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It is well known by now that John McGahern was a scrupulous reviser of his own work, even if this insight into his compositional methods has not been accompanied by a substantial body of research on the archive and the revisions themselves. This essay aims to address this anomaly by focusing on the genetic evolution of McGahern's short story ‘Doorways’. Specifically, it will concentrate on the earliest handwritten drafts when the work is at its most provisional. The consensus view of McGahern's writing practices is of an artist committed to ideals of Flaubertian perfectionism, but implicit in this view is a bias towards the more granular work of late-stage refinement. The approach this essay takes shows McGahern at his most distant from the Flaubertian perfectionist that he is best known as, thus opening up new ways to reconsider how those works achieve their distinctive appearance of refined delicacy and simplicity.

This study sought to understand how the programmed death ligand 1 (PD-L1) inhibitor durvalumab and the immunomodulatory agent pomalidomide regulate immune cell activation and function in patients with relapsed/refractory (RR) multiple myeloma (MM). Immunologic changes in peripheral blood and bone marrow of patients treated with durvalumab as monotherapy or in combination with pomalidomide with/without dexamethasone were characterized by assessing subsets of immune cells and gene signatures to understand the immunomodulatory effect of the treatment. Soluble PD-L1 levels were elevated at screening in patients with RRMM but did not correlate with response to durvalumab combination therapy. Immune cell subsets were increased in peripheral blood during treatment with durvalumab and pomalidomide, and combination therapy induced significant gene expression changes in the MM tumor microenvironment versus durvalumab alone. Estimation of cell populations based on RNA sequencing data revealed increased monocytes, neutrophils, and natural killer cells with the combination therapy, but not with durvalumab alone. Additionally, multiplex immunofluorescence of bone marrow demonstrated that immune populations were different in responders versus nonresponders to durvalumab plus pomalidomide with dexamethasone therapy. Overall, durvalumab effectively blocked soluble PD-L1; however, durvalumab monotherapy was not associated with immunologic changes, which were observed with combination therapy.

Hydration plays an important role in performance, injury prevention, and recovery for athletes engaged in competitive sports. Therefore, it is important that strength and conditioning coaches understand an athlete’s hydration needs to prevent illness and enhance performance. The purpose of this study was to identify hydration knowledge, attitudes, and behaviors of collegiate track and field throwers, as well as identify barriers to hydration and sources of nutritional information. The Rehydration and Refueling in Collegiate Track and Field Throwers Survey was sent to 271 track and field thrower coaches with a request to forward the email to current track and field throwers. Pearson correlation coefficients were calculated regarding knowledge, attitude, and behavior scores among the participants in this sample. Differences among response patterns were assessed via Chi-square analysis. Alpha level was set at p = .05. Results demonstrated that 97.3% (n = 287) of respondents knew that dehydration would decrease performance, but 50.5% (n = 149) erroneously believed thirst was the best indicator of dehydration. Chi-square analysis demonstrated a significant difference in reported values between participants who intended to eat a performance-enhancing diet and those who consumed less fluid than recomended values (207 – 295 m)l in the 2-3 hours prior to competition (χ2 = 10.87, p < .05). Pearson correlation coefficients demonstrated a large association between knowledge and behavior (r = .70, p < .05), a medium association between knowledge and attitude (r = .41, p < .05), and a small association between attitude and behavior (r = .21, p < .05). This suggests that strength and conditioning coaches and health staff need to educate and monitor hydration behaviors among collegiate track and field throwers to optimize performance.

Researchers suggest that sport participation among athletes with disabilities promotes healthier lifestyles, increases self-esteem, and enhances peer acceptance. Ideally, coaches should be confident in teaching skills, tactics, and sportsmanship, while exhibiting appropriate leadership behaviors in order to positively impact the psychosocial development of any athlete. Thus, the present research examined sources of coaching efficacy that predict leadership behaviors in coaches who work with athletes who have physical disabilities. Seventy international Paralympic coaches of female and male sport teams completed a modified version of the Coaching Success Questionnaire-2, the Coaching Efficacy Scale and the Leadership Scale for Sports. Regression models indicated that total coaching efficacy was a significant predictor of instructional and positive feedback leadership behaviors, with prior success also being a significant predictor of instructional behavior.

Background The tumor microenvironment (TME) is increasingly appreciated as an important determinant of cancer outcome, including in multiple myeloma (MM). However, most myeloma microenvironment studies have been based on bone marrow (BM) aspirates, which often do not fully reflect the cellular content of BM tissue itself. To address this limitation in myeloma research, we systematically characterized the whole bone marrow (WBM) microenvironment during premalignant, baseline, on treatment, and post-treatment phases. Methods and findings Between 2004 and 2019, 998 BM samples were taken from 436 patients with newly diagnosed MM (NDMM) at the University of Arkansas for Medical Sciences in Little Rock, Arkansas, United States of America. These patients were 61% male and 39% female, 89% White, 8% Black, and 3% other/refused, with a mean age of 58 years. Using WBM and matched cluster of differentiation (CD)138-selected tumor gene expression to control for tumor burden, we identified a subgroup of patients with an adverse TME associated with 17 fewer months of progression-free survival (PFS) (95% confidence interval [CI] 5–29, 49–69 versus 70–82 months, χ2 p = 0.001) and 15 fewer months of overall survival (OS; 95% CI –1 to 31, 92–120 versus 113–129 months, χ2 p = 0.036). Using immunohistochemistry-validated computational tools that identify distinct cell types from bulk gene expression, we showed that the adverse outcome was correlated with elevated CD8+ T cell and reduced granulocytic cell proportions. This microenvironment develops during the progression of premalignant to malignant disease and becomes less prevalent after therapy, in which it is associated with improved outcomes. In patients with quantified International Staging System (ISS) stage and 70-gene Prognostic Risk Score (GEP-70) scores, taking the microenvironment into consideration would have identified an additional 40 out of 290 patients (14%, premutation p = 0.001) with significantly worse outcomes (PFS, 95% CI 6–36, 49–73 versus 74–90 months) who were not identified by existing clinical (ISS stage III) and tumor (GEP-70) criteria as high risk. The main limitations of this study are that it relies on computationally identified cell types and that patients were treated with thalidomide rather than current therapies. Conclusions In this study, we observe that granulocyte signatures in the MM TME contribute to a more accurate prognosis. This implies that future researchers and clinicians treating patients should quantify TME components, in particular monocytes and granulocytes, which are often ignored in microenvironment studies., Author summary Why was this study done? The cells around a tumor, also known as the tumor microenvironment (TME), can help a tumor grow by suppressing the immune system or fight a tumor by mounting an immune response. Most studies of multiple myeloma (MM) have focused on the tumor itself, rather than the bone marrow (BM) TME in which the tumor is growing. We hypothesized that the MM TME held clues that could help us better treat patients. What did the researchers do and find? We used a gene-expression–based computational technique to determine which cell types were present in patient BM. Patients with BM lacking a family of innate immune cells called granulocytes presented with worse outcomes compared to other patients. As MM progresses from a predisease to a cancerous state, the percentage of granulocytes decreases; the patients with the fewest granulocytes had more serious diseases. What do these findings mean? If granulocytes help myeloma patients respond to therapy, then addressing the decline in granulocytes may improve MM treatment. Patients with MM and few granulocytes in their BM should be watched for worse outcomes.

