Myeloid Infiltration Research Articles

An Unusual Extramedullary Presentation of an NPM1-Mutated Acute Myeloid Leukemia (AML) in a Young Adult

Abstract Introduction/Objective Pleural effusion presentation of AML, particularly without leukemic disease, has been rarely described in the literature. This case serves to investigate the value of an identified NPM1 mutation. Methods/Case Report A 25-year-old male with Down syndrome presented with dyspnea. Computerized tomography scan identified a moderate pleural effusion and pelvic lytic bone lesions. A complete blood count was normal. Pleural fluid cytologic evaluation showed a spectrum of immature myeloid precursors, and flow cytometry identified a 23% CD117 positive myeloblast population expressing CD33, CD13, and dim CD45 but lacking CD34 and HLA-DR. Fluorescence in-situ hybridization testing was negative for a PML-RARA fusion. Given the phenotype, molecular testing was initiated and confirmed an NPM1 mutation. Testing revealed additional pathogenic variants in DNMT3A and IDH1 with no FLT3 mutations. Peripheral blood smear and bone marrow evaluations yielded no clear leukemic involvement, and cytogenetics from bone marrow showed 47,XY,+21c[20]. The significance of this myeloid infiltrate was initially not entirely clear with no established leukemic involvement or tumor-forming mass as required for the World Health Organization diagnosis of myeloid sarcoma. However, the finding of the NPM1 mutation lent high confidence for a diagnosis of an extramedullary presentation of acute myeloid leukemia, though the lytic bone lesions were not biopsied to possibly demonstrate a myeloid sarcoma. Results (if a Case Study enter NA) N/A. Conclusion The identified NPM1 mutation was instrumental in diagnosing an unusual presentation of AML where material was limited. Subsequent to this presentation the patient developed more overt leukemic disease at an outside institution, additionally supporting this diagnosis.

Endothelial caveolin-1 regulates cerebral thrombo-inflammation in acute ischemia/reperfusion injury.

SummaryBackgroundThrombo-inflammation is an important checkpoint that orchestrates infarct development in ischemic stroke. However, the underlying mechanism remains largely unknown. Here, we explored the role of endothelial Caveolin-1 (Cav-1) in cerebral thrombo-inflammation.MethodsThe correlation between serum Cav-1 level and clinical outcome was analyzed in acute ischemic stroke patients with successful recanalization. Genetic manipulations by endothelial-specific adeno-associated virus (AAV) and siRNA were applied to investigate the effects of Cav-1 in thrombo-inflammation in a transient middle cerebral artery occlusion (tMCAO) model. Thrombo-inflammation was analyzed by microthrombosis formation, myeloid cell infiltration, and endothelial expression of adhesion molecules as well as inflammatory factors.FindingsReduced circulating Cav-1, with the potential to predict microembolic signals, was more frequently detected in recanalized stroke patients without early neurological improvement. At 24 h after tMCAO, serum Cav-1 was consistently reduced in mice. Endothelial Cav-1 was decreased in the peri-infarct region. Cav-1−/− endothelium, with prominent barrier disruption, displayed extensive microthrombosis, accompanied by increased myeloid cell inflammatory infiltration after tMCAO. Specific enhanced expression of endothelial Cav-1 by AAV-Tie1-Cav-1 remarkably reduced infarct volume, attenuated vascular hyper-permeability and alleviated thrombo-inflammation in both wild-type and Cav-1−/− tMCAO mice. Transcriptome analysis after tMCAO further designated Rxrg as the most significantly changed molecule resulting from the knockdown of Cav-1. Supplementation of RXR-γ siRNA reversed AAV-Tie1-Cav-1-induced amelioration of thrombo-inflammation without affecting endothelial tight junction.InterpretationEndothelial Cav-1/RXR-γ may regulate infarct volume and neurological impairment, possibly through selectively controlling thrombo-inflammation coupling, in cerebral ischemia/reperfusion.FundingThis work was supported by National Natural Science Foundation of China.

Abstract IA012: Intratumoral activation of double-stranded RNA sensors induces systemic immune response to irradiated sarcoma

