing ~ 990 kb and ~ 4.7 Mb respectively. The mutation panel includes exons of 228 genes and the translocation panel investigates the structural and copy number changes. The two panels are manufactured separately and combined in a 5:1 ratio to prevent over-sequencing of the larger translocation panel. To validate the MM panel, we performed targeted sequencing on 110 MM patient samples and compared the results to different orthogonal technologies. Libraries were prepared from the DNA extracted from CD138+ bone marrow cells and non-tumor tissue (peripheral blood or saliva) using the KAPA workflow. Following hybridization and amplification the libraries were sequenced using 75 bp paired end sequencing. Sequences were aligned to hg19 and mutations and translocations were identified using Strelka and Manta. Copy number was determined using the ratio of non-tumor to tumor reads in each targeted region. Data was further validated using clinical FISH (copy number and translocations), MLPA (copy number), known standards (mutations), or ddPCR (mutations). Canonical IgH translocations were detected in 33.6% of patients by sequencing which agreed with FISH except for one patient in which a t(11;14) was detected by FISH only. The overall sensitivity and specificity for translocation detection was 97.4% and 100%, respectively. Non-canonical translocations were detected in 14.5% of samples, 43% of which were to the MYC locus. MYC abnormalities were detected in 46.4% of samples. Copy number was determined by sequencing and MLPA for 22 regions that were directly comparable in 103 patient samples and the concordance between the technologies was R2=0.969. For the important prognostic regions, the concordance was R2=0.962 (CDKN2C), R2=0.986 (CKS1B), and R2=0.973 (TP53). Mutation detection validation was performed using samples with known variant allele frequencies (VAF) for common mutations. We were able to determine the sequencing VAF for 74 mutations across 5 samples with a concordance of R2=0.9849 between the expected and observed frequencies. The minimum detected VAF was 1.3% at an average depth of 891x. We also performed ddPCR on 6 patient samples with the common KRAS, NRAS and BRAF mutations with a VAF concordance of R2=0.9983. In summary, we have developed and validated a sequencing panel which is being deployed to clinical laboratories that is relevant to MM patient prognosis, risk stratification, and treatment. Citation Format: Brian Andrew Walker, Aarif Ahsan, Parvathi Sudha, Tasneem Kausar, Sushmita Rane, Erin Flynt, Anjan Thakurta. A targeted capture panel to identify translocations, copy number abnormalities, and mutations in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 450.

Introduction: Multiple myeloma (MM) is a cancer of the plasma cells in the bone marrow, and its clinical course depends on a complex interplay of clinical traits and molecular characteristics of the plasma cells. Since risk-adapted therapy is becoming standard of care, there is an urgent need for a precise risk stratification model to assist in therapeutic decision-making. While progress has been made, there remains a significant opportunity to improve patient stratification to optimize treatment and to develop new therapies for high-risk patients. To accelerate the development and evaluation of such risk models in MM, we formed a DREAM Challenge, a crowd-sourced competition that engages large cross-disciplinary teams of experts to address complex problems in biomedicine. Methods and Data: In collaboration with Multiple Myeloma Genome Project (MGP), clinical variables, patient outcomes, genetic, and gene expression data from thousands of samples were curated and harmonized from multiple public and private studies. In preparation for the challenge, a team of data scientists was assembled to evaluate this data, benchmark public high-risk models, and assess established prediction metrics with regard to progression-free survival (PFS) and overall survival (OS), the clinical endpoints of the challenge. Docker containers will be used to validate submitted models on private data that would otherwise not be available and to facilitate the transition of the best performing predictive signature to a clinical application. The MM DREAM challenge is accessible at: synapse.org. Results: The international staging system (ISS) for myeloma was used as a baseline classifier for high-risk patients (PFS < 18mo). We evaluated published high-risk signatures - UAMS-5, UAMS-17, UAMS-70, and EMC92 - as benchmarks and observed that they consistently outperformed the baseline ISS predictor. High-risk prediction scores