Introduction The newly diagnosed pancreatic ductal adenocarcinoma (PDAC) population has a high unmet need for effective treatments, with median survival of < 1 year. The phase 3 APACT (Adjuvant Pancreatic Adenocarcinoma Clinical Trial) evaluated the use of adjuvant nab-paclitaxel plus gemcitabine vs. gemcitabine in 866 patients with surgically resected PDAC. We explored the tumor microenvironment (TME) of >500 baseline resected APACT tumors to identify TME features that are associated with adverse outcomes. Methods Biopsy analyses included RNA-seq, DNA-seq, and multiplexed immunohistochemistry (mIHC). We quantified, for 533 patient biopsies, via mIHC the spatial arrangement of 7 immune cell types and 2 checkpoint markers relative to tumor and nontumor regions, and pairwise colocalization relative to each other. We identified combinations of biomarkers that correlate significantly with molecular signatures, predefined patient subsets, and overall survival (OS). Hazard ratios (HR) and p-values were computed via a Cox proportional hazards regression model which included resection and lymph node status as covariates. Significantly differentially-expressed transcripts were associated with biomarkers derived from the mIHC via unpaired t-test. Results Higher densities of CD8+ T cells within tumor regions correlated with longer OS (hazard ratio HR=0.76; p=0.03), and higher overall densities of CD163+ macrophages correlated with shorter OS (HR=1.44; p=0.006). We furthermore identified a patient subset (n=72) with a combination of higher CD8+ T cell and lower CD163+ macrophage densities that had a strikingly significant decreased HR of 0.46 (p=0.009). Moreover, patients with a high degree of spatial colocalization between CD8+ T cells and dual-positive CMAF+CD163+ M2-like macrophages observed a significant increased HR of 1.51 (p=0.0006), a biomarker only surpassed by nodal status in significance of correlation with OS (HR=1.9; p=0.00015). While this cellular colocalization was computed to be independent of cell densities, we found that the association of this colocalized pair of cell types with shorter OS was most significant for patients with lower overall CD8+ T cell densities (HR=1.84; p=0.0004). We further identified differentially regulated transcripts associated with this interaction and found specific putative ligand-receptor pairs that were also associated with lower OS. Conclusion A combination of greater infiltration of CD8+ cytotoxic T cells and lower infiltration of macrophages is associated with longer OS in the APACT trial. Moreover, the spatial colocalization between CD8+ T cells and CMAF+ M2 macrophages is associated with shorter OS. These findings were observed across both treatment arms of the study. Future investigation of this apparent interaction, and associated differentially expressed transcripts, may inform management of patients with pancreatic adenocarcinoma and increase effectiveness of PDAC therapies. Citation Format: David J. Reiss, Andrew Browne, Brian Fox, Alexander V. Ratushnyy, Maria Wang, Andrew V. Biankin, Thomas Lila. Spatial arrangements of immune cells of the pancreatic ductal adenocarcinoma tumor microenvironment correlated with outcomes in the phase 3 APACT trial [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C085.

The ability to predict individual differences in motor learning has significant implications from both theoretical and applied perspectives. However, there is high variability in the methodological and analytical strategies employed as evidence for such predictions. Here, we critically examine the evidence for predictions of individual differences in motor learning by reviewing the literature from a 20-year period (2000-2020). Specifically, we examined four factors: (i) the predictor and predicted variables used, (ii) the strength of the prediction and associated sample size, (iii) the timescale over which the prediction was made, and (iv) the type of motor task used. Overall, the results highlight several issues that raise concerns about the quality of the evidence for such predictions. First, there was a large variation in both predictor and predicted variables, suggesting the presence of a large number of researcher degrees of freedom. Second, sample sizes tended to be small and the strength of the correlation showed an inverse relation with sample size. Third, the timescale of most predictions was very short, mostly constrained to a single day. Last, most studies were largely restricted to two experimental paradigms - adaptation and sequence learning. Based on these issues, we highlight recommendations for future studies to improve the quality of evidence for predicting individual differences in motor learning.

The purpose of this study was to test the perceived effects of psychological skills training (PST) on anxiety and anaerobic performance in college-aged students. Fifty-five college students (44 females, 11 males) volunteered to complete an online survey instrument. Participants were recruited via convenience sampling methods to answer questions built through a Qualtrics online survey. The questionnaire was split into two sections. The first section asked participants to identify psychological skills, their familiarity with them, and their frequency of use. The second section consisted of categorizing each skill so participants could mark the extent to which it influences their training goal. Data analysis showed males were more familiar with five out of six psychological skills when compared to females. This is result is likely due to males showing higher stress levels during training and performance compared to females. The findings of this study confirm the importance of mental health education and PST in college students.

This paper describes several aspects of the physical layer and over the air interface of Loon. Loon utilizes stratospheric balloon-based high-altitude platforms (HAPs) that use Long-Term Evolution (LTE) to connect people with standard User Equipment (UEs) to the Internet. In particular, topics covered include the Loon prototype eNodeB (eNB) antenna pattern, the observed channel, UE battery life, and coexistence with terrestrial networks using the same spectrum. While channel models from a HAP to the ground have been well studied in the past, the use of polarization diversity to establish Multi-Input Multi-Output (MIMO) communication to real UEs below 1 GHz has not. In addition, a theoretical analysis of terrestrial coexistence and an analysis of the estimated impact on UE battery life when communicating with HAPs are presented. Finally, results from several measurement campaigns and from experiments with polarization diversity are presented as a spot check of theory.

Summary Idiopathic pulmonary fibrosis (IPF) is a progressive fatal disease affecting over 100 000 people in Europe with an increasing incidence. Available treatments offer only slowing of disease progression and are poorly tolerated by patients leading to cessation of therapy. Lung transplant remains the only cure. Therefore, alternative treatments are urgently required. The pathology of IPF is complex and poorly understood and thus creates a major obstacle to the discovery of novel treatments. Additionally, preclinical assessment of new treatments currently relies upon animal models where disparities with human lung biology often hamper drug development. At a cellular level, IPF is characterized by persistent and abnormal deposition of extracellular matrix by fibroblasts and alveolar epithelial cell injury which is seen as a key event in initiation of disease progression. In-depth investigation of the role of alveolar epithelial cells in health and disease has been impeded due to difficulties in primary cell isolation and culture ex vivo. Novel strategies employing patient-derived induced pluripotent stem cells engineered to produce type 2 alveolar epithelial cells (iAEC2) cultured as three-dimensional organoids have the potential to overcome these hurdles and inform new effective precision treatments for IPF leading to improved survival and quality of life for patients worldwide.

Background and Objectives: National Collegiate Athletic Association (NCAA) soccer coaches implement numerous warm-up and flexibility strategies to prepare athletes for training and competition. The F&eacute, d&eacute, ration Internationale de Football Association (FIFA) developed the 11+ injury prevention program to reduce non-contact injuries. This study aimed to analyze the level of familiarity with and implementation of the evidence-based FIFA 11+ amongst NCAA Division I (DI) and Division III (DIII) men&rsquo, s and women&rsquo, s soccer coaches. Materials and Methods: NCAA soccer coaches in the United States received an Institutional Review Board&mdash, approved survey hyperlink. A total of 240 coaches completed the survey. The respondents represented 47.5% men&rsquo, s and 52.5% women&rsquo, s teams distributed within DI and DIII programs. Descriptive statistics are reported as frequency counts and mean &plusmn, standard deviation where applicable. Pearson&rsquo, s chi-square tests were performed to assess potential differences with a significance level set at &alpha, &lt, 0.05. Results: The results indicated that approximately 62% of the respondents reported being familiar with the FIFA 11+ program. Of those coaches familiar with the program, 15.0% reported full implementation, 57.5% reported partial implementation, and 27.5% reported no implementation. Chi-square analyses revealed significant differences in FIFA 11+ implementation based upon division level (&chi, 2 = 4.56, p = 0.033) and coaching certification levels (&chi, 2 = 13.11, p = 0.011). Conclusions: This study indicates that there is a gap between FIFA 11+ knowledge and actual implementation. To reduce the risk of non-contact injury, there is a need to educate coaches and athletic trainers on the purpose of the FIFA 11+ program and how to perform the exercises correctly.