Abstract Background: Undifferentiated pleomorphic sarcoma (UPS) is a common aggressive sarcoma subtype for which existing therapies are ineffective. Immune checkpoint blockade (ICB) is effective in a minority of patients. Most UPS tumors have a predominantly myeloid infiltrate, a highly plastic cellular population with the potential to serve as a substrate for immunotherapy. Radiotherapy (RT), a standard therapy for UPS, can also drive myeloid cells into the tumor. Based on abundant expression of pattern recognition receptors (PRRs) by myeloid cells, we hypothesized that intratumoral injection of a PRR agonist would leverage pre-existing and RT-associated myeloid cells by redirecting them to prime adaptive anti-tumor immunity. Methods: We used BO-112, a nanoplexed double-stranded ribonucleic acid that activates PRRs, most of which are not typically activated by RT. We used an ICB-resistant mouse model, in which tumor cells derived from a KrasG12D/−P53−/− model of UPS are used to establish large tumors in flanks of C57BL/6J mice. Tumor-bearing mice were treated with RT (8 Gy x 3 fractions daily) ± BO-112 (30 ug/dose). Tumor volume and survival between groups were compared by ANOVA and log-rank test, respectively. To test the role of lymphocytes, the study was repeated in RAG−/−gc−/− mice. Kinetic analysis of myeloid and T cells was performed in the tumors and lymph nodes (LN) on days 15, 21, 26 using high-dimensional flow cytometry. To evaluate the fate of monocytes, LysM-eGFP monocytes were intratumorally transferred following treatment, with subsequent analysis of tumors and LNs on days 16, 18, 21. Results: We detected significantly improved tumor control starting from day 16 with mean tumor volume of 240 mm3 in the combination group compared to 490 mm3 in BO112, 790 mm3 in RT, and 875mm3 in mock groups (p<0.0001) which translated into a maximal survival advantage in the combination group (p<0.005), which was associated with both local and systemic increase in IFNg+ CD8 T cells. In RAG−/−gc−/− mice, the benefit of combination therapy was completely negated. Despite the lymphocyte-dependent effect, myeloid cells were preferentially targeted by BO-112. In the combination therapy group (compared to mock), we observed an early reduction in Ly6ChiMHCII+ cells (p<0.05) within the tumor followed by a progressive increase of the same population in the draining LN over time (p=0.01). In parallel, we observed trafficking of adoptively transferred intratumoral eGFP+ monocytes from the tumor to dLNs in response to BO-112 and RT. Conclusions: Local-only therapy of BO-112 and RT yields a lymphocyte-dependent anti-tumor immune response in an immunotherapy-resistant model of UPS. Longitudinal analysis of locoregional immune responses and adoptive transfer experiments indicate that combination BO-112 and RT induces tumor-to-dLN myeloid trafficking that may bridge an adaptive response and confer therapeutic effect. An ongoing phase 1 dose escalation study testing the combination of RT, BO-112 and nivolumab is currently accruing. Citation Format: Anusha Kalbasi. Intratumoral activation of double-stranded RNA sensors induces systemic immune response to irradiated sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA012.

454O A phase (Ph) I/II trial of the CXCR2 antagonist AZD5069 in combination with enzalutamide (ENZA) in patients (pts) with metastatic castration resistant prostate cancer (mCRPC)

Tumour-infiltrating myeloid cells drive mCRPC growth and therapeutic resistance, in part through IL-23 and NRG1 secretion. Inhibition of myeloid infiltration by disrupting the CXCR2 receptor axis can reverse ENZA resistance in PC models. AZD5069 is a selective CXCR2 antagonist.

Myeloid Wls expression is dispensable for skin wound healing and blood vessel regeneration.

Wnt signaling controls blood vessel growth, regression and patterning during embryonic and postnatal life. Macrophages are major producers of Wnt ligands and angiogenic growth factors. It regulates vascular development and specification during embryogenesis and wound healing. Macrophage dysregulation in wound healing impairs vessel regeneration and delay wound closure. During cutaneous wound healing, the endovascular progenitors (EVPs) proliferate and differentiate into mature endothelial (D) cells in response to signals produced by perivascular cells, including macrophages, governing blood vessels regeneration. However, the role of macrophage’s Wnt production on endothelial cells, especially the EVPs during wound healing is currently unknown. Here we used a cutaneous excisional wound model in mice with conditional deletion of Wnt secretion by myeloid cells (Wlsfl/flLysM-Cre+ ) to assess the kinetics of endothelial subpopulations (including EVP), myeloid infiltration, collagen deposition and wound closure. Deletion of Wls expression by myeloid cells did not affect wound closure and collagen deposition, indicating that myeloid Wls expression does not promote wound healing and regeneration. Myeloid-specific Wls deletion elevated the EVP population during the peak of angiogenesis, yet without affecting blood vessel density. Wounds in Wlsfl/flLysM-Cre+ animals showed unperturbed myeloid infiltration and differentiation. Overall, our data indicate that macrophage Wnt production shapes EVP kinetics without major relevance to wound healing. These findings extend the knowledge of macrophage and endothelial molecular crosstalk and position myeloid-derived Wnt production as a regulator of endovascular progenitor.

MuSyC dosing of adjuvanted cancer vaccines optimizes antitumor responses.

With the clinical approval of T-cell–dependent immune checkpoint inhibitors for many cancers, therapeutic cancer vaccines have re-emerged as a promising immunotherapy. Cancer vaccines require the addition of immunostimulatory adjuvants to increase vaccine immunogenicity, and increasingly multiple adjuvants are used in combination to bolster further and shape cellular immunity to tumor antigens. However, rigorous quantification of adjuvants’ synergistic interactions is challenging due to partial redundancy in costimulatory molecules and cytokine production, leading to the common assumption that combining both adjuvants at the maximum tolerated dose results in optimal efficacy. Herein, we examine this maximum dose assumption and find combinations of these doses are suboptimal. Instead, we optimized dendritic cell activation by extending the Multidimensional Synergy of Combinations (MuSyC) framework that measures the synergy of efficacy and potency between two vaccine adjuvants. Initially, we performed a preliminary in vitro screening of clinically translatable adjuvant receptor targets (TLR, STING, NLL, and RIG-I). We determined that STING agonist (CDN) plus TLR4 agonist (MPL-A) or TLR7/8 agonist (R848) as the best pairwise combinations for dendritic cell activation. In addition, we found that the combination of R848 and CDN is synergistically efficacious and potent in activating both murine and human antigen-presenting cells (APCs) in vitro. These two selected adjuvants were then used to estimate a MuSyC-dose optimized for in vivo T-cell priming using ovalbumin-based peptide vaccines. Finally, using B16 melanoma and MOC1 head and neck cancer models, MuSyC-dose–based adjuvating of cancer vaccines improved the antitumor response, increased tumor-infiltrating lymphocytes, and induced novel myeloid tumor infiltration changes. Further, the MuSyC-dose–based adjuvants approach did not cause additional weight changes or increased plasma cytokine levels compared to CDN alone. Collectively, our findings offer a proof of principle that our MuSyC-extended approach can be used to optimize cancer vaccine formulations for immunotherapy.

