Intratumoral Neutrophils Research Articles

Abstract 1342: Evaluating the role of antibody in pancreatic tumorigenesis

Abstract Introduction: Infiltrating pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of 10%. The PDAC tumor microenvironment (TME) is characterized by a fibroinflammatory response mediated by a complex mix of cancer-associated fibroblast (CAF) subsets resulting significant deposition of extracellular matrix (ECM), poor vascularization, T cell exclusion, and recruitment of immune-suppressive myeloid cells. Collectively, these factors contribute to a resistance to aggressive chemotherapeutics and failure of checkpoint inhibitors. Preclinical studies as well as clinical efforts designed to deplete the dense tumor stroma or ECM have been successful, but unexpectedly accelerated tumor progression underscoring critical gaps in our understanding of the dynamics between tumor, stromal elements, and infiltrating immune cell subsets. Rationale: In some cancer subtypes, B cells have been shown to suppress cancer progression through secretion of antibodies targeted to aberrantly expressed self-, stress-associated-, or tumor-specific antigens on neoplastic cells. A considerable body of literature also indicates that B cells may play a role in promotion of pancreatic tumor progression, however, the role of antibodies in PDAC progression is unclear. The major objective of our studies is to understand the significance of anti-tumor antibody responses in pancreatic tumorigenesis. Methods: To address this question, we crossed the spontaneous mouse model of pancreatic cancer where Pdx1 drives Cre-mediated activation of a mutant KrasG12D allele on a heterozygous Tp53+/FL mutant background (KPC-WT mice) with animals that retain B cells that lack the ability to secrete antibody of any immunoglobulin (Ig) isotype (KPC-μS-AID mice). Results: Kaplan-Meier survival studies demonstrate that loss of all circulating antibody accelerates progression to lethal PDAC in KPC-μS-AID versus KPC-WT mice (median survival of 3- versus 5-months, respectively), with increased incidence of lung and liver metastases. Further, loss of circulating antibodies increased the density of intratumoral neutrophils, reduced numbers of podoplanin+ CAFs and reduced ECM density. IgG subclass antibodies were localized at higher densities within stroma/ECM in pancreata from both mice and surgically resected tumor specimens from human PDAC patients. Loss of AID-dependent class switched antibodies similarly accelerated lethal PDAC in KPC-AID versus KPC-WT mice. Conclusions: These findings implicate class-switched antibodies in delaying pancreatic tumor progression through potential regulation of intratumoral neutrophil density and ECM production within the PDAC TME. Citation Format: Jeremy Foote. Evaluating the role of antibody in pancreatic tumorigenesis [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 1342.

Abstract 6142: Gain-of-function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy

Abstract The immune microenvironment plays a critical role in cancer development, progression and resistance to therapy. Specific tumor genotypes can profoundly influence the composition of the immune microenvironment. After oncogenic KRAS, TP53 is the most frequently mutated gene in pancreatic ductal adenocarcinoma (PDAC). KRASG12D mutations in tumor cells are known to modify the immune landscape of PDAC, however the immune effects of neomorphic TP53 mutations have not been defined. Focusing on the most common neomorphic TP53 mutation in human PDAC, we sought to evaluate the non-cell-autonomous role of gain-of-function mutant Trp53R172H in modulating the immune microenvironment of pancreatic tumors by using KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) genetically engineered to express the Trp53R172H gain-of-function. Orthotopically implanted tumors derived from CRISPR-generated KrasG12D/+;Trp53R172H/+ PDEC had a distinct immune profile in comparison to KrasG12D/+;Trp53+/+ tumors, characterized by an influx of intratumoral CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 (Th1) cells. Analysis of publicly available human PDAC cohorts revealed enrichment of genes in neutrophil-related pathways in TP53-mutated tumors. Knockdown of CXCL2, a neutrophil chemoattractant, in the tumor epithelial compartment of CRISPR KrasG12D/+;Trp53R172H/+ PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing CRISPR KrasG12D/+;Trp53R172H/+ PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link gain-of-function mutant Trp53R172H to the presence of intratumoral neutrophils in pancreatic cancer and suggests that tumor genotypes could inform patient selection for immunotherapy. Citation Format: Despina Siolas, Emily Vucic, Emma Kurz, Cristina Hajdu, Dafna Bar-Sagi. Gain-of-function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy [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 6142.

Neutrophil infiltration combined with necrosis in the primary tumor is a useful prognostic indicator for three-year disease-free survival time in patients with colorectal cancer.