Introduction: Mutations in TP53 occur in 10% of patients (pts) with AML and HR-MDS and have been associated with worse outcomes and an immunosuppressive phenotype. To define the immune and epigenetic landscape in TP53-M advanced myeloid neoplasms (MN), we compared data from 61 pts with HR-MDS or AML with TP53 mutations (TP53-M) to 143 TP53 wildtype (TP53-WT) pts who were followed prospectively with serial samples in a well-annotated clinical trial in which all pts received hypomethylating agent (HMA)-based therapy. Methods: The FUSION trial (NCT02775903) was a large, randomized phase 2 study comparing azacitidine (AZA) monotherapy with AZA + anti-PD-L1 antibody durvalumab in 2 separate cohorts of previously untreated unfit AML and HR-MDS pts (Zeidan A et al, ASH 2019). Responses were classified by IWG 2003 and 2006 criteria for AML and MDS, respectively. Survival was estimated using Kaplan-Meier techniques. Samples from peripheral blood (PB) and bone marrow (BM) were obtained at baseline and serially on trial. A 38-targeted mutation analysis was performed at Munich Leukemia Laboratory. Only level 1 pathogenic TP53 mutations with variant allele frequency (VAF) ≥2% were included. DNA methylation was assessed using Illumina's Infinium Human Methylation EPIC methylation array. Immunophenotyping and immune checkpoint expression was performed using flow cytometry. Gene expression profiles were studied by RNA-sequencing. Results: Of 129 AML and 84 HR-MDS pts enrolled in FUSION trial, 37 had TP53-M AML, 88 TP53-WT AML, 24 TP53-M HR-MDS, and 55 TP53-WT HR-MDS pts. The average VAF for TP53 mutations were 37%, and 90% had ≥10% VAF. TP53-M AML pts were more likely to have poor-risk cytogenetics, therapy-related disease, and lower BM blast percentage compared to TP53-WT AML pts. TP53-M HR-MDS were more likely to have secondary MDS, very poor risk cytogenetics by IPSS-R, and very high risk IPSS-R score. There were no statistically significant differences in overall response rate (ORR) between TP53-M and TP53-WT pts (AML cohort: ORR: 35.1% [95% CI: 21%-53%] vs. 34.1% [CI: 26%-45%]; HR-MDS cohort: ORR: 41.7% [CI: 23%-63%] vs. 60% [CI: 46%-73%]). Median OS was 8.1 [95% CI: 5.4 - 13] months (mos) among TP53-M AML pts and 16.6 [95% CI: 13 - 21] mos for TP53-WT AML pts [Figure 1A]. Median OS was 9.8 [95% CI: 9.3 - 20+] mos for TP53-M HR-MDS pts and 23.5 [95% CI: 12 - 25+] mos for TP53-WT HR-MDS pts [Figure 1B]. Global DNA methylation was independent of TP53 mutation status in both AML (global methylation score x10^5: TP53-M: 4.9 [SD: 0.23] vs TP53-WT: 4.8 [SD: 0.33]; p=0.35) and HR-MDS pts (global methylation score: TP53-M: 4.8 [SD: 0.20] vs TP53-WT: 4.7 [SD: 0.25]; p=0.24) at baseline. DNA methylation changes after one cycle of AZA treatment were similar in both cohorts (AML: TP53-M:4.4 [SD: 0.38] vs TP53-WT: 4.5 [SD: 0.41, p=0.33]; HR-MDS: TP53-M: 4.3 [SD: 0.35] vs TP53-WT: 4.3 [SD: 0.36]; p=0.52). In RNA sequencing (Figure 2), TP53-M pts had higher expression of T-cell genes (e.g. IL7R) in both AML and HR-MDS compared to TP53-WT pts. Compared to TP53-WT pts, IFN alpha signature genes were reduced only in TP53-M AML pts but were increased in TP53-M HR-MDS pts. PD-L1 (CD274) expression was correlated with the T-cell gene signature and had a higher expression in TP53-M samples. TP53-M pts showed lower expression of tumor associated genes (e.g. CD34) consistent with the tumor cell percentages seen by BM flow cytometry. However, in gene set enrichment analysis, MYC target genes, MTORC1, and E2F were enriched in TP53-M samples consistent with the higher expression of proliferation genes (e.g., MKI67). In the bone marrow flow cytometry of AML pts, more T-cells were seen in TP53-M pts (Figure 3A), and PDL1 positive tumor cells were trending higher in TP53-M pts while the total abundance of tumor cells was slighter higher in TP53-WT (Figure 3B). Discussion: We confirm here that achieving a response to AZA therapy in AML or HR-MDS pts is not impacted by presence of TP53 mutations, however as expected median OS was substantially shorter among TP53-M pts for both AML and HR-MDS. In analyzing the epigenetic landscape, there were no differences in baseline global DNA methylation by TP53 status. RNA sequencing showed enrichment of T-cell genes and PD-L1, and an increase in gene expression of proliferation genes in TP53-M pts. Taken together, these findings support the presence of immunosuppressive microenvironment among TP53-M pts with advanced MN. Figure 1 Figure 1. Disclosures Zeidan: Janssen: Consultancy; Jasper: Consultancy; Epizyme: Consultancy; Acceleron: Consultancy, Research Funding; Gilead: Consultancy, Other: Clinical Trial Committees; Genentech: Consultancy; Kura: Consultancy, Other: Clinical Trial Committees; BMS: Consultancy, Other: Clinical Trial Committees, Research Funding; Loxo Oncology: Consultancy, Other: Clinical Trial Committees; Jazz: Consultancy; BioCryst: Other: Clinical Trial Committees; Astex: Research Funding; Novartis: Consultancy, Other: Clinical Trial Committees, Travel support, Research Funding; ADC Therapeutics: Research Funding; Incyte: Consultancy, Research Funding; Agios: Consultancy; AbbVie: Consultancy, Other: Clinical Trial Committees, Research Funding; Pfizer: Other: Travel support, Research Funding; BeyondSpring: Consultancy; Cardiff Oncology: Consultancy, Other: Travel support, Research Funding; Geron: Other: Clinical Trial Committees; Daiichi Sankyo: Consultancy; AstraZeneca: Consultancy; Amgen: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy, Research Funding; Aprea: Consultancy, Research Funding; Ionis: Consultancy; Astellas: Consultancy. Hasle: Bristol Myers Squibb: Current Employment. Thompson: Bristol Myers Squibb: Current Employment. Lopes de Menezes: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Rose: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Boss: Bristol Myers Squibb: Current Employment. Fox: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Introduction: The transmembrane protein CD47 is ubiquitously expressed on normal cells and often overexpressed on cancer cells, including NHL. Binding of CD47 to signal regulatory protein-α (SIRPα) on macrophages induces an anti-phagocytic signal enabling tumor cells to escape the innate immune response. Macrophage antitumor activity can be restored by simultaneously blocking the CD47-SIRPα signaling axis and inducing a pro-phagocytic signal with the use of tumor-opsonizing antibodies. Targeting SIRPα on monocytes and macrophages rather than the ubiquitously expressed CD47 may overcome some toxicities associated with anti-CD47 therapies. Here, we report the discovery and characterization of a fully human anti-SIRPα antibody and its preclinical activity in combination with the opsonizing antibody rituximab in CD20+ diffuse large B-cell lymphoma (DLBCL) cell lines. Methods: A total of ~ 10 10 fully human immunoglobulin G antibodies were screened for binding to the extracellular domain of recombinant human SIRPα using a yeast display platform. Surface plasmon resonance was used to determine CC-95251 binding coverage across SIRPα haplotypes. The ability of CC-95251 to block CD47-SIRPα interaction was measured using Octet ® and Biacore™ assays. We determined the crystal structure of SIRPα in complex with the CC-95251 Fab to characterize its epitope and to define the structural basis for CD47-SIRPα interaction blockade. To identify tumor types likely susceptible to CD47-SIRPα axis disruption, expression levels of CD47-SIRPα and CD163 were assessed in bulk tumor samples using The Cancer Genome Atlas (TCGA) data. The antitumor effects of CC-95251 in combination with rituximab were examined by measuring the percentage of phagocytic macrophages in co-culture experiments of differentiated macrophages and CD20+ DLBCL cell lines (OCI-Ly3, RIVA, Pfeiffer, and Karpas 422). To confirm CC-95251 binding to monocytes, immunophenotyping of peripheral blood mononuclear cells from healthy donors and cynomolgus monkeys was performed using multiparameter flow cytometry. Lastly, pharmacokinetics and hematologic effects were analyzed in cynomolgus monkeys after treatment with 10, 30, or 100 mg/kg CC-95251. Results: Initial screening by yeast display yielded ~ 350 candidates. The top 24 clones were characterized fully and CC-95251 was selected as the lead monoclonal antibody exhibiting high binding affinity across the 6 most prevalent SIRPα human haplotypes. CC-95251 potently blocked CD47-SIRPα binding in a dose-dependent manner, with a concentration of 100 nM inhibiting CD47 binding almost completely. Co-crystallization modeling showed that CC-95251 engages SIRPα in a region overlapping the CD47 binding site, demonstrating a mechanism for CD47-SIRPα blockade. DLBCL was identified as a suitable tumor type for CC-95251 treatment based on CD47-SIRPα expression and macrophage infiltration. Co-culture experiments of donor macrophages and several DLBCL cell lines showed that CC-95251 monotherapy had weak-to-moderate antitumor activity. However, when combined with rituximab, the levels of phagocytic macrophages were markedly increased in a CC-95251 dose-dependent manner, suggesting that inhibition of the CD47-SIRPα anti-phagocytic axis with CC-95251 and activation of pro-phagocytic signaling with rituximab provide an enhanced antitumor effect in DLBCL cell lines. CC-95251 predominantly bound to cells of myeloid origin, including monocytes and, to a lesser extent, myeloid dendritic cells, whereas no binding to natural killer cells was observed. Toxicology studies in cynomolgus monkeys showed safe intravenous delivery of CC-95251 at therapeutic doses, with no evidence of white blood cell, monocyte, lymphocyte, or red blood cell depletion. Conclusions: CC-95251 is a novel, high-affinity, fully human monoclonal anti-SIRPα antibody that blocks the binding of CD47 to SIRPα. When combined with the therapeutic opsonizing antibody rituximab, CC-95251 enhances macrophage phagocytic activity against DLBCL cell lines in co-culture models. These results support the clinical evaluation of CC-95251 + rituximab for relapsed or refractory NHL. A phase 1 dose-escalation and -expansion study of CC-95251 for the treatment of advanced solid and hematologic malignancies is underway (NCT03783403). Disclosures Chan: Bristol Myers Squibb: Current Employment. Trout: Bristol Myers Squibb: Ended employment in the past 24 months. Mikolon: Bristol Myers Squibb: Current Employment. Adams: Bristol Myers Squibb: Current Employment. Guzman: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Fenalti: Bristol Myers Squibb: Current Employment. Mavrommatis: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Abbasian: Bristol Myers Squibb: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Dearth: Bristol Myers Squibb: Current Employment. Fox: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Sivakumar: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Hariharan: Bristol Myers Squibb: Current Employment.