Comprehensive characterization of immune landscape of Indian and Western triple negative breast cancers

PurposeTriple-negative breast cancer (TNBC) is a heterogeneous disease with a significant challenge to effectively manage in the clinic worldwide. Immunotherapy may be beneficial to TNBC patients if responders can be effectively identified. Here we sought to elucidate the immune landscape of TNBCs by stratifying patients into immune-specific subtypes (immunotypes) to decipher the molecular and cellular presentations and signaling events of this heterogeneous disease and associating them with their clinical outcomes and potential treatment options. Experimental DesignWe profiled 730 immune genes in 88 retrospective Indian TNBC samples using the NanoString platform, established immunotypes using non-negative matrix factorization-based machine learning approach, and validated them using Western TNBCs (n=422; public datasets). Immunotype-specific gene signatures were associated with clinicopathological features, immune cell types, biological pathways, acute/chronic inflammatory responses, and immunogenic cell death processes. Responses to different immunotherapies associated with TNBC immunotypes were assessed using cross-cancer comparison to melanoma (n=504). Tumor-infiltrating lymphocytes (TILs) and pan-macrophage spatial marker expression were evaluated. ResultsWe identified three robust transcriptome-based immunotypes in both Indian and Western TNBCs in similar proportions. Immunotype-1 tumors, mainly representing well-known claudin-low and immunomodulatory subgroups, harbored dense TIL infiltrates and T-helper-1 (Th1) response profiles associated with smaller tumors, pre-menopausal status, and a better prognosis. They displayed a cascade of events, including acute inflammation, damage-associated molecular patterns, T-cell receptor-related and chemokine-specific signaling, antigen presentation, and viral-mimicry pathways. On the other hand, immunotype-2 was enriched for Th2/Th17 responses, CD4+ regulatory cells, basal-like/mesenchymal immunotypes, and an intermediate prognosis. In contrast to the two T-cell enriched immunotypes, immunotype-3 patients expressed innate immune genes/proteins, including those representing myeloid infiltrations (validated by spatial immunohistochemistry), and had poor survival. Remarkably, a cross-cancer comparison analysis revealed the association of immunotype-1 with responses to anti-PD-L1 and MAGEA3 immunotherapies. ConclusionOverall, the TNBC immunotypes identified in TNBCs reveal different prognoses, immune infiltrations, signaling, acute/chronic inflammation leading to immunogenic cell death of cancer cells, and potentially distinct responses to immunotherapies. The overlap in immune characteristics in Indian and Western TNBCs suggests similar efficiency of immunotherapy in both populations if strategies to select patients according to immunotypes can be further optimized and implemented.

Combined MEK and STAT3 Inhibition Uncovers Stromal Plasticity by Enriching for Cancer-Associated Fibroblasts With Mesenchymal Stem Cell-Like Features to Overcome Immunotherapy Resistance in Pancreatic Cancer

We have shown that reciprocally activated rat sarcoma (RAS)/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and Janus kinase/signal transducer and activator of transcription 3 (STAT3) pathways mediate therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC), while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance and alters stromal architecture. We now determine whether MEKi+STAT3i reprograms the cancer-associated fibroblast (CAF) and immune microenvironment to overcome resistance to immune checkpoint inhibition in PDAC. CAF and immune cell transcriptomes in MEKi (trametinib)+STAT3i (ruxolitinib)-treated vs vehicle-treated Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) tumors were examined via single-cell RNA sequencing (scRNAseq). Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 silencing of CAF-restricted Map2k1/Mek1 or Stat3, or both, enabled interrogation of CAF-dependent effects on immunologic remodeling in orthotopic models. Tumor growth, survival, and immune profiling via mass cytometry by time-of-flight were examined in PKT mice treated with vehicle, anti-programmed cell death protein 1 (PD-1) monotherapy, and MEKi+STAT3i combined with anti-PD1. MEKi+STAT3i attenuates Il6/Cxcl1-expressing proinflammatory and Lrrc15-expressing myofibroblastic CAF phenotypes while enriching for Ly6a/Cd34-expressing CAFs exhibiting mesenchymal stem cell-like features via scRNAseq in PKT mice. This CAF plasticity is associated with M2-to-M1 reprogramming of tumor-associated macrophages, and enhanced trafficking of cluster of differentiation 8+ T cells, which exhibit distinct effector transcriptional programs. These MEKi+STAT3i-induced effects appear CAF-dependent, because CAF-restricted Mek1/Stat3 silencing mitigates inflammatory-CAF polarization and myeloid infiltration invivo. Addition of MEKi+STAT3i to PD-1 blockade not only dramatically improves antitumor responses and survival in PKT mice but also augments recruitment of activated/memory T cells while improving their degranulating and cytotoxic capacity compared with anti-PD-1 monotherapy. Importantly, treatment of a patient who has chemotherapy-refractory metastatic PDAC with MEKi (trametinib), STAT3i (ruxolitinib), and PD-1 inhibitor (nivolumab) yielded clinical benefit. Combined MEKi+STAT3i mitigates stromal inflammation and enriches for CAF phenotypes with mesenchymal stem cell-like properties to overcome immunotherapy resistance in PDAC.

P-Rex1 Signaling Hub in Lower Grade Glioma Patients, Found by In Silico Data Mining, Correlates With Reduced Survival and Augmented Immune Tumor Microenvironment.

