Increased Apolipoprotein A1 Expression Correlates with Tumor-Associated Neutrophils and T Lymphocytes in Upper Tract Urothelial Carcinoma.

An increased neutrophil-to-lymphocyte ratio (NLR) is a poor prognostic biomarker in various types of cancer, because it reflects the inhibition of lymphocytes in the circulation and tumors. In urologic cancers, upper tract urothelial carcinoma (UTUC) is known for its aggressive features and lack of T cell infiltration; however, the association between neutrophils and suppressed T lymphocytes in UTUC is largely unknown. In this study, we examined the relationship between UTUC-derived factors and tumor-associated neutrophils or T lymphocytes. The culture supernatant from UTUC tumor tissue modulated neutrophils to inhibit T cell proliferation. Among the dominant factors secreted by UTUC tumor tissue, apolipoprotein A1 (Apo-A1) exhibited a positive correlation with NLR. Moreover, tumor-infiltrating neutrophils were inversely correlated with tumor-infiltrating T cells. Elevated Apo-A1 levels in UTUC were also inversely associated with the population of tumor-infiltrating T cells. Our findings indicate that elevated Apo-A1 expression in UTUC correlates with tumor-associated neutrophils and T cells. This suggests a potential immunomodulatory effect on neutrophils and T cells within the tumor microenvironment, which may represent therapeutic targets for UTUC treatment.

Effect of tumor-infiltrating immune cells (mast cells, neutrophils and lymphocytes) on neoadjuvant chemotherapy response in breast carcinomas.

INTRODUCTION: Despite screening, the incidence of breast cancer is increasing worldwide. Neoadjuvant chemotherapy (NAC) response is one of the most important parameters taken into consideration in surgery, optimal adjuvant chemotherapy planning and prognosis prediction. Research on predictive markers for the response to NAC is still ongoing. In our study, we investigated the relationship between tumor-infiltrating neutrophils/mast cells/lymphocytes and NAC response in breast carcinomas. MATERIAL AND METHOD: Study included 117 patients who were diagnosed with invasive breast carcinoma using core needle biopsy. In these biopsies tumor-infiltrating neutrophils/mast cells/lymphocytes were evaluated and Miller Payne Score was used for NAC response. RESULT: 53 patients exhibited high TILs, 36 had high TINs, and 46 showed high TIMs. While pathological complete response was 27 % in all patients, it was 38 % in high TINs patients, 35 % in high TILs patients, and 28 % in high TIMs patients. High TIMs were observed to be statistically associated with survival. TILs, TINs, nuclear grade, ER, PR and HER2 expression, Ki-67 proliferation index were found to be associated with the Miller - Payne score. In multivariate analysis, TINs, nuclear grade, pathological stage, and molecular subtype were found to be independent risk factors for treatment response. CONCLUSION: TINs have better prognostic value to predict neoadjuvant treatment than TILs. High TIMs are associated with increased overall survival. The inclusion of TINs in NAC response and TIMs in overall survival in pathology reports and treatment planning is promising in breast carcinomas as they are simple to use and reproducible markers.

Tumor-Intrinsic Enhancer of Zeste Homolog 2 Controls Immune Cell Infiltration, Tumor Growth, and Lung Metastasis in a Triple-Negative Breast Cancer Model.

Triple-negative breast cancer (TNBC) is an aggressive and highly metastatic type of tumor. TNBC is often enriched in tumor-infiltrating neutrophils (TINs), which support cancer growth in part by counteracting tumor-infiltrating lymphocytes (TILs). Prior studies identified the enhancer of zeste homolog 2 (EZH2) as a pro-tumor methyltransferase in primary and metastatic TNBCs. We hypothesized that EZH2 inhibition in TNBC cells per se would exert antitumor activity by altering the tumor immune microenvironment. To test this hypothesis, we used CRISPR to generate EZH2 gene knockout (KO) and overexpressing (OE) lines from parent (wild-type-WT) 4T1 cells, an established murine TNBC model, resulting in EZH2 protein KO and OE, respectively. In vitro, EZH2 KO and OE cells showed early, transient changes in replicative capacity and invasiveness, and marked changes in surface marker profile and cytokine/chemokine secretion compared to WT cells. In vivo, EZH2 KO cells showed significantly reduced primary tumor growth and a 10-fold decrease in lung metastasis compared to WT cells, while EZH2 OE cells were unchanged. Compared to WT tumors, TIN:TIL ratios were greatly reduced in EZH2 KO tumors but unchanged in EZH2 OE tumors. Thus, EZH2 is key to 4T1 aggressiveness as its tumor-intrinsic knockout alters their in vitro secretome and in vivo primary tumor growth, TIN/TIL poise, and metastasis.