from these models were moderately correlated, suggesting published classifiers capture non-overlapping determinants of risk. Development of de novo classifiers by our team integrating clinical and molecular data highlighted opportunities for model refinement and supports rationalization of a crowd-sourced challenge to advance the field. Conclusion: Preliminary analysis of the challenge data suggests there is an opportunity to significantly improve risk stratification models in MM. In addition to the robust benchmarking of existing classifiers, we anticipate new, more accurate models will be proposed through a MM challenge given the scale of the combined data sets. We hope to uncover novel clinical and molecular traits that may yield insight into the pathology of MM and provide direction for follow-up studies. Importantly, this challenge will illustrate the advantages of leveraging public data and crowdsourcing to address therapeutically relevant questions in oncology. In addition, this challenge establishes a community resource for future research and benchmarking of novel classifiers. Citation Format: Michael Mason, Michael Amatangelo, Daniel Auclair, Doug Bassett, Hongyue Dai, Andrew Dervan, Erin Flynt, Hartmut Goldschmidt, Dirk Hose, Konstantinos Mavrommatis, Gareth Morgan, Nikhil Munshi, Alex Ratushny, Dan Rozelle, Mehmet Samur, Frank Schmitz, Ken Shain, Anjan Thakurta, Fadi Towfic, Matthew Trotter, Brian Walker, Brian S. White, Thomas Yu, Justin Guinney. Multiple Myeloma DREAM Challenge: A crowd-sourced challenge to improve identification of high-risk patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4725. doi:10.1158/1538-7445.AM2017-4725

IMiDs® immunomodulatory drugs Lenalidomide (LEN) and Pomalidomide (POM) are antineoplastic agents that have a significant clinical impact in the treatment of patients with multiple myeloma (MM). Recent studies indicate that the anti-proliferative activity of LEN and POM in MM cells is associated with ubiquitination and degradation of the Ikaros family zinc finger protein transcription factors, Aiolos (IKZF3) and Ikaros (IKZF1), the substrates of the Cereblon (CRBN)-dependent Cullin 4 E3-ligase complex. Measurement of Aiolos and Ikaros protein levels in MM patient samples is critical for future correlative studies. Here we describe the development and validation of dual IHC Aiolos/CD138 and Ikaros/CD138 assays using the highly specific Aiolos rabbit monoclonal antibody CGN-9-9-7, and a commercial Ikaros rabbit polyclonal antibody H-100 in combination with a commercial CD138 antibody. Aiolos and Ikaros immunoreactivity in CD138+ MM cells is scored using the semi-quantitative H-score method. H-scores range from 0 to 300 and are the sum of the products of the percentage of tumor cells (0-100%) and the intensity of staining (0-3). The assays were specific and detected high Aiolos and Ikaros expression in the positive control OCI-Ly10 lymphoma cell line, and low Aiolos and Ikaros levels in POM treated OCI-Ly10 cells. These results were confirmed via Western Blot analysis of Aiolos and Ikaros expression data in these cells lines. Assay precision was examined via staining serial sections of the same MM tumor across three different days and deemed acceptable. The dual IHC assays were used to evaluate bone marrow core biopsies or aspirate clots from 22 MM patients and H-scores were determined by a board-certified pathologist. The dual IHC assays detected a wide range of Aiolos or Ikaros immunoreactivity in the 22 MM cases, with nuclear H-scores ranging from 10 to 260 for Aiolos and 40 to 240 for Ikaros. In conclusion, the dual Aiolos/CD138 and Ikaros/CD138 IHC assays were developed to provide a specific and reliable semi-quantitative method to evaluate Aiolos and Ikaros protein levels in both bone marrow core biopsies and clots from MM patients. These assays are currently being used in clinical trials to assess the importance of Aiolos and Ikaros as biomarkers in MM patients treated with IMiDs® immunomodulatory drugs. Citation Format: Yan Ren, Maria Wang, Suzana Couto, Donna Hansel, Anita K. Gandhi, Patrick Hagner, Anjan Thakurta, Rajesh Chopra, Mike Breider. Dual-color immunohistochemistry assays for measuring Aiolos and Ikaros proteins in multiple myeloma patient samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5445. doi:10.1158/1538-7445.AM2015-5445