Systematic analysis of tumor transcriptomes, combined with deep genome sequencing and detailed clinical assessment of hundreds of patients, constitutes a powerful strategy aimed to identify potential biomarkers and therapeutic targets to guide personalized treatments. Oncogenic signaling cascades are integrated by multidomain effector proteins such as P-Rex1, a guanine nucleotide exchange factor for the Rac GTPase (RacGEF), known to promote metastatic dissemination of cancer cells. We hypothesized that patients with high P-Rex1 expression and reduced survival might be characterized by a particular set of signaling proteins co-expressed with this effector of cell migration as a central component of a putative signaling hub indicative of poor prognosis. High P-Rex1 expression correlated with reduced survival of TCGA Lower Grade Glioma (LGG) patients. Thus, guided by PREX1 expression, we searched for signaling partners of this RacGEF by applying a systematic unbiased in silico data mining strategy. We identified 30 putative signaling partners that also correlated with reduced patient survival. These included GPCRs such as CXCR3, GPR82, FZD6, as well as MAP3K1, MAP2K3, NEK8, DYRK3 and RPS6KA3 kinases, and PTPN2 and PTPN22 phosphatases, among other transcripts of signaling proteins and phospho-substrates. This PREX1 signaling hub signature correlated with increased risk of shorter survival of LGG patients from independent datasets and coincided with immune and endothelial transcriptomic signatures, indicating that myeloid infiltration and tumor angiogenesis might contribute to worsen brain tumor pathology. In conclusion, P-Rex1 and its putative signaling partners in LGG are indicative of a signaling landscape of the tumor microenvironment that correlates with poor prognosis and might guide the characterization of signaling targets leading the eventual development of immunotherapeutic strategies.

Abstract 5545: Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration

Abstract Immunotherapies such as anti-CTLA-4 immune checkpoint blockade (ICB) have revolutionized cancer treatment, yet quality of life and continuation of therapy can be constrained by off-target tissue damage or immune-related adverse events (irAEs). At present, there is limited understanding of irAE mechanisms, hampering development of approaches to mitigate their damage. We addressed this problem by generating animal models of intestinal irAE. Our results show that disruption of homeostatic immunity by genetic predisposition to intestinal inflammation or acute gastrointestinal infection sensitizes mice to anti-CTLA-4-mediated intestinal toxicity. Inflammation-prone mice treated with anti-CTLA-4 showed neutrophil accumulation, systemic interleukin-6 (IL-6) release, and dysbiosis. Significantly, IL-6 blockade combined with antibiotic treatment improved anti-CTLA-4 therapeutic efficacy and reduced intestinal irAEs. Immune signatures were validated in biopsies from patients who developed colitis during ICB, supporting the utility of our models. This study provides new pre-clinical models, mechanistic insight into irAEs, and potential approaches to enhance ICB efficacy while mitigating irAEs. Citation Format: Yifan Zhou, Yusra B. Medik, Bhakti Patel, Daniel B. Zamler, Sijie Chen, Thomas Chapman, Sarah Schneider, Rachel L. Babcock, Taylor T. Chrisikos, Laura M. Kahn, Allison M. Dyevoich, Elizabeth M. Park, Alexandria P. Cogdill, Daniel H. Johnson, Sarah B. Johnson, Khalida M. Wani, Debora A. Ledesma, Courtney W. Hudgens, Jingjing Wang, Md Abdul Wadud Khan, Aron Y. Joon, Weiyi Peng, Haiyan S. Li, Reetakshi Arora, Ximing Tang, Maria Gabriela Raso, Xuegong Zhang, Wai Chin Foo, Michael T. Tetzlaff, Gretchen E. Diehl, Karen Clise-Dwyer, Elizabeth M. Whitley, Matthew M. Gubin, James P. Allison, Patrick Hwu, Nadim J. Ajami, Adi Diab, Jennifer A. Wargo, Stephanie S. Watowich. Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5545.

Abstract 3446: Radiation and intratumoral activation of double-stranded RNA sensors redirects myeloid cells and primes adaptive immunity