Gastric Tumor Microenvironment.

A compelling body of evidence has demonstrated that gastric cancer has a very particular tumor microenvironment, a signature very suitable to promote tumor progression and metastasis. Recent investigations have provided new insights into the multiple molecular mechanisms, defined by genetic and epigenetic mechanisms, supporting a very active cross talk between the components of the tumor microenvironment and thus defining the fate of tumor progression. In this review, we intend to highlight the role of very active contributors at gastric cancer TME, particularly cancer-associated fibroblasts, bone marrow-derived cells, tumor-associated macrophages, and tumor-infiltrating neutrophils, all of them surrounded by an overtime changing extracellular matrix. In addition, the very active cross talk between the components of the tumor microenvironment, defined by genetic and epigenetic mechanisms, thus defining the fate of tumor progression, is also reviewed.

Urine Lactoferrin as a Potential Biomarker Reflecting the Degree of Malignancy in Urothelial Carcinoma of the Bladder.

Urothelial carcinoma of the bladder (UCB) is potentially life-threatening; therefore, we aimed to discover a novel urine biomarker for diagnosis and prognostication of UCB. This is a retrospective case-control study. Exploration of a new biomarker using urine from 20 UCB patients in the present study revealed that urinary level of lactoferrin (LF), a multifunctional glycoprotein released from neutrophils, was higher in 11 of 15 with invasive/high-grade UCB than 5 with non-invasive one, and 2 healthy adults. We therefore focused on LF and assessed the value of urine LF normalized by urine creatinine concentration (LF/Cr) using an enzyme-linked immunosorbent assay. Diagnostic performance of urine LF/Cr was examined using urine from 92 patients with primary (newly diagnosed) untreated UCB and 166 controls without UCB, including 62 patients with pyuria, and 104 subjects without pyuria consisting of 84 patients and 20 healthy adults. However, the diagnostic accuracies were accompanied by the risk of bias. In 92 primary UCB patients, both pyuria and tumor-infiltrating neutrophils (TINs) were independent predictors for urine LF/Cr. In contrast, TINs or urine LF/Cr were independent predictors for invasive histology, whereas pyuria was not. In terms of prognostication, urine LF/Cr and nodal metastasis were independent predictors of disease-specific survival in 22 patients with muscle-invasive bladder cancer, characterized by a high mortality rate, in the Cox proportional hazards model. In conclusion, urine LF/Cr linked to TINs was a predictor of both invasive histology and prognosis in UCB. Urine LF/Cr is a potential biomarker reflecting the degree of malignancy in UCB.

Tumor-infiltrating CD8+ T cells combined with tumor-associated CD68+ macrophages predict postoperative prognosis and adjuvant chemotherapy benefit in resected gastric cancer.

BACKGROUND: Tumor-infiltrating immune cells are present in various malignant tumors, but their clinical significance in gastric cancer (GC) remains unclear. This study aimed to investigate the prognostic significance of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). METHODS: Using a prospective database containing 401 cases of GC, we evaluated TIL (cluster of differentiation 8 (CD8) expression) and TAM (cluster of differentiation 68 (CD68) expression) statuses via immunohistochemical staining. RESULTS: Compared with CD8+ TIL-negative cases (n = 196, 48.6%), CD8+ TIL-positive cases (n = 205, 51.1%) showed significantly better recurrence-free survival (RFS) [log-rank p<0.001; multivariate HR: 0.372; 95% confidence interval (CI): 0.239-0.579, p<0.001]. In contrast, compared with CD68+ TAM-negative cases (n = 217, 54.1%), CD68+ TAM-positive cases (n = 184, 45.9%) had significantly poor RFS [log-rank p<0.001; multivariate HR: 2.182; 95% CI: 1.435-3.318, p<0.001]. Thus, patients with a positive CD8+ TIL and negative CD68+ TAM status exhibited significantly increased RFS. Multivariate analysis demonstrated that CD8+ TILs and CD68+ TAMs may serve as independent prognostic markers for RFS. Incorporating CD8+ TIL and CD68+ TAM statuses into the AJCC TNM system generated a predictive model with better predictive accuracy for RFS. More importantly, patients with a positive TIL and negative TAM status showed a tendency of improved RFS after postoperative adjuvant chemotherapy (PAC). Similar results were obtained by overall survival (OS) analysis. CONCLUSIONS: CD8+ TIL and CD68+ TAM statuses were identified as independent prognostic factors that may be integrated into the current TNM staging system to refine risk stratification and to better predict the survival benefit from PAC in patients with GC. TRIAL REGISTRATION: The current controlled trial was registered at ClinicalTrials.gov (ID: NCT02327481 ) on December 30, 2014.