Tumor cells engage in bidirectional interactions with stroma and immune cells to promote disease progression and immune evasion. Stroma-specific gene signatures have been associated with outcome in diffuse large B-cell lymphoma (DLBCL), but their immunobiology has been understudied. To characterize the stromal landscape in lymphoma, we performed high-dimensional imaging mass cytometry analysis of the major stroma subsets and revealed a marked expansion and remodeling of the immuno-specialized fibroblastic reticular cells (FRCs) in human DLBCL biopsies (n=53). The FRC network was similarly remodeled in tumors from the IμBcl6 transgenic model of lymphoma, and aberrant fibroblasts were in close proximity to cancer cells. Modelling the interactions between murine and patient FRCs and tumor cells, using 2D and 3D cultures, showed that lymphoma drives the acquisition of an inflammatory-like, pro-tumoral (upregulation of fibroblast activating protein-α, FAP) phenotype and associated functional capabilities. Comparative bulk transcriptomic analysis revealed that lymphoma-FRCs undergo transcriptional reprogramming and activate gene pathways associated with inflammatory responses. Moreover, single-cell RNA-seq revealed an expansion of activated FRC clusters expressing B cell supporting genes, while T cell-associated FRCs were contracted. Altered chemokine signaling pathways in DLBCL-FRCs were functionally linked to reduced attraction of T cells and impeded migration along the lymphoma-reticular network. Moreover, lymphoma-FRCs upregulated expression of inhibitory PD-1 ligands that reduced the anti-tumor cytolytic activity of CD8+ T cells, a T cell bispecific antibody (CD20-TCB, glofitamab) and anti-CD19 CAR T cells in our coculture models. To overcome the immunosuppressive activity of DLBCL-FRCs, we investigated the use of CD20-TCB in combination with stroma-targeting immunocytokine fusion protein drug (FAP-IL2v, RG7461) or costimulatory fusion protein (FAP-4-1BBL, RG7827). Functional cytotoxicity assays using human and murine primary DLBCL patient samples revealed that both stroma-targeting drugs paired effectively with the CD20-TCB to enhance the cytotoxic activity of autologous CD8+ T cells. In addition, the ability of immune-/stroma- targeted combination immunotherapy to trigger anti-tumor activity and CD8+ T cell retention within the FRC-TME was demonstrated using 3D precision-cut lymph node slice-based organotypic cultures of DLBCL and other B cell malignancies. In conclusion our data reveal that lymphoma cells actively reprogram FRCs that acquire altered immunoregulatory function which prevents effective T cell motility and suppresses the anti-tumor function of cytolytic T cells. Importantly, we demonstrate that combination immunotherapy incorporating fibroblast-targeting fusion proteins could effectively recover anti-tumor T cell activity. Citation Format: Benedetta Apollonio, Nedyalko Petrov, Filomena Spada, Peter Jarvis, Domenico Cozzetto, Shichina Kannambath, David Kuo, Mansoor Saqui, Rose-Marie Amini, Gunilla Enblad, Graham Charlotte, Reuben Benjamin, Anna Vardi, Elisabeth Phillips, Jon Salisbury, Eric N. Olson, Brian Fox, Patrick Hagner, Anita Gandhi, Ruth F. Jarrett, Sylvia Herter, Marina Bacac, Christina Klaus, Christian Klein, Alexander Deutsch, Alan G. Ramsay. Stroma-immune landscape in lymphoma: new mechanisms of immunosuppression and therapeutic targeting [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 3165.

Background and aim Institute of Obstetricians and Gynaecologists (RCPI) guideline for preterm prelabour rupture of the membranes (PPROM) recommends the administration of antenatal corticosteroids for women who are 24 to 36 weeks pregnant with anticipated preterm labour. It reduces the risks of respiratory distress syndrome, intraventricular haemorrhage and necrotizing enterocolitis. Recommended steroid is 12 mg of intramuscular Betamethasone given 24 hours apart and at least 24 hours before delivery. This study determined the level of adherence to the guidelines. Methodology Mothers who presented in preterm prelabour rupture of membranes between 1/1/2015 and 31/12/2018 were incorporated into this study. Mothers were evaluated for maternal age, parity, body mass index, ethnicity and demographic distribution. Babies were evaluated for gender, gestational age, weight, corticosteroid type and time of administration prior to delivery, number of doses, type of respiratory support, comorbidities and length of hospital stay. Data was recorded on a collection sheet. Results 103 mothers with PPROM were eligible to receive antenatal steroids, of whom 44(43%) received recommended treatment and 59(57%) received incomplete or no treatment. Out of infants who received complete course of antenatal steroids, 7(16%) required surfactant and intubation, 3 (7%) required CPAP and surfactant, 16(36.4%) required only CPAP, 1 (2%) required high flow oxygen and 17(38.6%) did not need any respiratory support. Of the 59 infants who had an incomplete course or no steroids, 12(20%) required surfactant and intubation, 4(6.8%) required surfactant and CPAP, 20(34%) required CPAP, 1 (2%) required low flow oxygen and 22(37.2%) did not require any support. Of those who received complete treatment, 26 were early preterm (28- Conclusion It was found that adherence to guideline is satisfactory. Imminent preterm delivery prevented 100% adherence to the protocol. This is a risk reduction strategy and parents should be made aware of the possible outcomes.

Failure to clear antigens causes CD8+ T cells to become increasingly hypo-functional, a state known as exhaustion. We combined manually extracted information from published literature with gene expression data from diverse model systems to infer a set of molecular regulatory interactions that underpin exhaustion. Topological analysis and simulation modeling of the network suggests CD8+ T cells undergo 2 major transitions in state following stimulation. The time cells spend in the earlier pro-memory/proliferative (PP) state is a fixed and inherent property of the network structure. Transition to the second state is necessary for exhaustion. Combining insights from network topology analysis and simulation modeling, we predict the extent to which each node in our network drives cells towards an exhausted state. We demonstrate the utility of our approach by experimentally testing the prediction that drug-induced interference with EZH2 function increases the proportion of pro-memory/proliferative cells in the early days post-activation.