Abstract Background: Undifferentiated pleomorphic sarcoma (UPS) is a common aggressive sarcoma subtype for which existing therapies are ineffective. Immune checkpoint blockade (ICB) is effective in a minority of patients whose tumors harbor a pre-existing lymphocytic response. However, most UPS tumors have a myeloid infiltrate, a highly plastic cellular population with the potential to serve as a substrate for immunotherapy. Radiotherapy (RT), a standard therapy for UPS, can also drive myeloid cells into the tumor. Based on abundant expression of pattern recognition receptors (PRRs) by myeloid cells, we hypothesized that intratumoral injection of a PRR agonist would leverage pre-existing and RT-associated myeloid cells by redirecting them to prime adaptive anti-tumor immunity. Methods: We used BO-112, a nanoplexed double-stranded ribonucleic acid that activates PRRs, most of which are not typically activated by RT. We used an ICB-resistant mouse model, in which tumor cells derived from a KrasG12D/-P53-/- model of UPS are used to establish large tumors in flanks of C57BL/6J mice. Tumor-bearing mice were treated with RT (8 Gy x 3 fractions daily) starting on day 8 ± BO-112 (30 ug/dose) on days 8 and 10. Tumor growth and survival between groups were compared by ANOVA and log-rank test, respectively. To test the role of lymphocytes, the study was repeated in RAG-/-gc-/- mice. Kinetic analysis of myeloid and T cells was performed in the tumors and lymph nodes (LN) on days 15, 21, 26 using high-dimensional flow cytometry. To evaluate the fate of monocytes, LysM-eGFP monocytes were intratumorally transferred following treatment, with subsequent analysis of tumors and LNs on days 16, 18, 21. Results: Tumor control was significantly improved in the combination group starting from day 16 with mean tumor volume of 240 mm3 in the combination group compared to 490 mm3 in BO112, 790 mm3 in RT, and 875mm3 in mock groups (p<0.0001) which translated into a maximal survival advantage in the combination group (p<0.005). However, in RAG-/-gc-/- mice, this benefit of combination therapy was completely negated. This therapeutic combination appeared to drive an early reduction in Ly6ChiMHCII+ cells (p<0.05) within the tumor followed by a progressive increase of the same population in the draining LN over time (p=0.01). In parallel, we observed trafficking of adoptively transferred intratumoral eGFP+ monocytes from the tumor to dLNs in response to BO-112 and RT. Conclusions: Local-only therapy of BO-112 and RT yields a lymphocyte-dependent anti-tumor immune response in an immunotherapy-resistant model of UPS. Longitudinal analysis of locoregional immune responses and adoptive transfer experiments indicate that combination BO-112 and RT induces tumor-to-dLN myeloid trafficking that may bridge an adaptive response and confer therapeutic effect. Citation Format: Jie Deng, Scott C. Chin, Mito Tariveranmoshabad, Danielle S. Graham, Hailey R. Lee, Marisol Quintero, Dorthe Schaue, Anusha Kalbasi. Radiation and intratumoral activation of double-stranded RNA sensors redirects myeloid cells and primes adaptive immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3446.

Abstract 1712: A single-cell RNA atlas of innate and adaptive intratumoral immunity in triple negative breast cancer during chemo- and immunotherapies

Abstract Checkpoint inhibitors (CIs) such as anti-PD-1 and anti PD-L1 have shown combinatorial clinical activity with chemotherapy in triple negative breast cancer (TNBC), but only in a minority of patients and only for a limited period of time. Moreover, it is currently unclear what chemotherapeutic drug is the most appropriate to combine with CIs in TNBC patients. We have investigated at the single cell level the transcriptome and the trajectories of more than 50,000 innate and adaptive intratumoral immune cells in two syngeneic, immune competent, orthotopic murine models of local and metastatic TNBC. Mice injected with 4T1 cells had a predominant lymphoid infiltrate, mice injected with EMT6 cells had a predominant myeloid infiltrate. Mice were treated with CIs and several different types of chemotherapeutics, alone or in combinations. In both models, capecitabine (alone or with CIs) was the less effective drug. Platinum, doxorubicin and taxanes showed synergy with CIs and had superimposable activity. Intermittent, medium dosage cyclophosphamide (CTX) plus vinorelbine and CIs was the most active combinatorial therapy (Falvo et al, Cancer Research 2021). Vinorelbine activated antigen presenting cells and CTX generated new T cell clones including stem cell-like TCF1+ CD8+ T cells. Treatments with most in vivo efficacy were associated to a decrease of regulatory T cells and of gamma delta T cells, which were found to have a pro-tumoral activity in these murine models, likely due to IL-17 expression in the neoplastic microenvironment. An increase of several different clusters of exhausted-like CD8+ T cells was observed in pre-clinical treatments with low efficacy; an opposite trend was found for several clusters of proliferative CD8+ T cells in treatments with high in vivo efficacy. Regarding macrophages, M2-like cells were enriched after treatments with low efficacy, while an opposite behaviour was found in M1-like macrophages. Interestingly, we observed a significant increase of an M1-like cluster with high expression of the Ly6c1/Ly6c2 gene in mice successfully treated with vinorelbine, CTX and CIs. For both cell lines the percentage of plasma B cells increased after in vivo treatments with high efficacy. In particular, the most effective treatment significantly increased the frequency of germinal B cells, which were absent in untreated tumors. These data can lead to new insights on the diagnosis and treatment of TNBC and to possible clinical applications. Citation Format: Laura Carpen, Paolo Falvo, Stefania Orecchioni, Giulia Mitola, Roman Hillje, Saveria Mazzara, Patrizia Mancuso, Stefano Pileri, Alessandro Raveane, Francesco Bertolini. A single-cell RNA atlas of innate and adaptive intratumoral immunity in triple negative breast cancer during chemo- and immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1712.