1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol treatment inhibits abnormal tumor growth by regulating neutrophil infiltration in a non-small cell lung carcinoma mouse model.

Excessive neutrophil infiltration into the tumor microenvironment (TME) is an important factor that contributes to tumor overgrowth and limited immunotherapy efficacy. Neutrophils activate various receptors involved in tumor progression, while suppressing the infiltration and activity of cytotoxic T cells and creating optimal conditions for tumor growth. Therefore, the appropriate control of neutrophil infiltration is an effective strategy for tumor treatment. In the present study, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) inhibited tumor overgrowth by suppressing excessive neutrophil infiltration, resulting in >74.97 % reduction in tumor size in a Lewis lung carcinoma (LLC-1) mouse model. All subjects in the positive control group died during the 90-day survival period, whereas only four subjects in the PLAG treatment group survived. PLAG had a significantly higher tumor growth inhibitory effect and survival rate than other neutrophil infiltration-targeting inhibitors (e.g., Navarixin, lymphocyte antigen 6 complex locus G6D antibody [aLy6G]). The ability of PLAG to regulate neutrophil infiltration and inhibit tumor growth depends on thioredoxin-interacting protein (TXNIP). In tumors lacking TXNIP expression, PLAG failed to control neutrophil infiltration and infiltration-related factor release, and the inhibitory effect of PLAG on tumor growth was reduced. PLAG-mediated inhibition of neutrophil infiltration enhances the efficacy of immune checkpoint inhibitors (ICIs), increasing the antitumor efficacy and survival rate by 30 %. In conclusion, PLAG could be a novel alternative to anti-tumor drugs that effectively targets excessive neutrophil infiltration into cancer tissues.

Prognostic significance and immune escape implication of tumor-infiltrating neutrophil plasticity in human head and neck squamous cell carcinoma.

Tumor-infiltrating neutrophils play a crucial role in the progression of head and neck squamous cell carcinoma (HNSCC). Here, we aimed to statistically quantify the plasticity of HNSCC-infiltrating N2/N1 neutrophils and examine its impacts on survival and immune infiltration landscape. A retrospective study of 80 patients who underwent curative surgical resection for HNSCC between 2014 and 2017 was conducted in this study. HNSCC-infiltrating neutrophil phenotypes were classified using immunofluorescence staining, and the N2/N1 neutrophil plasticity was evaluated via the ratio of N2/N1 neutrophils. We then assessed the correlations between N2/N1 neutrophil plasticity, clinicopathological characteristics, and immune infiltration landscape using rigorous statistical methods. Infiltration variations of N1 and N2 neutrophils were observed between the tumor nest (TN) and tumor stroma (TS), with TN exhibiting higher N2 neutrophil infiltration and lower N1 neutrophil infiltration. High ratios of N2/N1 neutrophils were correlated with advanced TNM stage, large tumor size and invasion of adjacent tissue. High infiltration of N2 neutrophils was associated with decreased overall and relapse-free survival, which were opposite for N1 neutrophils. The independent prognostic role of N2/N1 neutrophil plasticity, particularly within the TN region, was confirmed by multivariate analyses. Moreover, the ratio of N2/N1 neutrophils within the TN region showed correlations with high CD8+ T cells infiltration and low FOXP3+ Tregs infiltration. We identify HNSCC-infiltrating N2/N1 neutrophil plasticity as a crucial prognostic indictor which potentially reflects the tumor microenvironment (TME) and immune escape landscape within HNSCC tissues. Further investigations and validations may provide novel therapeutic strategies for personalized immunomodulation in HNSCC patients.

EFHD1 expression is correlated with tumor-infiltrating neutrophils and predicts prognosis in gastric cancer.