Histopathological evaluation plays a key role in the diagnosis of colorectal cancer (CRC). Tumor-related local inflammation is regarded as a novel prognostic parameter. Neutrophils constitute one of the main types of inflammatory cells. The aim of the present study was to evaluate the prognostic value of intratumoral tumor-associated neutrophils (intraTANs), stromal TANs (stromaTANs) and necrosis, as well as their combined parametric value in formalin-fixed paraffin-embedded tissue sections from patients with CRC. For this purpose, a retrospective study of 160 patients with CRC who underwent surgery was conducted. The association of intraTANs, stromaTANs, necrosis and their combined parametric value with the clinicopathological features of patients with CRC was examined. The Kaplan-Meier method and the log-rank test were used to compare survival curves. To identify independent prognostic factors, uni- and multivariate Cox proportional hazards regression models were used. StromaTANs were associated with lymph node metastasis (P=0.049) and tumor deposits (P=0.041). In addition, necrosis was found to be associated with venous (P=0.003), lymphatic (P=0.007) and perineural (P=0.015) invasion, as well as with lymph node metastasis (P=0.033), the number of invaded lymph nodes (P=0.012), and lymph node pouch invasion (P=0.043). Furthermore, necrosis was found to be associated with the white blood cell count (P=0.030), neutrophil count (P=0.011), the combined neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (NLR-PLR) (P=0.038), and the combined platelet and NLR (PLT-NLR status) (P=0.030), as well as with the serum carcinoembryonic antigen (CEA) levels following surgery (P=0.011) and the monocyte-to-lymphocyte ratio (P=0.023). The combined parametric value was found to be associated with pT stage (P=0.049), venous (P=0.034) and lymphatic (P=0.026) invasion, and with serum CEA levels prior to surgery (P=0.029). The analysis of the 3-year disease-free survival (DFS) time revealed that tumor growth [hazard ratio (HR), 2.070; 95% CI, 1.837-3.808; P=0.003] and the combined parametric value (intraTANs, stromaTANs and necrosis, HR, 1.577; 95% CI, 1.372-3.032; P=0.028) were independent factors for patients with CRC. Taken together, the findings of the present study demonstrated that the combined value of neutrophils and necrosis examined in the cancerous tissue may be used as a prognostic factor for the 3-year DFS time in patients with CRC.

NAP1051, a Lipoxin A4 Biomimetic Analogue, Demonstrates Antitumor Activity Against the Tumor Microenvironment.

Resolving tumor-associated inflammation in the tumor microenvironment (TME) may promote antitumor effects. Lipoxin A4 (LXA4) is a short-lived endogenous bioactive lipid with potent anti-inflammatory and pro-resolving properties. Here, a biomimetic of LXA4, NAP1051, was shown to have LXA4-like in vitro properties and antitumor activity in colorectal cancer xenograft models. NAP1051 inhibited neutrophil chemotaxis toward fMLP and dose-dependently promoted dTHP-1 efferocytosis which was equipotent to aspirin-triggered lipoxin A4 (ATLA). In dTHP-1 cells, NAP1051 induced strong phosphorylation on ERK1/2 and AKT similar to formyl peptide receptor 2 (FPR2/ALX) agonists. In two mouse xenograft colorectal cancer models, NAP1051 significantly inhibited tumor growth when given orally at 4.8 to 5 mg/kg/day. Flow cytometric analyses showed that NAP1051 reduced splenic and intratumoral neutrophil and myeloid-derived suppressor cell populations, which correlated to the antitumor effect. In addition, NAP1051 reduced NETosis in the TME while stimulating T-cell recruitment. Overall, these results show that NAP1051 possesses key lipoxin-like properties and has antitumor activity against colorectal cancer via modulation of neutrophils and NETosis in the TME.

Tumor-Derived Factors Differentially Affect the Recruitment and Plasticity of Neutrophils.

Simple SummaryNeutrophils are the most abundant type of circulating leukocytes and play an important role in tumor biology. In the context of cancer, the origin of tumor-associated neutrophils (TANs) as well as the interplay between circulating neutrophil subpopulations (NDN and LDN) are still not clear. Our results show for the first time that TANs can originate from both NDN and LDN, with LDN infiltrating at a higher extent than NDN. CXCL1 and CXCL2 chemokines strongly affect NDN and LDN migration, and tumor-conditioned media differently impact their chemotaxis. Moreover, NDN display tumor-induced phenotypical plasticity, transitioning towards a low-density state (LD-NDN). We found that tumor-conditioned media and CXCL1 promote LD-NDN formation, suggesting that multiple factors mediate neutrophils’ plasticity. Newly formed LD-NDN present similar characteristics to LDN in terms of morphology, functional activity, and surface receptor expression, indicating that a portion of LDN could originate from NDN undergoing phenotypical changes.Neutrophils play a key role in cancer biology. In contrast to circulating normal-density neutrophils (NDN), the amount of low-density neutrophils (LDN) significantly increases with tumor progression. The correlation between these neutrophil subpopulations and intratumoral neutrophils (TANs) is still under debate. Using 4T1 (breast) and AB12 (mesothelioma) tumor models, we aimed to elucidate the source of TANs and to assess the mechanisms driving neutrophils’ plasticity in cancer. Both NDN and LDN were found to migrate in response to CXCL1 and CXCL2 exposure, and co-infiltrate the tumor site ex vivo and in vivo, although LDN migration into the tumor was higher than NDN. Tumor-derived factors and chemokines, particularly CXCL1, were found to drive neutrophil phenotypical plasticity, inducing NDN to transition towards a low-density state (LD-NDN). LD-NDN appeared to differ from NDN by displaying a phenotypical profile similar to LDN in terms of nuclear morphology, surface receptor markers, decreased phagocytic abilities, and increased ROS production. Interestingly, all three subpopulations displayed comparable cytotoxic abilities towards tumor cells. Our data suggest that TANs originate from both LDN and NDN, and that a portion of LDN derives from NDN undergoing phenotypical changes. NDN plasticity resulted in a change in surface marker expression and functional activity, gaining characteristics of LDN.