Abstract 1328: Deciphering the role of CREB1 in shaping the tumor immune landscape of pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) remains a major therapeutic challenge with hallmarks including oncogenic mutations, desmoplastic architecture, and an immunosuppressive tumor microenvironment (TME). Previously, we have identified Cyclic AMP Response Element Binding protein 1 (CREB) as an oncogenic transcriptional factor downstream of KRAS that promotes disease aggressiveness, poor survival, and immune exclusion. Based on these, we sought to determine the impact of tumor intrinsic CREB deletion in shaping the tumor immune microenvironment. Methods: We have generated a novel genetically engineered mouse model (GEMM) of pancreas-specific CREB deletion (CREBfl/fl) in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice that phenocopy human PDAC disease. CRISPR/CAS9-based genomic editing was utilized to ablate CREB (CREBKO) in KPC tumor cells. RNA-sequencing analysis was performed in KPC CREB wild type (CREBEV) vs. CREBKO tumor cells to identify CREB-mediated transcriptomic changes. Syngeneic orthotopic tumor implantation of these cells was performed into the pancreata of mice. High dimensional immunophenotyping was accomplished to assess changes in the immune subsets with CREB deletion in murine tumors. Additionally, these tissues were processed for immunohistochemical and qPCR-based analysis to assess changes in fibroinflammatory and immune mediators. Results: Pancreas-specific CREB deletion in the KPC GEMM led to a significant reduction in the primary tumor burden, liver metastases and improved overall survival compared to wild-type KPC. Additionally, CREB deletion significantly remodeled the tumor stroma, as evidenced by the reduction in the expression of fibroinflammatory and immunosuppressive markers. In assessing the immune repercussions of CREB deletion in pancreatic tumors, we observed a decreased infiltration of CD11b+ myeloid-derived suppressor cells (MDSCs) and granulocytic-PMN MDSCs (CD11b+ Ly6Ghigh Ly6Clow F4/80-), with a concomitant increase in the antigen-presenting M1-like macrophages (F4/80+ MHC-II high CD86 high). Also, CREB ablation in these tumors further facilitated increased infiltration of activated effector CD8+ T cells resulted in enhanced anti-tumor immune response within the PDAC TME. Mechanistically, RNA transcriptomic and secretome analysis in CREBKO Vs. the wild type KPC tumor cells identified several differentially expressed immunomodulatory soluble mediators responsible for shaping CREB dependent immunogenic landscape in PDAC. Conclusion: Overall, depleting CREB reshapes the tumor immune landscape to reduce innate immunosuppressive myeloid infiltration and reinvigorate the antitumor T cell immune responses to improve overall survival in PDAC. Citation Format: Siddharth Mehra, Vanessa Garrido, Samara Singh, Iago D Silva, Luis Alberto Nivelo, Anna Bianchi, Shrey Modi, Zhiqun Zhou, Austin Dosch, Nilesh Deshpande, Supriya Srinivasan, Christine Rafie, Ifeanyichukwu Ogobuiro, Xi Chen, Alejandro Villarino, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. Deciphering the role of CREB1 in shaping the tumor immune landscape of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1328.

Abstract 950: Role of epithelial-mesenchymal plasticity in immune remodeling during metastasis

Abstract Introduction: Breast cancer is the most prevalent type of cancer in women, accounting for 12% of all annual cancer cases worldwide with most deaths attributed to metastasis. It displays both inter- and intra-tumoral heterogeneity which is linked to epithelial-mesenchymal transition (EMT). This program involves the acquisition of a mesenchymal-like phenotype allowing cells to disseminate from the primary tumor and metastasize. However, recent studies show that tumor cells do not need to undergo complete EMT to gain invasive potential; instead, co-expression of both epithelial and mesenchymal markers is critical for metastasis. Such tumor cells are thought to display epithelial-mesenchymal plasticity (EMP) which bestows them with enhanced fitness and flexibility to fulfill diverse requirements of the metastatic cascade. Additionally, hybrid EM phenotype has been linked to immunesuppression but has been understudied due to lack to suitable models with intact immune system. Thus, this necessitates the mechanistic dissection of tumor immune microenvironment (TIME) in models with EM plasticity. To characterize the contribution of EMP to metastasis, we utilized syngeneic mouse mammary tumor cell lines, which are hybrid EM and investigated their proteomic and cytokine profile and identified several novel cytokines. Methods: Reverse Phase Protein Array (RPPA) was performed to determine the proteomic profile of tumors with EM plasticity. In addition, Luminex assay was performed to assess the cytokine/chemokine profile in above tumor models. Results: The RPPA involved a panel of 450 antibodies and showed that tumors with EM plasticity were enriched in specific immunesuppressive proteins. At the same time, Luminex analysis was suggestive of recruitment of tumor-associated macrophages (TAMs) in models with EM plasticity. Additionally, cells with EM plasticity showed the ability to induce tumor-promoting/M2-like macrophages (in vitro). Conclusions: Our data indicates that cells/tumors with EM plasticity show differential protein expression than E and M counterparts and are enriched in specific immunesuppressive proteins. Moreover, such cells have immune networks (cytokines/chemokines) that promote recruitment of TAMs - the largest myeloid infiltrate in the microenvironment of solid tumors. Thus, our study invites more attention to investigate the mechanisms of immunesuppression and metastasis in tumors with EM plasticity. Citation Format: Tanvi H. Visal. Role of epithelial-mesenchymal plasticity in immune remodeling during metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 950.

Abstract 3415: The neutrophil-to-lymphocyte ratio (NLR) reflects intratumor myeloid derived suppressor cell (MDSC) infiltration in metastatic castration-resistant prostate cancers (mCRPC)