Pancreatic ductal adenocarcinoma (PDAC) remains a difficult disease to treat, with few therapies available that target specific patient subgroups. Translational studies in pancreatic cancer can be technically challenging due to biopsy specimen characteristics including low tumor cellularity and dense fibrotic stroma. Resected primary pancreatic tumors obtained during the phase 3 APACT clinical trial (NCT01964430 adjuvant nab-paclitaxel + gemcitabine versus gemcitabine monotherapy n = 866) are a unique resource for characterizing molecular and immune subtypes among PDAC tumors and their association with treatment outcomes in the adjuvant chemotherapy setting. Tumor-infiltrating immune cells were assessed in serial sections of 453 tumors by dual-chromogen immuno-histochemical (IHC) staining (CD4 CD8 CD20 CD163 CMAF CD56 FoxP3 PD-1 PD-L1). Image alignment, segmentation, and spatial localization of stained cells relative to a pan-cytokeratin staining-based tumor epithelial mask was performed using a commercial analysis platform. Expression profiles were obtained for 515 macrodissected tumor biopsy regions by RNA-seq. Transcriptional subtypes were assigned based on schema previously reported by Moffit and Bailey, and molecular pathway correlates were characterized using gene set enrichment analysis. Based on immunochemical staining, higher intratumoral CD8+ or lower CD163+ cell densities were associated with modestly longer disease-free or overall survival in patients treated with nab-paclitaxel plus gemcitabine. The combination of both high CD8+ and low CD163+ cell density was notably associated with longer overall survival compared to other subjects treated with the combination regimen (mOS>55months versus 36 months HR=0.46 p= Citation Format: Thomas Lila, Andrew Biankin, Andrew Browne, David J. Reiss, Brian Lu, Daniel Pierce, Alexander Ratushny, Kao-tai Tsai, Sneh Lata, Sitharthan Kamalakaran, Tomas Babak, Brian Fox, Sam Danziger, Konstantinos Mavrommatis, Matthew W. B. Trotter, David Chang, Fadi Towfic. Multi-omic Profiling of primary pancreatic adenocarcinomas obtained from the APACT adjuvant trial of nab-paclitaxel + gemcitabine vs gemcitabine [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-008.

Introduction: It is strikingly difficult to develop successful treatments for PDAC; even with curative resection, most patients die from early occult metastases. Prior studies identified the presence of tumor-infiltrating lymphocytes (TILs) in primary PDAC tumors as having prognostic significance in the PDAC adjuvant setting, sharpening the questions of what fraction of patients have immune-infiltrated tumors and what therapeutic strategies should be pursued in these patients vs. the non-infiltrated group. The phase 3 APACT trial evaluated the use of adjuvant nab-paclitaxel plus gemcitabine vs. gemcitabine in 866 patients with PDAC who had undergone primary tumor resection, with the primary endpoint of disease-free survival evaluated by independent review. We extended studies of the tumor microenvironment of PDAC to a large set of resected APACT primary tumors in an effort to further refine features of tumor or immune infiltrate that influence disease progression and to determine if chemotherapy regimen–specific predictive signatures are identifiable. Tissue analyses for a large subset of APACT samples included RNA-seq, DNA-seq, multiplexed immunohistochemistry (IHC), and proteomics. Methods: We imaged and quantified markers for tumor cells, 7 different immune cells, and 2 immune checkpoint markers using bright-field chromogenic multiplexed IHC from pretreatment samples for more than 500 APACT primary tumor samples. We computationally defined the tumor, tumor margin, and distal stromal (> 150 μm from tumor boundary) regions, and quantified densities and distributions of immune cells in these regions. As part of an initial analysis of more than 400 samples, we applied both unsupervised clustering and supervised classification to these IHC measurements to identify patient subgroups with similar spatial arrangements of immune cells relative to tumor regions. Results: The preliminary analysis of normalized cell densities across all 3 tissue regions revealed 3 patient subgroups: one in which immune cells are mixed within the tumor regions; a second where immune cells approach the tumor boundary but are depleted within the tumor; and a third in which immune cells are depleted in both tumor and its margin, remaining at high densities only in the distal stromal regions. Within these latter subgroups, CD20+, CD4+, and CD8+ cells were more prevalently depleted from tumor and/or margin, whereas CD163+ and CD163+CMAF+ cells showed less of this arrangement. Nearly 85% of patients fell in the second or third patient group. Conclusions: We are pursuing analyses of these data in conjunction with upcoming molecular and genetic profiling data to further elucidate the association of the immune cell populations and these subgroups with clinical outcomes. These data will provide an unprecedented opportunity for exploratory analysis and discovery of immune, molecular, and genetic biomarkers for PDAC patient stratification. Citation Format: David J. Reiss, Thomas Lila, Suzana Couto, Sitharthan Kamalakaran, Yan Ren, Doug Bowman, Amber Ortiz, Maria Wang, Clifton Drew, Kao-Tai Tsai, Mathieu Marella, Brian Fox, Garth McGrath, Matthew Trotter, Fadi Towfic, Ian Cushman, Alexander Ratushny, Brian Lu, Daniel Pierce, Jim Cassidy. Spatial organization of pancreatic ductal adenocarcinoma (PDAC)–associated immune cells from the Adjuvant Pancreatic Adenocarcinoma Clinical Trial (APACT) study [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A43.

Background: The availability of chimeric antigen receptor (CAR)-modified T cells (CAR T) has profoundly increased therapeutic options for patients (pts) with B cell malignancies, including DLBCL. Liso-cel is an investigational, anti-CD19, defined composition, 4-1BB, CAR T cell product administered at a target dose of CD4+ and CD8+ CAR T cells. To understand tumor microenvironmental (TME) factors affecting short-term and durable responses in pts with R/R DLBCL who received liso-cel in the TRANSCEND NHL 001 study, we conducted multiplexed IF analyses of 111 DLBCL biopsies for 83 pts obtained at baseline (n=58) and approximately 11 days (D11) (n=53; 28 paired) after liso-cel infusion (NCT02631044). Methods: We employed three 5-plex IF panels, consisting of antibodies detecting (1) B cell (CD19, CD20) and T cell lineage (CD4, CD8, EGFR) markers, (2) immunosuppressive markers (CD163, FoxP3, CD73, IDO1, PD-L1), and (3) functional markers (CD3, Ki67, GZMB, PD-1, EGFR). Liso-cel expresses a truncated EGFR (EGFRt), and fluorescent anti-EGFR was used to identify CAR T cells within the tumor biopsies. We also performed bulk tumor RNA profiling for an overlapping subset of 50 baseline biopsies and 37 D11 biopsies (11 paired). We investigated the association of differences in marker densities for pts with best overall response (BOR) of complete response (CR), and progressive disease (PD). Baseline and D11 biopsy findings were correlated with early responses at ~1 month (M1) posttreatment (PD n=16; CR n=42) and durable responses at ~9 months (M9) posttreatment (PD n=76; CR n=32; 55 pts evaluated at both M1 and M9). We investigated how baseline and D11 densities, with spatial distinction between tumoral and peritumoral regions, correlated with early and durable responses. All comparisons describe differences in median densities, and have statistical significance reported with uncorrected P values assessed via the (unpaired) Wilcoxon-Mann-Whitney nonparametric test. Results: Signals in baseline biopsies that correlated with early (M1) response differed from those that correlated with durable (M9) CR. A 21% higher baseline presence of PD-1+ T cells was associated with pts who achieved early CR at M1 vs pts who had PD at M1 (P=0.007). Pts with durable CR at M9 had 39% lower baseline levels of CD163+ macrophages (P=0.019) and 270% higher levels of CD73+ cells (P=0.028) than those with PD at M9. On-treatment (D11) tumors of pts with both early and durable CR had 28% higher levels of EGFRt+ (CAR T) CD8+ T cells (P=0.022), and 810% higher EGFRt- (non-CAR T) CD4+ (but notably, not CD8+; P=0.28) T cells (P=0.009). We also investigated changes in marker densities between baseline and on-treatment (D11) biopsies, and found that pts with durable CR at M9 had decreased on-treatment B cell densities (P=0.029), and increased densities of CD8+ GZMB+, Ki67+, and/or PD-1+ CAR (P=0.001) as well as non-CAR T (P=0.017) cells. Pts with durable CR also had a 29% increase in tumor-associated CD163+ macrophages at D11 relative to baseline (P=0.033). While the accessibility of spatial arrangements and multilabeled cells from IF enables a more nuanced picture of the TME, many of the general trends described above are concordant with those observed in bulk tumor RNA sequencing. Lower baseline expression of CD163 (P=0.021) and higher expression of CD73 (P=0.054) were seen in pts with durable CR. Additionally, elevated on-treatment (D11) expression of CD3E, CD4, and liso-cel (P Conclusions: Overall, these data suggest that increased infiltration of tumor-specific CAR T cells upon initial treatment with liso-cel helped establish an active immune response, and that recruitment of additional functional endogenous (particularly CD4+) T cells correlated with durable response. Higher numbers of activated/functional T cells and lower numbers of macrophages prior to treatment also correlated with durable response to liso-cel. Thus, tumors in responders may already have had a baseline TME in which T cells could infiltrate and respond to antigen. This may have promoted the success of CAR T cell entry into tumors and the subsequent recruitment and activation of endogenous lymphocytes that support their function. Disclosures Reiss: Celgene Corporation: Employment, Equity Ownership. Do:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Kuo:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gray:Celgene Corporation: Employment, Equity Ownership. Olson:Celgene Corporation: Employment, Equity Ownership. Lee:Celgene Corporation: Employment, Equity Ownership. Young:Celgene Corporation: Employment, Equity Ownership. Srinivasan:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Gray:Celgene: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership. Dubovsky:Celgene: Employment. Schmitz:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership.