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA In preclinical and mostly in vitro studies, pomalidomide (Pom) has been shown to mediate direct anti-proliferative effects on tumor cells, as well as immune-modulatory effects on T cells, NK cells and monocytes. Cereblon (CRBN), a direct cellular target for Pom has been involved in the anti-proliferative effects in tumor cells via selective degradation of Ikaros (IKZF1) and Aiolos (IKZF3). Depletion of IKZF1/IKZF3 has also been implicated in Len-mediated amplification of anti-CD3-induced IL2 production in human T cells in culture. However the impact of pomalidomide on tumor proliferation and immune activation in vivo is unknown. Here we have evaluated the clinical and pharmacodynamic effects of continuous or intermittent dosing strategies of pomalidomide/dexamethasone in lenalidomide-refractory myeloma in a randomized trial. 39 eligible patients with relapsed myeloma were randomized to therapy with Pom/Dexamethazone (following Pom alone for cycle 1), utilizing either continuous Pom dosing (2 mg-28/28 days, cohort 1, n = 19) or an intermittent dosing schedule (4 mg-21/28 days, cohort 2, n = 20). Dexamethazone was administered at 40 mg weekly at cycle 2 and beyond. Intermittent dosing strategy, despite having frequent adverse events, led to greater tumor reduction. Both cohorts experienced similar event-free and overall survival. Both regimens led to a distinct pattern but similar degree of mid-cycle immune activation as manifest by increased expression of cytokines and lytic genes in T and NK cells. Pomalidomide induced polyfunctional T cell activation, with increased proportion of co-inhibitory receptor BTLA+ T cells and Tim-3+ NK cells. Baseline levels of cereblon, ikaros and aiolos protein in tumor cells using validated IHC assay on marrow biopsies, did not correlate with response or survival. Pomalidomide treatment led to a rapid decline in Ikaros in T and NK cells in vivo as measured by intranuclear flow staining, and therapy-induced activation of CD8+ T cells correlated with clinical response. These data demonstrate that pomalidomide leads to strong and rapid immunomodulatory effects involving both innate and adaptive immunity, even in heavily pre-treated MM, which correlate with clinical anti-tumor effects. Clinicaltrials.gov-[NCT01319422][1]. Citation Format: Rituparna Das, Kartik Sehgal, Lin Zhang, Rakesh Verma, Yanhong Deng, Mehmet Kocoglu, Juan Vasquez, Srini Koduru, Yan Ren, Maria Wang, Suzana Couto, Mike Breider, Donna Hansel, Stuart Seropian, Dennis Cooper, Anjan Thakurta, Xiaopan Yao, Kavita M. Dhodapkar, Madhav V. Dhodapkar. Comparison of pomalidomide dosing strategies in lenalidomide-refractory myeloma: Impact on clinical outcome, immune activation and cereblon targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1354. doi:10.1158/1538-7445.AM2015-1354 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01319422&atom=%2Fcanres%2F75%2F15_Supplement%2F1354.atom

mTOR signaling, which plays a central role in controlling cell cell proliferation, survival, mobility and angiogenesis, is considered an important target for new anticancer drugs development. The involvement of the CD40/CD154 signaling pathway has also been reported to play an important role in regulating anti-tumor immune responses. In this study, using a first-in-class orally bioavailable mTOR kinase inhibitor, AZD8055, which, unlike rapamycin, can inhibit both mTORC1 and mTORC2, we evaluated the anti-tumor efficacy of mTOR kinase inhibitor treatment alone in a murine model of metastatic renal cell carcinoma (Renca) and compared biological responses achieved by administering AZD8055 as a single agent to those obtained in combination with agonistic anti-CD40 antibody. In vitro survival assays showed that Renca cell apoptosis could be induced by AZD8055. In vivo, although both agents administered alone had some anti-tumor efficacy, the combination of AZD8055 and CD40 Ab induced a significant anti-tumor response in both intra-renal and intra-splenic inoculations model of renal cell carcinoma, as compared to single agent treatments. The combination treatment, also resulted in an increase and activation of tumor- and liver-associated macrophages, NK cells and CD8 T cells in vivo. AZD8055/anti-CD40 treatment also increased the level of systemic Th1 cytokines, including IL-12, IFN-γ, TNFα. IFN-γ production by CD8+ T cells and TNFα production by macrophages were also found to be elevated in the AZD8055/anti-CD40 combination treated-group, compared with any single treatment group. Furthermore, the expression of Th1-associated chemokines, RANTES and IP10, were significantly induced in the combination treated-group. These data suggest that the combination of mTOR kinase targeting agents with immunotherapy could be an area of further development in renal cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5596.