Gain-of-function p53R172H mutation drives accumulation of neutrophils in pancreatic tumors, promoting resistance to immunotherapy

Tumor genotype can influence the immune microenvironment, which plays a critical role in cancer development and therapy resistance. However, the immune effects of gain-of-function Trp53 mutations have not been defined in pancreatic cancer. We compare the immune profiles generated by KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDECs) engineered genetically to express the Trp53R172H mutation with their p53 wild-type control. KrasG12D/+;Trp53R172H/+ tumors have a distinct immune profile characterized by an influx of CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ Tcells, CD8+ Tcells, and CD4+ T helper 1 cells. Knockdown of CXCL2, a neutrophil chemokine, in the tumor epithelial compartment ofCRISPR KrasG12D/+;Trp53R172H/+ PDEC tumors reverses the neutrophil phenotype. Neutrophil depletion of mice bearing CRISPR KrasG12D/+;Trp53R172H/+ tumors augments sensitivity to combined CD40 immunotherapy and chemotherapy. These data link Trp53R172H to the presence of intratumoral neutrophils in pancreatic cancer and suggest that tumor genotypes could inform selection of affected individuals for immunotherapy.

Abstract 2697: Prognostic significance of intratumoral neutrophil lymphocyte ratio (NLR) in breast cancer patients

Abstract Background: The elevation of neutrophil lymphocyte ratio (NLR) in the blood are reported to associate with poor prognosis in breast cancer based on the notion that neutrophils represent pro-cancer, and lymphocytes represent anti-cancer immune cells. Tumor immune microenvironment has been demonstrated to play critical roles in the outcome of breast cancer patients. However, there is scarce evidence on the clinical relevance of intratumoral NLR in breast cancer patients. In the current study, we hypothesized that intratumoral NLR high tumors are associated with worse survival particularly in TNBC that is known to have high immune cell infiltration. Methods: We obtained a total of 1904 breast cancer patients' data from METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) and analyzed. NLR was calculated by the gene expressions of CD66b (CEACAM8) and CD8 (CD8A). NLR high and low were divided by the median. Overall Survival (OS) and Disease-Free Survival were calculated utilizing Kaplan Meier method between intratumoral NLR high and low groups. xCell algorithm was used to analyze the infiltrated immune cells within the tumor immune microenvironment as we have previously published. Results: Intratumoral NLR high group was associated with worse OS in whole, ER-positive/HER2-negative, and triple negative (TN) subtypes, in agreement with the previous studies. TN subtype alone demonstrated worse DFS of NLR high group. Surprisingly, gene set enrichment analysis (GSEA) demonstrated no gene set enrichment to NLR high group, which implicates that there is no distinctive mechanism that associate with worse survival. Whereas, immune response-related gene sets significantly enriched to NLR low group in TN subtype. This enrichment was consistent in ER-positive/HER2-negative subtype. Compared with ER-positive/HER2-negative subtype, anti-cancer immune cells such as CD4+ T cells, CD8+ T cells, M1 macrophage, and helper T helper type 1 cells were significantly infiltrated in TN patients (p < 0.001 for all genes), where M2 macrophages and neutrophils were less and regulatory T cells and T helper type 2 cells were more infiltrated in TN subtype. Furthermore, intratumoral NLR was significantly lower in TN compared with ER-positive/HER2-negative subtype (p < 0.001). These results suggest that intratumoral NLR low group is associated with better survival due to favorable tumor immune microenvironment in TN subtype rather than NLR high group has worse survival. Conclusions: Intratumoral NLR low tumor demonstrated more favorable OS in whole cohort, ER-positive/HER2-negative, and more favorable DFS in TN patients alone. Intratumoral NLR low breast cancer was associated with enhanced immune response and higher infiltration of anti-cancer immune cells were observed in TN subtype compared to ER-positive/HER2-negative which may contribute to the favorable outcome of in TN breast cancer. Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Vijayashree V. Murthy, Eriko Katsuta, Nobuhisa Matsuhashi, Manabu Futamura, Kazuhiro Yoshida, Kazuaki Takabe. Prognostic significance of intratumoral neutrophil lymphocyte ratio (NLR) in breast cancer patients [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 2697.

Activated neutrophils polarize protumorigenic interleukin-17A-producing T helper subsets through TNF-α-B7-H2-dependent pathway in human gastric cancer.