Introduction: Targeting immune cells in the tumor microenvironment is an attractive approach to improving antitumor activity of standard therapy in r/r hematologic malignancies. Durvalumab is a monoclonal antibody that blocks programmed cell death ligand-1 (PD-L1), allowing T cells to recognize and kill tumor cells. Methods: The FUSION NHL-001 study (NCT02733042) is a phase 1/2 study assessing safety and efficacy of durvalumab as monotherapy or in combination. Eligible patients (pts) must have had r/r DLBCL or FL after ≥1 systemic therapy requiring therapeutic intervention. Other inclusion criteria include ECOG performance status 0-2 and ≥1 CT-measurable lesion. Pretreatment tumor biopsies were collected to assess biomarkers of response to durvalumab combination therapies. Pts with r/r B-cell neoplasms were enrolled into 1 of 4 arms that included durvalumab monotherapy (Arm D) or in combination with lenalidomide ± rituximab (Arm A), ibrutinib (Arm B), or rituximab ± bendamustine (Arm C). Durvalumab was given at a fixed dose of 1500 mg every 4 weeks. The study consisted of 2 parts: dose finding (except for Arm D) to establish the recommended phase 2 dose (RP2D) for each combination and dose confirmation. We present final subset analyses for pts with DLBCL and FL treated in Arms A, C, and D. Results: A total of 38 DLBCL and 22 FL pts were enrolled. Baseline characteristics are presented in Tables 1 and 2. Arm A was prematurely closed after an FDA announcement regarding safety concerns with combination of lenalidomide and checkpoint inhibitors in multiple myeloma; therefore, RP2D for lenalidomide ± rituximab could not be defined. However, 4 pts (DLBCL, n=1; FL, n=3) experienced dose-limiting toxicities (DLTs): febrile neutropenia (lenalidomide 10 mg + rituximab), headache, hepatitis, and thrombocytopenia (lenalidomide 20 mg + rituximab). One pt with DLBCL experienced DLT in Arm C (neutropenia) when treated with bendamustine 90 mg/m2 + rituximab. RP2D for bendamustine was established as 70 mg/m2. Durvalumab treatment (13 infusions) was completed for 10 (DLBCL, n=4; FL, n=6) pts (17%), 2 in Arm A and 8 in Arm C; the main reason for durvalumab withdrawal was disease progression. During the study, 38 pts (63%) experienced 81 serious adverse events (SAEs), most frequently related to infections (Figure 1). A total of 22 immune-mediated AEs (imAEs) related to durvalumab were reported in 13 pts (grade 1, 2, and 3 in 9, 7, and 6 cases, respectively): transaminitis and increased bilirubin (9 events), diarrhea (5 events), rash and pruritus (5 events), thyroid disorder (2 events), and infusion-related reaction (1 event). Two pts were treated with systemic steroids, both for grade 3 transaminitis. Others received symptomatic treatment. Ten pts experienced AEs that led to any drug discontinuation: 4 in Arm A (cerebral ischemia, febrile neutropenia, myalgia, hyponatremia), 3 in Arm C (transaminitis; neutropenia in 2 pts) and 3 in Arm D (drug reaction with eosinophilia and systemic symptoms, gastrointestinal perforation, prolonged QT). There were 6 grade 5 AEs, none considered related to study drugs. For pts with DLBCL (Table 3), overall response rate (ORR) was 18% and complete response rate (CRR) was 8%. Median progression-free survival (PFS) (Figure 2) was 2.5 months (95% CI, 1.25-5.13). There were 30 deaths on study (2, 19, and 9 pts in Arms A, C, and D, respectively), 24 (80%) related to disease progression. Median overall survival (OS) was 7.9 months (2.66-15.31). For pts with FL (Table 3), ORR was 59% and CRR was 27%. Median PFS (Figure 3) was 10.6 months (4.63-NE). There were 6 deaths on study (2 and 4 pts in Arms A and D, respectively); 3 (50%) were related to disease progression and 1 to second primary malignancy (bladder cancer, Arm A). Median OS was not reached. An interferon-γ signature comprising 4 genes, IFN-γ, CD274, LAG3, and CXCL9, trended higher with best ORR in pts with r/r DLBCL (N=30, P=0.06) and FL (N=18, P=0.01) independent of treatment arm (Arms A, C, and D included in analysis). Conclusions: Durvalumab as monotherapy or in combination in DLBCL and FL is tolerable without unexpected safety signals but requires close monitoring. Durvalumab alone or in combination appeared to add limited benefit to therapy for r/r DLBCL or FL. However, use of an interferon-γ gene signature may serve as a biomarker by which to enrich for r/r NHL pts that may be more responsive to anti-PD-L1-based therapy and will require further investigation. Disclosures Casulo: Gilead: Honoraria, Other: Travel, accommodation, expenses; Roche: Other: Travel, accommodation, expenses; Celgene: Research Funding. Santoro:MSD: Speakers Bureau; Lilly: Speakers Bureau; Sandoz: Speakers Bureau; BMS: Consultancy; Takeda: Speakers Bureau; BMS: Speakers Bureau; Abb-Vie: Speakers Bureau; Amgen: Speakers Bureau; Celgene: Speakers Bureau; AstraZeneca: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Arqule: Consultancy, Speakers Bureau; Eisai: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Servier: Consultancy, Speakers Bureau; Roche: Speakers Bureau; Gilead: Consultancy, Speakers Bureau. Ando:Eisai: Research Funding. Le Gouill:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche-Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support. Ruan:Celgene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie company: Research Funding; Juno: Consultancy; Kite: Consultancy. Radford:Novartis: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Research Funding; BMS: Consultancy, Honoraria; AstraZeneca: Equity Ownership, Research Funding; Seattle Genetics: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; GSK: Equity Ownership. Arcaini:Celgene, Roche, Janssen-Cilag, Gilead: Other: Travel expenses; Gilead Sciences: Research Funding; Celgene: Speakers Bureau; Bayer, Celgene, Gilead Sciences, Roche, Sandoz, Janssen-Cilag, VERASTEM: Consultancy. Pinto:Roche: Speakers Bureau; Roche, Takeda: Other: Travel grants; EDO-Mundipharma: Patents & Royalties; Roche, MSD, Bristol-Myers Squibb, Servier: Honoraria; Servier, Roche, Bristol-Myers Squibb, MSD: Membership on an entity's Board of Directors or advisory committees. Izutsu:Eisai, Chugai, Zenyaku: Honoraria; Eisai, Symbio, Chugai, Zenyaku: Research Funding; Celgene: Consultancy; Chugai, Celgene, Daiichi Sankyo, Astra Zeneca, Eisai, Symbio, Ono, Bayer, Solasia, Zenyaku, Incyte, Novartis, Sanofi, HUYA Bioscience, MSD, Astellas Amgen, Abbvie, ARIAD, Takeda, Pfizer: Research Funding; Kyowa Kirin, Eisai, Takeda, MSD, Chugai, Nihon Medi-physics, Janssen, Ono, Abbvie, Dainihon Sumitomo, Bayer, Astra Zeneca, HUYA Japan, Novartis, Bristol-Byers Squibb, Mundi, Otsuka, Daiichi Sankyo, Astellas, Asahi Kasei: Honoraria. Rule:Astra-Zeneca: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; TG Therapeutics: Consultancy, Honoraria; Napp: Consultancy; Kite: Consultancy. Munoz:Pharmacyclics /Janssen: Consultancy, Research Funding, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Merck: Consultancy; Kyowa: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene/Juno: Consultancy, Research Funding; Genentech: Consultancy, Research Funding, Speakers Bureau; Kite/Gilead: Consultancy, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy; Alexion: Consultancy; Pfizer: Consultancy; Fosunkite: Speakers Bureau; AstraZeneca: Speakers Bureau; Portola: Research Funding; Incyte: Research Funding. Casadebaig:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Rettby:Celgene Corporation: Employment, Equity Ownership. Dell'Aringa:Celgene Corporation: Employment, Equity Ownership. Delarue:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Czuczman:Celgene Corporation: Employment, Equity Ownership. Cartron:Roche, Celgene: Consultancy; Sanofi, Gilead, Janssen, Roche, Celgene: Honoraria. OffLabel Disclosure: Durvalumab is a PD-L1 blocking antibody indicated for the treatment of patients with 1) locally advanced or metastatic urothelial carcinoma who have disease progression during or following platinum-containing chemotherapy, or who have disease progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy, or 2) unresectable, stage 3 NSCLC whose disease has not progressed following concurrent platinum-based chemotherapy and radiation therapy.