Abstract Background: High neutrophil-to-lymphocyte ratio (NLR) associates with worse overall survival (OS) from prostate cancer (PC) and low response rates to abiraterone and taxane chemotherapy and may reflect systemic inflammatory changes. A direct link between high NLR and intratumoral myeloid infiltration has not been demonstrated to date. We evaluated the relationship between NLR and intratumor immune cell densities in mCRPC biopsies. Design: mCRPC biopsies from 71 patients treated at The Institute of Cancer Research/Royal Marsden Hospital (ICR/RMH, UK) were stained by immunofluorescence for CD11b, CD15, and DAPI and by immunohistochemistry for CD3 to algorithmically quantify PMN-MDSC and T cell densities in the tumor and stromal compartments. NLR (neutrophil count divided by lymphocyte count), ALP, LDH, and albumin were obtained from blood collected contemporaneous with mCRPC biopsies. Clinical data were retrospectively collected. Negative binomial regression determined the association between leucocyte densities and log-transformed NLR. Modified Poisson regression estimated the risk ratio (RR) for the presence of PMN-MDSCs in relation to higher NLR (>3). OS was analysed using Cox regression and Kaplan-Meier. RNAseq data from Stand Up 2 Cancer/Prostate Cancer Foundation (SU2C/PCF, n=159) and RMH cohorts (n=98) were analysed by Spearman’s correlation. Results: NLR positively associated with the density of PMN-MDSCs infiltrating the tumor (p=0.0004) and stromal (p=0.0007) compartments of mCRPC biopsies as determined by Halo࣪ computational modelling. Tumors with high NLR were more likely to be infiltrated by PMN-MDSCs (RR: 1.41; 95% confidence interval [CI]: 1.04-1.92; p=0.03). NLR independently associated with worse OS from the time of mCRPC biopsy after adjusting for LDH, ALP, metastasis at the time of diagnosis, and albumin (HR: 1.71; 95% CI: 1.20-2.46). Patients with low NLR and an absence of tumor-infiltrating PMN-MDSC had longer OS than those with a high NLR, tumor-infiltrating PMN-MDSCs or a combination of both (p=0.015). Whilst there was no association between NLR or PMN-MDSC density and CD3 T cell density, RNAseq analyses of two independent CRPC cohorts showed that MDSC signatures strongly and positively associated with signatures of T cell terminal exhaustion (SU2C/PCF: rs=0.74; p<1x10-6; RMH: rs=0.64; p<1x10-6) providing credence for the impact of MDSCs on T effector function. Conclusion: Peripheral blood NLR was positively correlated with prostate tumor-infiltrating PMN-MDSC density supporting the interaction between the circulating myeloid compartment and intratumoral myeloid infiltration in patients with advanced PC. Citation Format: Christina Guo, Jan Rekowski, Mateus Crespo, Bora Gurel, Wei Yuan, Adam Sharp, Rafael Grochot, Khobe Chandran, Semini Sumanasuriya, Ana Ferreira, Andrea Alimonti, Johann S. de Bono. The neutrophil-to-lymphocyte ratio (NLR) reflects intratumor myeloid derived suppressor cell (MDSC) infiltration in metastatic castration-resistant prostate cancers (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3415.

Abstract 3615: TREM2+ macrophages mediate immunosuppression in tumors

Abstract Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer’s disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy. Citation Format: Martina Molgora, Blanda Di Luccia, Darya Khantakova, Natalia Jaeger, Rafael Sanguinetti Czepielewski, Cristiane Secca da Silva, Jennifer Bando, Susan Gilfillan, Marina Cella, Robert Schreiber, Marco Colonna. TREM2+ macrophages mediate immunosuppression in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3615.

MODL-26. Development of humanized immune system, posterior fossa A ependymoma patient-derived xenograft model

Abstract Cellular interactions between tumor and immune cells are critical in ependymoma biology. We have shown distinct immunobiology phenotypes by ependymoma molecular subgroups, with PFA2 developing an anti-tumor immune phenotype and in contrast PFA1 tumor immune cells being pro-tumor. We recently established two fully characterized pediatric PFA1 intracranial xenograft models in NSG mice. These models, while critical for advancing PFA studies, lack the ability to make lymphocytes. To address this we have established a humanized orthotopic model of PFA1 ependymoma that are grafted to produce functional human lymphocytes. To do this, CD34+ human umbilical cord blood was injected into the cranial facial vein of newborn, irradiation immunodepleted, BRGS mice. Human immune chimerism was determined at 10 weeks by flow cytometry of peripheral blood. Next, we injected existing PDX model MAF-928_XF cells into the 4th ventricle at 12 weeks age and tumors were monitored by MRI. Initial scanning found the tumors were delayed in developing, consistent with other humanized solid tumor mouse models. Mice were euthanized between 32-34 weeks age and necropsies were performed to isolate brain, lymph nodes, blood and spleen. Human chimerism was detectable, by flow cytometry, in spleen, lymph nodes and blood, and most cells were T-cells. Human T-cells were detectable in all tumors, in proportions consistent with human disease (0.05-0.5% of CD45+). Tumors also had 35-50% mouse myeloid infiltration. Tumors were MHC I negative and MHC II positive, and PD-L1 low. Histology was consistent with human ependymoma. We are using spatial proteomics to determine cellular location and phenotype of infiltrating immune cells. Initial studies indicate this model will be usable for modeling the critical tumor-immune interactions and pre-clinically testing the next therapies in PFA ependymoma and can be easily adapted to other pediatric brain tumors in which immune factors have a critical role.

DIPG-11. DIPGs show robust MHC class I expression but low lymphoid and high myeloid infiltration partly reversible by DNA methyltransferase inhibition