RationaleNeutrophils constitute massive cellular constituents in inflammatory human gastric cancer (GC) tissues, but their roles in pathogenesis of inflammatory T helper (Th) subsets are still unknown.MethodsFlow cytometry analysis and immunohistochemistry were used to analyze the responses and phenotypes of neutrophils in different samples from 51 patients with GC. Kaplan‐Meier plots and Multivariate analysis for the survival of patients were used by log‐rank tests and Cox proportional hazards models. Neutrophils and CD4+ T cells were purified and cultured for ex vivo, in vitro and in vivo regulation and function assays.ResultsGC patients exhibited increased tumoral neutrophil infiltration with GC progression and poor patient prognosis. Intratumoral neutrophils accumulated in GC tumors via CXCL6/CXCL8‐CXCR1‐mediated chemotaxis, and expressed activated molecule CD54 and co‐signaling molecule B7‐H2. Neutrophils induced by tumors strongly expressed CD54 and B7‐H2 in both dose‐ and time‐dependent manners, and a close correlation was obtained between the expressions of CD54 and B7‐H2 on intratumoral neutrophils. Tumor‐derived tumor necrosis factor‐α (TNF‐α) promoted neutrophil activation and neutrophil B7‐H2 expression through ERK‐NF‐κB pathway, and a significant correlation was found between the levels of TNF‐α and CD54+ or B7‐H2+ neutrophils in tumor tissues. Tumor‐infiltrating and tumor‐conditioned neutrophils effectively induced IL‐17A‐producing Th subset polarization through a B7‐H2‐dependent manner ex vivo and these polarized IL‐17A‐producing Th cells exerted protumorigenic roles by promoting GC tumor cell proliferation via inflammatory molecule IL‐17A in vitro, which promoted the progression of human GC in vivo; these effects could be reversed when IL‐17A is blocked. Moreover, increased B7‐H2+ neutrophils and IL‐17A in tumors were closely related to advanced GC progression and predicted poor patient survival.ConclusionWe illuminate novel underlying mechanisms that TNF‐α‐activated neutrophils link B7‐H2 to protumorigenic IL‐17A‐producing Th subset polarization in human GC. Blocking this pathological TNF‐α‐B7‐H2‐IL‐17A pathway may be useful therapeutic strategies for treating GC.

Can Intratumoral neutrophil lymphocyte ratio (NLR) be a prognostic biomarker in breast cancer patients?

e12573 Background: In breast cancer patients, it is well known that the elevation of neutrophil lymphocyte ratio (NLR) in the blood are reported to associate with poor prognosis based on the notion that neutrophils represent pro-cancer, and lymphocytes represent anti-cancer immune cells. Tumor immune microenvironment has been demonstrated to play critical roles in the outcome of breast cancer patients. However, there is scarce evidence on the clinical relevance of intratumoral NLR in breast cancer patients. In the current study, we hypothesized that intratumoral NLR high tumors are associated with worse survival particularly in TNBC that is known to have high immune cell infiltration. Methods: A total of 1904 breast cancer patients’ data from METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) and analyzed. NLR was calculated by the gene expressions of CD66b (CEACAM8) and CD8 (CD8A). NLR high and low were divided by the median. Overall Survival (OS) and Disease-Free Survival were calculated utilizing Kaplan Meier method between intratumoral NLR high and low groups. xCell algorithm was used to analyze the infiltrated immune cells within the tumor immune microenvironment as we have previously published. Results: Intratumoral NLR high group was associated with worse OS in whole, ER-positive/HER2-negative, and triple negative (TN) subtypes, in agreement with the previous studies. TN subtype alone demonstrated worse DFS of NLR high group. Surprisingly, gene set enrichment analysis (GSEA) demonstrated no gene set enrichment to NLR high group, which implicates that there is no distinctive mechanism that associate with worse survival. Whereas, immune response-related gene sets significantly enriched to NLR low group in TN subtype. This enrichment was consistent in ER-positive/HER2-negative. Compared with ER-positive/HER2-negative subtype, anti-cancer immune cells such as CD4+ T cells, CD8+ T cells, M1 macrophage, and helper T helper type 1 cells were significantly infiltrated in TN patients (p < 0.001 for all genes), where M2 macrophages and neutrophils were less and regulatory T cells and T helper type 2 cells were more infiltrated in TN subtype. Furthermore, intratumoral NLR was significantly lower in TN compared with ER-positive/HER2-negative subtype (p < 0.001). These results suggest that intratumoral NLR low group is associated with better survival due to favorable tumor immune microenvironment in TN subtype rather than NLR high group has worse survival. Conclusions: Intratumoral NLR low tumor demonstrated more favorable OS and more favorable DFS in TN patients. Intratumoral NLR low breast cancer was associated with enhanced immune response and higher infiltration of anti-cancer immune cells were observed in TN subtype compared to ER-positive/HER2-negative which may contribute to the favorable outcome of in TN breast cancer.

Expression, regulation and clinical significance of B7-H3 on neutrophils in human gastric cancer

Neutrophils are conspicuous components of gastric cancer (GC) tumors, increasing with tumor progression and poor patient survival. However, the phenotype, regulation and clinical relevance of neutrophils in human GC are presently unknown. Most intratumoral neutrophils showed an activated CD54+ phenotype and expressed high level B7-H3. Tumor tissue culture supernatants from GC patients induced the expression of CD54 and B7-H3 on neutrophils in time-dependent and dose-dependent manners. Locally enriched CD54+ neutrophils and B7-H3+ neutrophils positively correlated with increased granulocyte-macrophage colony stimulating factor (GM-CSF) detection ex vivo; and in vitro GM-CSF induced the expression of CD54 and B7-H3 on neutrophils in both time-dependent and dose-dependent manners. Furthermore, GC tumor-derived GM-CSF activated neutrophils and induced neutrophil B7-H3 expression via JAK-STAT3 signaling pathway activation. Finally, intratumoral B7-H3+ neutrophils increased with tumor progression and independently predicted reduced overall survival. Collectively, these results suggest B7-H3+ neutrophils to be potential biomarkers in GC.