BACKGROUND: Loss of immune surveillance is critical in the pathogenesis of multiple myeloma (MM) and the progression from smoldering to symptomatic MM. To date, no clear efficacy signal has been observed with programmed-death 1 and programmed death ligand-1 inhibitors in patients with MM. General immune dysfunction in MM is well documented, but the evolving immune landscape in relapsed/refractory MM (RRMM) vs newly diagnosed MM (NDMM) is less well characterized. This study aimed to characterize immune profiles in peripheral blood and bone marrow from patients with NDMM and RRMM. METHODS: Peripheral blood samples were collected from 35 NDMM and 146 RRMM patients and 36 age-matched healthy volunteers (HVs). Cell surface and intracellular antigen staining using fluorochrome labeled antibodies was performed on a BD FACSCanto II flow cytometer. Bone marrow aspirates were collected from 26 NDMM and 73 RRMM patients, and the transcriptome was assessed by mRNA-Seq. RESULTS: In peripheral blood, T-cell populations differed between HVs and NDMM and RRMM patients. Absolute numbers of lymphocytes were higher in HVs than in NDMM and RRMM, regardless of the MM disease state. Absolute numbers of total CD4+ T cells and naïve CD4+ T cells were lower in RRMM patients, whereas CD4+ effector memory T cells as a proportion of total CD4+ T cells were increased in RRMM patients. Blood from RRMM patients also contained increased levels of proliferating CD4+ T cells, as evidenced by Ki67, ICOS, and HLA-DR, compared with blood from NDMM patients; HVs had values much closer to those from NDMM than from RRMM patients, suggesting a trend influenced by disease state or therapeutic intervention. In bone marrow, immunologic gene expression signatures were elevated in NDMM vs RRMM patients; the differences were similar to those in peripheral blood. Using limma to model the differential expression of all measured genes between NDMM and RRMM, we identified 367 genes that were elevated in NDMM patients vs 52 in RRMM patients. Gene set analyses using Molecular Signatures Database immunologic signatures (C7) applied to those 367 genes showed that naïve T-cell genes were increased in the bone marrow of NDMM vs RRMM patients. Gene set enrichment analysis with limma, using 489 gene sets from xCell representing 64 cell types and controlling for differences in tumor burden, indicated that macrophage, monocyte, and neutrophil genes were upregulated and T cells, particularly naïve CD4+ T cells, were downregulated in RRMM patients. Immunohistochemistry results from bone marrow biopsies showed increased programmed death-ligand 1 expression on tumor and infiltrating immune cells and increased CD8 infiltration into bone marrow in RRMM vs NDMM patients. Multiparameter immunofluorescence is underway to confirm these findings and further understand the tumor immune microenvironment in patient subsets. As expected, baseline RRMM immune cell populations depended on prior lines of therapy. Daratumumab-exposed RRMM patients had elevated total CD8+ T cells in peripheral blood but decreased CD38+, CD4+, and CD8+ T cells, as well as decreased total natural killer cells, compared with the daratumumab-naïve patients. Transcriptome analyses of bone marrow from daratumumab-exposed RRMM patients revealed increased T-cell gene expression signatures relative to marrow from daratumumab-naïve patients. Additionally, pomalidomide-exposed RRMM patients had increased activated CD4+ and CD8+ T cells vs pomalidomide-naïve patients. CONCLUSIONS: These data indicate that RRMM patients have peripheral blood and bone marrow environments with highly differentiated T-cell populations, whereas NDMM patients show elevated T-cell levels with proliferative capacity. Furthermore, the bone marrow of RRMM patients is enriched with neutrophils and macrophages; investigation is ongoing to determine if these cell types contribute to an immunosuppressive tumor microenvironment. Understanding immune system function based on disease progression, patient segments, and prior lines of therapy is imperative as treatment of MM improves, and it may inform the administration and sequence of next generation immunotherapeutics and identify predictive biomarkers for optimal treatment selection. Disclosures Pietz: Celgene Corporation: Employment. Tometsko:Celgene Corporation: Employment, Equity Ownership. Copeland:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Thompson:Celgene Corporation: Employment, Equity Ownership. Agarwal:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Buchholz:Celgene Corporation: Employment. Komashko:Celgene Corporation: Employment. Dell'Aringa:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment.