Background: Gastric cancer (GC) ranks third in terms of mortality worldwide. The tumor microenvironment is critical for the progression of gastric cancer. This study investigated the association between EF-hand domain containing 1 (EFHD1) expression and its clinical significance in the tumor microenvironment (TME) of gastric cancer. Methods: We used bioinformatic analyses to assess the relevance of EFHD1 mRNA in the TME of gastric carcinoma tissues and its relationship with clinical features. Therefore, we performed multiplex immunohistochemistry analyses to determine the potential role of the EFHD1 protein in the TME of gastric cancer. Results: EFHD1 expression increased dramatically in gastric cancer tissues compared to levels in non-cancerous tissue samples (t = 6.246, P < 0.001). The EFHD1 protein presentation was associated with invasion depth (χ2 = 19.120, P < 0.001) and TNM stages (χ2 = 14.468, P = 0.002). Notably, EFHD1 protein expression was significantly related to CD66b + neutrophil infiltration of the intratumoral (r = 0.420, P < 0.001) and stromal (r = 0.367, P < 0.001) TME in gastric cancer. Additionally, Cox regression analysis revealed that EFHD1 was an independent prognostic predictor (hazard ratio [HR] = 2.262, P < 0.001) in patients with gastric cancer. Conclusions: Our study revealed the pattern of EFHD1 overexpression in the TME of patients with gastric cancer and demonstrated its utility as a biomarker for unfavorable clinical outcomes, thereby providing a potential immunotherapy target.

CXCL5-mediated accumulation of mature neutrophils in lung cancer tissues impairs the differentiation program of anticancer CD8 T cells and limits the efficacy of checkpoint inhibitors.

Lung tumor-infiltrating neutrophils are known to support growth and dissemination of cancer cells and to suppress T cell responses. However, the precise impact of tissue neutrophils on programming and differentiation of anticancer CD8 T cells in vivo remains poorly understood. Here, we identified cancer cell-autonomous secretion of CXCL5 as sufficient to drive infiltration of mature, protumorigenic neutrophils in a mouse model of non-small cell lung cancer (NSCLC). Consistently, CXCL5 transcripts correlate with neutrophil density and poor prognosis in a large human lung adenocarcinoma compendium. CXCL5 genetic deletion, unlike antibody-mediated depletion, completely and selectively prevented neutrophils accumulation in lung tissues. Depletion of tumor-infiltrating neutrophils promoted expansion of tumor-specific CD8 T cells, differentiation into effector cells and acquisition of cytolytic functions. Transfer of effector CD8 T cells into neutrophil-rich tumors, inhibited IFN-ϒ production, indicating active suppression of effector functions. Importantly, blocking neutrophils infiltration in the lung, overcame resistance to checkpoint blockade. Hence, this study demonstrates that neutrophils curb acquisition of cytolytic functions in lung tumor tissues and suggests targeting of CXCL5 as a strategy to restore anti-tumoral T cell functions.

Prolonged survival of anaplastic thyroid carcinoma is associated with resectability, low tumor-infiltrating neutrophils/myeloid-derived suppressor cells, and low peripheral neutrophil-to-lymphocyte ratio.

PURPOSE: Anaplastic thyroid carcinoma (ATC) is the most lethal form of thyroid cancer with most patients dying of their disease within a few months. Only a very small percentage of long-term survivors (LTS) are alive for 2 years or longer. In this retrospective case-control study, we provided a comprehensive comparison between 46 ATC LTSs and 75 ATC control patients who suffered disease-specific mortality within 2 years, aiming to identify factors that may be associated with prolonged survival in ATC. METHODS: A comprehensive clinicopathologic and molecular comparison was performed between 46 ATC LTSs and 75 ATC control patients. Peripheral neutrophil count and neutrophil-to-lymphocyte ratio (NLR) were recorded. The composition of the tumor microenvironment was compared using immunohistochemistry. RESULTS: Compared with ATC control patients, ATC LTSs were characterized by 1) higher frequency of (primary) resection as well as clinicopathologic parameters attributed to resectability; 2) lower rate of concurrent RAS/BRAF and TERT promoter mutations; 3) lower peripheral neutrophil count and NLR; and 4) lower number of tumor-infiltrating neutrophils/myeloid-derived suppressor cells (MDSC). The survival benefits of low peripheral neutrophil counts and low NLR persisted even when controlling for distant metastasis status at presentation. CONCLUSIONS: In addition to traditional beneficial prognostic factors, e.g., surgical resection, factors attributed to resectability, and absence of co-existing RAS/BRAF and TERT promoter mutations, we herein show that tumor-infiltrating and circulating neutrophils/MDSC are adverse prognostic factors in ATC.

Tea tree oil extract causes mitochondrial superoxide production and apoptosis as an anticancer agent, promoting tumor infiltrating neutrophils cytotoxic for breast cancer to induce tumor regression.