Abstract LT005: Tumor-intrinsic gain of function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment that promotes resistance to immunotherapy

Abstract KRASG12D mutations in tumor cells can modify the immune microenvironment of pancreatic ductal adenocarcinoma (PDAC), though the effects of secondary genetic alterations have been unexplored. After oncogenic KRAS, TP53 is the most frequently mutated gene in PDAC, however the immune effects of neomorphic TP53 mutations have not been defined. We sought to evaluate the non-cell-autonomous role of mutant p53 in modulating the immune microenvironment of pancreatic cancer by comparing the immune profiles generated by KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) genetically engineered to express the Trp53R172H gain-of-function mutation to their p53 wildtype control. We found orthotopically implanted tumors derived from KrasG12D;Trp53R172H PDEC had a distinct immune profile in comparison to KrasG12D/+;Trp53+/+ tumors, characterized by an influx of intratumoral CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 (Th1) cells. Analysis of publicly available human PDAC cohorts revealed enrichment of genes in neutrophil-related pathways in TP53-mutated tumors. Knockdown of CXCL2, a neutrophil chemoattractant, in the tumor epithelial compartment of KrasG12D;Trp53R172H PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing KrasG12D;Trp53R172H PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link mutant p53 to the presence of intratumoral neutrophils in pancreatic cancer and suggests that tumor genotypes could inform patient selection for immunotherapy. Citation Format: Despina Siolas, Emily Vucic, Emma Kurz, Cristina Hajdu, Dafna Bar-Sagi. Tumor-intrinsic gain of function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment that promotes resistance to immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT005.

Granulocyte-Macrophage Colony-Stimulating Factor-Activated Neutrophils Express B7-H4 That Correlates with Gastric Cancer Progression and Poor Patient Survival.

Neutrophils are prominent components of gastric cancer (GC) tumors and exhibit distinct phenotypes in GC environment. However, the phenotype, regulation, and clinical relevance of neutrophils in human GC are presently unknown. Here, immunohistochemistry, real-time PCR, and flow cytometry analyses were performed to examine levels and phenotype of neutrophils in samples from 41 patients with GC, and also isolated, stimulated, and/or cultured neutrophils for in vitro regulation assays. Finally, we performed Kaplan-Meier plots for overall survival by using the log-rank test to evaluate the clinical relevance of neutrophils and their subsets. In our study, neutrophils in tumor tissues were significantly higher than those in nontumor tissues and were positively associated with tumor progression but negatively correlated with GC patient survival. Most intratumoral neutrophils showed an activated CD54+ phenotype and expressed high-level immunosuppressive molecule B7-H4. Tumor tissue culture supernatants from GC patients induced neutrophils to express CD54 and B7-H4 in both time-dependent and dose-dependent manners. Locally enriched CD54+ neutrophils and B7-H4+ neutrophils positively correlated with increased granulocyte-macrophage colony-stimulating factor (GM-CSF) detection ex vivo, and in vitro GM-CSF induced the expression of CD54 and B7-H4 on neutrophils in a time-dependent and dose-dependent manner. Moreover, GC tumor-derived GM-CSF activated neutrophils and induced neutrophil B7-H4 expression via Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signaling pathway activation. Furthermore, higher intratumoral B7-H4+ neutrophil percentage/number was found in GC patients with advanced tumor node metastasis stage and reduced overall survival following surgery. Our results illuminate a novel regulating mechanism of B7-H4 expression on tumor-activated neutrophils in GC, suggesting that functional inhibition of these novel GM-CSF-B7-H4 pathways may be a suitable therapeutic strategy to treat the immune tolerance feature of GC.

Abstract PR-005: Tumor intrinsic p53 mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy

Abstract Intratumoral genetic alterations can cause remodeling of local immune cell populations through distinct cellular mechanisms. After oncogenic KRAS, TP53 is the gene most frequently mutated in pancreatic ductal adenocarcinoma (PDAC). These mutations in TP53 are neomorphic and confer phenotypes distinct from those due to homozygous TP53 deletion. We sought to evaluate the non-cell-autonomous role of mutant p53 protein in the immune microenvironment by studying KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) engineered using CRISPR/Cas gene editing to express the Trp53R172H gain-of-function mutation. We found distinct immune profiles associated with orthotopically implanted tumors derived from KrasG12D;Trp53R172H PDEC, characterized by an influx of intratumoral CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 (Th1) cells. Analysis of publicly available human PDAC cohorts revealed enrichment of genes in neutrophil-related pathways in TP53-mutated tumors. Knockdown of CXCL2, a neutrophil chemoattractant, in the tumor epithelial compartment of KrasG12D;Trp53R172H PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing KrasG12D;Trp53R172H PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link mutant p53 to the presence of intratumoral neutrophils in pancreatic cancer and suggests that tumor genotypes could inform patient selection for immunotherapy. Citation Format: Despina Siolas, Emily Vucic, Emma Kurz, Cristina Hajdu, Dafna Bar-Sagi. Tumor intrinsic p53 mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy [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 PR-005.