Introduction The multiple myeloma (MM) tumor microenvironment (TME) strongly influences patient outcomes as evidenced by the success of immunomodulatory therapies. To develop precision immunotherapeutic approaches, it is essential to identify and enumerate TME cell types and understand their dynamics. Methods We estimated the population of immune and other non-tumor cell types during the course of MM treatment at a single institution using gene expression of paired CD138-selected bone marrow aspirates and whole bone marrow (WBM) core biopsies from 867 samples of 436 newly diagnosed MM patients collected at 5 time points: pre-treatment (N=354), post-induction (N=245), post-transplant (N=83), post-consolidation (N=51), and post-maintenance (N=134). Expression profiles from the aspirates were used to infer the transcriptome contribution of immune and stromal cells in the WBM array data. Unsupervised clustering of these non-tumor gene expression profiles across all time points was performed using the R package ConsensusClusterPlus with Bayesian Information Criterion (BIC) to select the number of clusters. Individual cell types in these TMEs were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results Our deconvolution approach accurately estimated percent tumor cells in the paired samples compared to estimates from microscopy and flow cytometry (PCC = 0.63, RMSE = 9.99%). TME clusters built on gene expression data from all 867 samples resulted in 5 unsupervised clusters covering 91% of samples. While the fraction of patients in each cluster changed during treatment, no new TME clusters emerged as treatment progressed. These clusters were associated with progression free survival (PFS) (p-Val = 0.020) and overall survival (OS) (p-Val = 0.067) when measured in pre-transplant samples. The most striking outcomes were represented by Cluster 5 (N = 106) characterized by a low innate to adaptive cell ratio and shortened patient survival (Figure 1, 2). This cluster had worse outcomes than others (estimated mean PFS = 58 months compared to 71+ months for other clusters, p-Val = 0.002; estimate mean OS = 105 months compared with 113+ months for other clusters, p-Val = 0.040). Compared to other immune clusters, the adaptive-skewed TME of Cluster 5 is characterized by low granulocyte populations and high antigen-presenting, CD8 T, and B cell populations. As might be expected, this cluster was also significantly enriched for ISS3 and GEP70 high risk patients, as well as Del1p, Del1q, t12;14, and t14:16. Importantly, this TME persisted even when the induction therapy significantly reduced the tumor load (Table 1). At post-induction, outcomes for the 69 / 245 patients in Cluster 5 remain significantly worse (estimate mean PFS = 56 months compared to 71+ months for other clusters, p-Val = 0.004; estimate mean OS = 100 months compared to 121+ months for other clusters, p-Val = 0.002). The analysis of on-treatment samples showed that the number of patients in Cluster 5 decreases from 30% before treatment to 12% after transplant, and of the 63 patients for whom we have both pre-treatment and post-transplant samples, 18/20 of the Cluster 5 patients moved into other immune clusters; 13 into Cluster 4. The non-5 clusters (with better PFS and OS overall) had higher amounts of granulocytes and lower amounts of CD8 T cells. Some clusters (1 and 4) had increased natural killer (NK) cells and decreased dendritic cells, while other clusters (2 and 3) had increased adipocytes and increases in M2 macrophages (Cluster 2) or NK cells (Cluster 3). Taken together, the gain of granulocytes and adipocytes was associated with improved outcome, while increases in the adaptive immune compartment was associated with poorer outcome. Conclusions We identified distinct clusters of patient TMEs from bulk transcriptome profiles by computationally estimating the CD138- fraction of TMEs. Our findings identified differential immune and stromal compositions in patient clusters with opposing clinical outcomes and tracked membership in those clusters during treatment. Adding this layer of TME to the analysis of myeloma patient baseline and on-treatment samples enables us to formulate biological hypotheses and may eventually guide therapeutic interventions to improve outcomes for patients. Disclosures Danziger: Celgene Corporation: Employment, Equity Ownership. McConnell:Celgene Corporation: Employment. Gockley:Celgene Corporation: Employment. Young:Celgene Corporation: Employment, Equity Ownership. Schmitz:Celgene Corporation: Employment, Equity Ownership. Reiss:Celgene Corporation: Employment, Equity Ownership. Davies:MMRF: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; TRM Oncology: Honoraria; Abbvie: Consultancy; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria. Copeland:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Barlogie:Celgene: Consultancy, Research Funding; Dana Farber Cancer Institute: Other: travel stipend; Multiple Myeloma Research Foundation: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC. Trotter:Celgene Research SL (Spain), part of Celgene Corporation: Employment, Equity Ownership. Hershberg:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Dervan:Celgene Corporation: Employment, Equity Ownership. Ratushny:Celgene Corporation: Employment, Equity Ownership. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

Introduction Relapsed and refractory multiple myeloma (RRMM) remains a challenging disease to treat due to its heterogeneity and complexity. There is an urgent need for novel combination strategies, including immunotherapy. The study of the tumour and immune microenvironment before and after treatment with combination therapy is a crucial part of understanding the underpinning of disease response. Methods Longitudinal samples of bone marrow aspirates and whole blood were collected from a phase II clinical trial, MEDI4736-MM-003 (NCT02807454) where daratumumab and durvalumab naïve patients were exposed simultaneously to both these drugs. A combination of mass cytometry (CyTOF), RNAseq and flow cytometry were performed on a subset of samples from these subjects. Specifically, paired bone marrow mononuclear cells (BMMC) samples from nine patients taken at screening and six weeks post-treatment were analysed by mass cytometry (CyTOF) using a 37-marker pan-immune panel that included both lineage and functional intracellular/extracellular markers. In addition, whole blood sample specimens were collected at screening and on treatment (8, 15, 30, and 45 days after treatment) and analysed by flow cytometry. Flow cytometry panels were designed to allow interrogation of the abundance and activation status of immune cell subsets. Finally, RNA from bone marrow aspirates at screening and C2D15 were analysed by RNA sequencing. Expression profiles from the aspirates were used to estimate cell proportions by computational deconvolution. Individual cell types in these microenvironments were estimated using the DCQ algorithm and a gene expression signature matrix based on the published LM22 leukocyte matrix (Newman et al., 2015) augmented with 5 bone marrow- and myeloma-specific cell types. Results In a heavily pre-treated population with RRMM, treatment with durvalumab and daratumumab leads to shifts in a number of key immunological populations when compared to pre-treatment. In the bone marrow, CD8 and CD4 populations rise (by CyTOF and RNAseq), while NK, DC and B cell populations fall (by CyTOF). In the bone marrow within CD8+ T lymphocyte populations, we observed a post-treatment rise in markers of degranulation (granzyme p=0.0195, perforin p=0.0078, Wilcoxon signed-rank test). This is also accompanied by a fall in PD1 expression (p=0.0078) and rise in the co-stimulatory receptor DNAM1 (p=0.0273). These changes are most marked on cells with an effector memory CD45RA+ CD8+ T cell phenotype. In the blood, similar to the bone marrow, CD8+ T cells proliferate over the course of treatment (flow cytometry). A fall in both naïve and active NK cell populations is seen following treatment in bone marrow. NK cells express high levels of CD38 and are therefore depleted by daratumumab. Those NK cells which remain have an active phenotype with increased expression of TNFa (p=0.0039) and IFNg (p=0.0195) following treatment. Across the time points sampled in peripheral blood, NK cells were also decreased and those that remained were proliferating. Dendritic cells with a tolerogenic phenotype can be identified prior to treatment and are seen to fall in abundance following treatment with durvalumab and daratumumab. Conclusions The combination of durvalumab and daratumumab leads to several immune microenvironment changes that biologically portend clinical effect. We see increases in the abundance of cell populations with functional anti-tumour activity, including granzyme B+ CD8 T cells and a reduction in PD1high T cells. Despite the treatment expectedly reducing NK cell numbers, many functionally competent NK cells remain, as evidenced by the presence of anti-tumour cytokines. This combination strategy also reduces immunosuppressive tolerogenic DCs, which suppress CD4 and CD8 T cell activity. Taken together, this suggests that this chemotherapy free, doublet treatment has the potential to up-regulate anti-tumour immunological responses, which may restore immunosurveillance mechanisms critically needed in these highly refractory patients. Disclosures Seymour: Celgene: Research Funding. Young:Celgene Corporation: Employment, Equity Ownership. Tometsko:Celgene Corporation: Employment, Equity Ownership. Cavenagh:Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Thompson:Celgene Corporation: Employment, Equity Ownership. Whalen:Celgene Corporation: Employment, Equity Ownership. Danziger:Celgene Corporation: Employment, Equity Ownership. Fitch:Celgene Corporation: Employment, Equity Ownership. Fox:Celgene Corporation: Employment, Equity Ownership. Dervan:Celgene Corporation: Employment, Equity Ownership. Foy:Celgene Corporation: Employment, Equity Ownership. Newhall:Celgene Corporation: Employment, Equity Ownership. Gribben:Acerta Pharma: Honoraria, Research Funding; Cancer Research UK: Research Funding; TG Therapeutics: Honoraria; Roche: Honoraria; NIH: Research Funding; Medical Research Council: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria; Kite: Honoraria; Pharmacyclics: Honoraria; Novartis: Honoraria; Janssen: Honoraria, Research Funding; Wellcome Trust: Research Funding; Unum: Equity Ownership.

UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in anintegration independent manner. Efficient expression can bederived from a single copy of an integrated gene site resulting ina higher percentage of cells expressing the marker gene in theselected pool in comparison to standard non-UCOE containingvectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapidproduction of large quantities of protein in a short period oftime. Utilizing this system more than 300 mg of a recombinantantibody has been produced in less than 1 month from transfectionpools in shake flask. Selected subclones have been scaled intosmall bioreactors in less than 2 months, producing significantquantities of monoclonal antibody using a protocol generic for theparent cell line. The increased efficiency obtained with the UCOEvector reduces the number of transfectants which need to bescreened in order to obtain high productivity subclones.Transfection of a standard host cell line, preadapted to grow in alarge-scale setting, allows for rapid cell line developmentdecreasing the transition time from research into development andmanufacturing. Alternatively, the traditional approach of using aparent cell line which requires serum-free and suspensionadaptation after transfection further increases the need forscreening a large number of subclones, because many of thesubclones will not be able to grow under conditions that allowlarge-scale protein production. The use of a preadapted cell linecan reduce the time required to develop a cell line from months toweeks.