Diffuse intrinsic pontine gliomas (DIPGs) are the most aggressive tumors of the central nervous system in children. Median survival of patients is less than one year post-diagnosis. Radiotherapy remains the only standard treatment but is rarely curative. Immunotherapy is an emerging and promising treatment strategy for children with DIPG. However, general immunotherapy has not lived up to its promise to treat many cancers, including DIPGs, in part due to incomplete understanding of the barriers posed by the tumor microenvironment. We therefore evaluated the immune cell infiltration in a syngeneic mouse model of DIPG and in DIPG tumors collected from patients. We evaluated the expression profiles of T lymphocytes and myeloid cell markers in a cohort of 28 DIPG tumors compared to matched normal tissue specimens. Our data indicate that the expression of MHC I components in DIPG tumors is similar to that of matched normal tissue. Moreover, the well-known immune checkpoint, PD-L1, was not overexpressed in DIPG tumors. Using immunohistochemistry, we demonstrated only rare infiltration of lymphocytes but high enrichment of myeloid cells in tumor tissue. We found similar results in a syngeneic mouse model of DIPG. Collectively, our results indicate that DIPG tumors harbor rare lymphocytes and are enriched in myeloid cells. Recent studies have demonstrated that DNA methyltransferase inhibitors prime some tumors for more effective checkpoint blockade by activating expression of human endogenous retroviruses and sparking a T-cell mediated immune response through a process called “viral mimicry.” We tested the effect of decitabine in a syngeneic mouse model of DIPG. Decitabine reduced the tumor growth kinetics compared to vehicle but was unable to induce the recruitment of lymphoid cells in DIPG tumors. Importantly, we observed a noticeable reduction in the myeloid component of the DIPG microenvironment suggesting a possible role of myeloid cells in tumor growth and progression.

Estimating clear cell renal cell carcinoma transcriptomic signatures using machine learning and histopathology images.

4533 Background: Gene expression signatures derived from RNA sequencing data have been associated with treatment outcomes for renal cell carcinoma (RCC) patients. Incorporating these RNA biomarkers into clinical practice is promising, yet its real-world applicability is heavily limited as RNA profiling is expensive, time-consuming, and requires specialized expertise for data analysis. In this study, we applied a deep neural network framework to identify the correlation between standard pathology images and underlying RNA signatures using hematoxylin and eosin (H&E) stained formalin-fixed paraffin-embedded (FFPE) whole slides of clear cell kidney tumors from The Cancer Genome Atlas (TCGA). Methods: We collected 496 H&E stained FFPE clear cell RCC whole-slide images and the RNA gene signatures for 496 patients from the TCGA database. We partitioned 496 slides into training (N = 245, 50%), development (N = 49, 10%), and test (N = 202, 40%) sets. We used this dataset to train and evaluate our weakly-supervised deep learning model. The model was iteratively trained using extracted patches from a slide and processed the patches through a convolutional neural network (CNN), pre-trained for the RCC subtypes classification task, to represent features. The features were aggregated and summarized to predict angiogenesis, myeloid infiltration, and adenosine gene signature (AdenoSig) scores. Performance was assessed by computing Pearson’s correlation coefficients. 95% confident intervals (CI) are computed using the Fisher Z transformation. Results: The median angiogenesis score was 6.98 (range: 1.77-8.40), the median myeloid score was 0.54 (range: -4.59-5.96), and the median AdenoSig score was 268.31 (range: -4988.71-7621.57). A total of 202 slides were included in the test set. On this test set, the results of our weakly supervised method achieved a Pearson’s correlation of 0.65 (95% CI: 0.57-0.73), 0.10 (95% CI: -0.04-0.23), and 0.10 (95% CI: -0.04-0.23) with angiogenesis, myeloid, and AdenoSig scores from gold-standard RNA sequencing data, respectively. Conclusions: We proposed using deep learning-based AI techniques to process digitized histopathological images and estimate actionable signatures of angiogenesis, myeloid, and AdenoSig from H&E stained slides. Our model showed promising results for predicting angiogenesis scores compared to myeloid and AdenoSig scores. These results suggest the feasibility of this approach for estimating digital biomarkers from H&E histopathology images and offering a rapid and cost-effective alternative to conventional RNA sequencing.

Characterization of tumor antigen expression and myeloid immune profiles to inform the development of immune stimulating antibody conjugates (ISACs).

2557 Background: Immuno-oncology (IO) has historically focused on T cell-driven effects, but a growing class of myeloid therapies are under investigation. This class includes immune-stimulating antibody conjugates (ISACs), which comprise a tumor-targeting antibody conjugated to an immune-stimulating payload. ISACs may require both tumor-associated antigens and tumor-resident myeloid cells for activity. As new IO strategies such as TLR-activating ISACs are developed, understanding the myeloid landscape is relevant for cancer biology and drug development. Methods: Tumor microarrays with formalin-fixed paraffin embedded sections of breast (BC), colorectal (CRC), gastric (GC), head and neck squamous cell (HNSCC), and non-small cell lung (NSCLC) cancers were analyzed by immunohistochemistry for tumor antigens (HER2, CEA, PD-L1), myeloid cell markers (CD68/CD163/CD11c/BDCA2), and CD8+ T cells. Results: Tumor infiltrating myeloid cells and CD8+ T cells varied across indications (Table). Low densities of intra-tumoral CD8+ T cells (<100/mm2) were observed in CRC, with higher CD8+ T cell counts observed in other cohorts. In contrast to T cells, substantial myeloid infiltrates were observed across tumor types. While MSI status was associated with higher CD8+ T cell infiltration in CRC, myeloid cells were abundant in both MSI and MSS samples. Monocyte-derived (mDC) and conventional DCs (cDC), which respond strongly to TLR activation, were present in all indications, with highest numbers observed in NSCLC. HER2+ BC samples had high infiltration of plasmacytoid DCs, a key TLR7-responsive population. PD-L1 expression on immune cells was associated with higher CD8+ T cell and myeloid cell numbers. In contrast to HER2 and PD-L1, CEA expression was independent of immune infiltration. Conclusions: Abundant myeloid infiltrates were observed across solid tumors, independent of T-cell infiltration. The presence of myeloid cells in multiple tumor types offers broadscale therapeutic targets for ISACs and other myeloid-directed therapies that can activate the innate immune system as a bridge to adaptive immunity.[Table: see text]