S0191 Comparing Pathologic Features of Different Colonic Segments in Non-Hereditary Young Onset Colorectal Cancer

INTRODUCTION: Young patients with non-hereditary colorectal cancer (CRC) have unfavorable pathologic characteristics. The features and survival outcomes in different colonic segments are to be determined. METHODS: In this retrospective observational study we randomly selected patients (aged 18–50) with a histopathologic diagnosis of CRC at Carilion Clinic, Roanoke, from 2002 to 2017. Patients with inflammatory bowel disease and predisposing genetic syndromes were excluded. Demographics, polyp and tumor features, and mortality were obtained from the electronic medical record. Two pathologists reviewed histopathology to minimize diagnostic variability. The cumulative risk of mortality among patients with different pathologic features was estimated using Kaplan Meier curves. RESULTS: We identified 139 patients (Mean age, 41.6 ± 6.9 years; 53.2% males) with non-hereditary CRC. Thirty percent of tumors were located in the right colon (RC), 35.4% in the left colon (LC), and 34.5% in the rectum (Rec). In patients with resected tumors, a cancer-adjacent polyp was identified in 78.8% (RC), 73.7% (LC), and 47.4% (Rec). Lymphovascular invasion (LVI) was present in 59.0% compared to 38% (LC) and 25% (Rec). Perineural invasion (PNI) was present in 25.0% compared to 10.8% (LC) and 14.35% (Rec). RC tumor budding was (14%) compared to 13.1% (LC) and 2.9% (Rec), and tumor deposits in 30% compared 18.9% (LC) and 17.7% (Rec). Intratumoral lymphocytes were present in 32.1% (RC) compared to 15.8% (LC) and 3.03% (Rec). Intratumoral neutrophils were present in 3.6% in the RC compared to 10.5% (LC) and 3.03% (Rec). Thirty-two percent (RC) had Crohn’s like response compared to 26.3% (LC) and 3.0% (Rec). Microsatellite instability (MSI) accounted for 9.4% of (RC) tumors and 8.1% of (LC) tumors but was not present in rectal tumors. Among patients who had repeat colonoscopy for surveillance, about 90.0% of patients with RC cancer did not receive surveillance colonoscopy within 1 year of cancer diagnosis compared to 42% (LC) and 55.6% (Rec). Patients with Rec cancer had decreased survival compared to patients diagnosed with RC and LC cancers (P = 0.04) (Figure 1). CONCLUSION: Right colon tumors accounted for higher rates of unfavorable pathologic features compared to left colon and rectal tumors. Adherence to USMSTF surveillance recommendations is poor, especially in RC tumors. Patients with rectal cancer had the poorest survival outcomes compared to right and left colon tumors.Figure 1.: Interval time of diagnosis until last visit based on segment.

S0140 A Retrospective Analysis of Tumor Budding, Staging, Invasion, and Immune Response in Patients Diagnosed With Non-Hereditary Young Onset Colorectal Cancer

INTRODUCTION: Tumor budding is an adverse prognostic factor in colorectal cancer, has been studied extensively in patients with somatic, microsatellite stable variants, and is found in 20–40% of such cases. However, the presence and significance of this histopathologic phenomenon in young patients diagnosed with non-hereditary colon cancer is to be determined. In this study, our aim was to ascertain presence of tumor budding in young patients diagnosed with non-hereditary cancer and investigate correlations of this prognostic feature with other prognostic factors such as staging, invasion, and immune response trends. METHODS: In this retrospective observational study, we selected young patients (18–50 years old) with histopathologic diagnosis of non-hereditary colorectal adenocarcinoma at Carilion Clinic, Roanoke, from 2002 to 2017 and followed through 2011–2019. Patients with inflammatory bowel disease and those with predisposing genetic syndromes were excluded. Patient demographics, polyps’ features, cancer features, colonoscopy reports, and mortality were obtained from electronic medical record. Two pathologists reviewed pathology slides to minimize inter-pathologist variability in histologic diagnosis. The cumulative risk of mortality among patients with different pathologic features were estimated using Kaplan Meier curves. RESULTS: One hundred and thirty nine patients (mean age, 41.6 ± 6.9 years) with non-hereditary colorectal cancer were identified. One hundred and four of these cases displayed tumor budding on histology (74.8%). Tumor budding was significantly correlated with lymphovascular and perineural invasion (P < 0.01 for both). Tumor budding grade was also significantly correlated with high cancer stage (P < 0.01), with intratumoral lymphocyte and neutrophil presence (P < 0.01) and the presence of tumor deposits (P < 0.01). Lastly, the occurrence of a Crohn-like response, a histologic indicator for an immune reaction to the colon cancer, was noted to be significantly higher in neoplasms affected by higher grade tumor budding (P < 0.01). CONCLUSION: Tumor budding was found in the majority (74.8%) of young non-hereditary colorectal cancer patients, suggesting that this phenomenon is much more prevalent in this subpopulation than in the somatic, microsatellite stable colorectal cancer group. Tumor budding was also significantly correlated with low and high tumor stages as well as lymphovascular and perineural invasion in young patients with non-hereditary colon cancer.Table 1.: Table summarizing tumor budding correlations with staging, invasion, and immune response trends

Host genetics and tumor environment determine the functional impact of neutrophils in mouse tumor models.

BackgroundNeutrophils have been reported to have protumor, antitumor or neutral effects in cancer progression. The underlying causes for this functional variability are not clear.MethodsWe studied the role of neutrophils in six different mouse tumor models by intratumoral injection of antimicrobial peptides or vaccination. Changes in systemic and intratumoral immune cells were analyzed by flow-cytometry and mass-cytometry. The role of neutrophils was studied by antibody-mediated neutrophil depletion. Neutrophils from different mouse strains were compared by RNA sequencing.ResultsThe antimicrobial peptide Omiganan reduced the growth of TC-1 tumors in BL/6 mice and CT26 tumors in BALB/c mice. No significant effects were observed in B16F10, MC38 and 4T1 tumors. Growth delay was associated with increased abundance of neutrophils in TC-1 but not CT26 tumors. Systemic neutrophil depletion abrogated Omiganan efficacy in TC-1 but further reduced growth of CT26, indicating that neutrophils were required for the antitumor effect in TC-1 but suppressed tumor control in CT26. Neutrophils were also required for a therapeutic vaccine-induced T-cell mediated control of RMA tumors in BL/6 mice. Clearly, the circulating and intratumoral neutrophils differed in the expression of Ly6G and CD62L, between TC-1 and CT26 and between blood neutrophils of tumor-naïve BL/6 and BALB/c mice. RNA-sequencing revealed that neutrophils from BL/6 mice but not BALB/c mice displayed a robust profile of immune activation, matching their opposing roles in TC-1 and RMA versus CT26.ConclusionsNeutrophil functionality differs strongly between mouse strains and tumor types, with consequences for tumor progression and therapy.

Abstract 4480: Circulating and tumor associated neutrophil subtypes correlate with hyperprogressive disease (HPD) upon immunecheckpoint inhibitors (ICI) in advanced non-small cell lung cancer (aNSCLC) patients (pts) and in vivo models

Abstract Background: HPD was described in ≃10-25% of NSCLC pts upon single-agent ICI and correlated with poor prognosis. High circulating neutrophil count and neutrophils/lymphocytes ratio were associated with shorter survival and HPD in aNSCLC pts. In mouse lung cancer models, interleukin-17 (IL-17) promoted tumor growth upon ICI through increased intratumoral neutrophils. The role of specific circulating and/or tumor-associated neutrophil populations in driving HPD is unknown. Methods: aNSCLC pts treated with ICI and enrolled in a prospective monocentric study were assessed for HPD and circulating neutrophils' phenotype. HPD required 3 tumor measurements (2 before ICI, 1 upon ICI) and was defined as delta tumor growth rate (TGR) TGR (TGR upon ICI - TGR before ICI) &amp;gt;50% and/or TGR ratio (TGR upon ICI/ TGR before ICI) ≥2. Survival curves were estimated by Kaplan Meier and compared by log-rank, correlations with categorical/continuous variables were performed by Fisher/Mann-Whitney tests. The phenotype of circulating low density neutrophils (LDNs) was assessed by flow cytometry (FC) on peripheral blood mononuclear cells from fresh blood samples. Mature and immature LDNs were defined according to the expression of CD10 on CD66b+CD15+ cells, being immature the CD10− neutrophil population. The capacity of immune competent mice to undergo HPD upon anti-PD-1 treatment was further tested in C57BL/6 mice bearing Lewis Lung Carcinoma and treated with anti-murine PD-1. Tumor associated neutrophils' phenotype was assessed by FC analysis. Results: Of 46 aNSCLC, 85% were &amp;gt;65 years, 72% had adenocarcinoma histology, 89% stage IV, 22% had PD-L1 on tumor cells ≥50%, 72% received 1st line ICI. 19 (41%) pts had progression (PD) by RECIST, 4 (9%) pts were HPD by TGR ratio and/or delta-TGR. HPD [4.1 (95% CI 3.1-5) months] correlated with poorer median overall survival compared to non-HPD [6.9 (95% CI 6.1-7.8) months, p=0.03] and to PD pts [4.7 (95% CI 3.2-6.1) months, p=0.8]. Before ICI initiation, the median percentage of circulating immature CD10− CD15+ CD66b+ LDNs was significantly higher in HPD [36.6 (min 29.2; max 64.5)] compared to both non-HPD [8.9 (min 0; max 70.4); p=0.01] and PD non-HPD pts [10.1 (min 0; max 70.4), p=0.04]. HPD defined by TGR ratio and/or delta-TGR occurred in 3 (12.5%) out of 24 immune competent mice treated with anti-murine PD-1. The mean of Gr1highLy6Clow IL-17+ tumor associated neutrophils was significantly higher in HPD compared to non-HPD mice [0.25 (min 0.14; max 0.63) vs 0.06 (min 0.02; max 0.32), p=0.03]. Conclusions: Circulating immature CD10− LDNs and tumor associated IL-17+ neutrophils correlate with HPD upon ICI in aNSCLC pts and in vivo models, respectively. Characterization of neutrophils' functional properties in HPD pts and in vivo models is currently ongoing. Citation Format: Roberto Ferrara, Claudia Proto, Giuseppe Lo Russo, Diego Signorelli, Arsela Prelaj, Giulia Galli, Nicoletta Zilembo, Alessandro De Toma, Giuseppe Viscardi, Riccardo Lobefaro, Marta Brambilla, Monica Ganzinelli, Giuliano Molino, Giovanna Talarico, Marina Chiara Garassino, Mario Colombo, Sabina Sangaletti. Circulating and tumor associated neutrophil subtypes correlate with hyperprogressive disease (HPD) upon immunecheckpoint inhibitors (ICI) in advanced non-small cell lung cancer (aNSCLC) patients (pts) and in vivo models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4480.

Circulating and tumor-associated neutrophil subtypes discriminate hyperprogressive disease (HPD) from conventional progression (PD) upon immune checkpoint inhibitors (ICI) in advanced non-small cell lung cancer (NSCLC) patients (pts) and in vivo models.

9547 Background: HPD occurs in ≃10-25% of NSCLC pts upon single-agent ICI and correlates with poor prognosis. High circulating neutrophil count and neutrophils/lymphocytes ratio have been associated with shorter survival and HPD in NSCLC pts. In mouse lung cancer models, interleukin-17 (IL-17) promoted tumour growth upon ICI increasing intratumoral neutrophils. The role of specific circulating and/or tumour-associated neutrophils in driving HPD is currently unknown. Methods: NSCLC pts treated with single agent ICI were assessed for HPD and circulating neutrophils’ phenotype. Conventional PD was defined by RECIST 1.1. HPD required 3 tumour assessments (2 before ICI, 1 upon ICI) and was defined as delta tumour growth rate (TGR) (TGR upon ICI – TGR before ICI) &gt; 50% and/or TGR ratio (TGR upon ICI/ TGR before ICI) ≥2. Correlations with continuous variables were performed by Mann-Whitney test. Circulating low density neutrophils (LDNs) subtypes were assessed by flow cytometry (FC) on peripheral blood mononuclear cells (PBMCs) from fresh blood samples. LDNs were defined as CD66b+CD15+ cells among CD11b+ PBMCs. Immature subtypes were defined as CD10− and CD10−CD16− LDNs. The occurrence of HPD upon anti-PD-1 treatment was tested in C57BL/6 immune competent mice bearing Lewis Lung Carcinoma and treated with anti-murine PD-1. Tumour associated neutrophils’ phenotype was assessed by FC. Results: Of 52 NSCLC, 65% were &gt; 65 years, 83% had stage IV, 25% PD-L1 on tumour cells ≥50%, 67% received 1st line ICI. PD and HPD occurred in 21 (40%) and 5 (10%) pts, respectively. Before ICI start, HPD pts had higher circulating immature neutrophils measured as median percentage of CD10− LDNs [41.9 (min 26.7; max 83.5) vs 10.1 (min 0.69; max 79.3), p = 0.01] and of CD16− cells among CD10− LDNs [93 (min 89.5; max 98.4) vs 86.3 (min 24.2; max 99), p = 0.03] compared to conventional PD pts. PD and HPD occurred in 17 (71%) and 3 (12.5%) of 24 immune competent mice treated with anti-murine PD-1. The median percentage of IL-17+ tumour associated neutrophils (Gr1highLy6Clow) was significantly higher in HPD compared to PD mice [0.25 (min 0.14; max 0.63) vs 0.06 (min 0.02; max 0.32), p = 0.02]. Conclusions: Circulating immature (CD10− and CD10− CD16−) LDNs and IL-17+ tumour associated neutrophils discriminate HPD from conventional PD upon ICI in NSCLC pts and in vivo models, respectively. Functional characterization of specific neutrophil subsets is ongoing.

Preoperative β-Blockade with Propranolol Reduces Biomarkers of Metastasis in Breast Cancer: A Phase II Randomized Trial.

The majority of deaths from breast cancer occur following the development of metastatic disease, a process inhibited by β-blockers in preclinical studies. This phase II randomized controlled trial evaluated the effect of preoperative β-blockade with propranolol on biomarkers of metastatic potential and the immune cell profile within the primary tumor of patients with breast cancer. In this triple-blind placebo-controlled clinical trial, 60 patients were randomly assigned to receive an escalating dose of oral propranolol (n = 30; 80-160 mg daily) or placebo (n = 30) for 7 days prior to surgery. The primary endpoint investigated the effect of propranolol on prometastatic and proinflammatory gene expression within the primary tumor. Propranolol downregulated primary tumor expression of mesenchymal genes (P = 0.002) without affecting epithelial gene expression (P = 0.21). Bioinformatic analyses implicated downregulation of Snail/Slug (P = 0.03), NF-κB/Rel (P < 0.01), and AP-1 (P < 0.01) transcription factors in structuring the observed transcriptome alterations, and identified changes in intratumoral neutrophil, natural killer cell, and dendritic cell recruitment (all P < 0.01). Patients with clinical evidence of drug response (lowered heart rate and blood pressure) demonstrated elevated tumor infiltration of CD68+ macrophages and CD8+ T cells. One week of β-blockade with propranolol reduced intratumoral mesenchymal polarization and promoted immune cell infiltration in early-stage surgically-resectable breast cancer. These results show that β-blockade reduces biomarkers associated with metastatic potential, and support the need for larger phase III clinical trials powered to detect the impact of β-blockade on cancer recurrence and survival.See related commentary by Blaes et al., p. 1781.

Neutrophil expansion defines an immunoinhibitory peripheral and intratumoral inflammatory milieu in resected non-small cell lung cancer: a descriptive analysis of a prospectively immunoprofiled cohort

BackgroundThe biological underpinnings of the prognostic and predictive significance of a relative neutrophilia in patients with non-small lung cancer (NSCLC) are undefined. We sought to comprehensively examine the relationships between...