The antitumor activity of the tea tree oil (TTO) derived product, Melaleuca Alternifolia Concentrate (MAC) was characterized mechanistically at the molecular and cellular level. MAC was analyzed for its anticancer activity against human prostate (LNCaP) and breast (MCF-7) cancer cell lines growing in vitro. MAC (0.02-0.06% v/v) dose-dependently induced the intrinsic (mitochondrial) apoptotic pathway in both the LNCaP and MCF-7 cell lines, involving increased mitochondrial superoxide production, loss of mitochondrial membrane potential (MMP), caspase 3/7 activation, as well as the presence of TUNEL+ and cleaved-PARP+ cell populations. At concentrations of 0.01-0.04% v/v, MAC caused cell cycle arrest in the G0/1-phase, as well as autophagy. The in vivo anticancer actions of MAC were examined as a treatment in the FVB/N c-Neu murine model for spontaneously arising breast cancers. Intratumoral MAC injections (1-4% v/v) significantly suppressed tumor progression in a dose-dependent manner and was associated with greater levels of tumor infiltrating neutrophils exhibiting anticancer cytotoxic activity. Induction of breast cancer cell death by MAC via the mitochondrial apoptotic pathway was also replicated occurring in tumors treated in vivo. In conclusion, our data highlights the potential for the Melaleuca-derived MAC product inducing anticancer neutrophil influx, supporting its application as a novel therapeutic agent.

Characterization of CD66b and its relationship between immune checkpoints and their synergistic impact in the prognosis of surgically resected lung adenocarcinoma.

OBJECTIVES: CD66b positive tumor-infiltrating neutrophils (TINs) are key immunity cells in the tumor microenvironment (TME). However, their relationship with clinicopathological features, immune checkpoints (ICs), and prognostic value remains undetermined in lung adenocarcinoma (LUAD). In this study, we aimed to characterize the infiltration by TINs and the prognostic significance in patients with surgically resected LUAD. MATERIALS AND METHODS: Expression of CD66b and ICs, including PD-L1, PD-1, CTLA4, LAG3, TIM3, TIGIT, VISTA, and BTLA, in both cancer cell and tumor-infiltrating lymphocytes (TILs) were estimated by immunohistochemistry in resected LUAD. The associations between CD66b expression and clinicopathological characteristics in patient prognoses were analyzed. We also verified results in another cohort from 85 patients with untreated LUAD and further analyzed the correlation between CD66b expression and EGFR and KRAS mutation status in addition to the rearrangement of the anaplastic lymphoma receptor tyrosine kinase gene (ALK). RESULTS: A total of 240 patients were included in this study. CD66b expression was observed in 87 (36.2%) samples. ICs including PD-L1, PD-1, CTLA4, LAG3, TIM3, TIGIT, VISTA, and BTLA were observed in percentages that ranged from 23.8% to 59.4%. Positive CD66b expression significantly correlated with smoking history (p = 0.029), pathological stage (p = 0.040), and the positive expression of LAG-3 (p < 0.001), PD-1 (p = 0.008), CTLA-4 (p = 0.013), TIM-3 (p = 0.025), TIGIT (p = 0.002), PD-L1 in TILs (p = 0.015), and PD-L1 in tumor cells (p = 0.010). CD66b positivity was significantly associated with worse recurrence-free survival (RFS) (hazard ratio, HR, 1.687; 95% confidence interval, CI, 1.058-2.690, p = 0.028) and overall survival (OS) (HR, 1.667; 95% CI, 1.097-2.534, p = 0.017). Subgroup analysis revealed that the CD66b+/LAG-3 + group had the worst RFS (5-year rate: 39.5%,) and OS (5-year rate: 53.7%,), while the CD66b-/LAG-3 - group had the best RFS (5-year rate: 65.6%) and OS (5-year rate: 78.8%). The p value in analysis of RFS and OS was 0.005 and 0.008, respectively. In the verification set, high expression of CD66b was also significantly correlated with the positive expression of LAG-3 (p < 0.001), PD-1 (p = 0.002), CTLA-4 (p = 0.034), TIM-3 (p = 0.049), PD-L1 in TILs (p = 0.003), and PD-L1 in tumor cells (p = 0.045). There was no correlation between CD66b expression and positive TIGIT expression (p = 0.077), EGFR mutation (p = 0.223), KRAS mutation (p = 0.151), and ALK fusion (p = 0.310). CONCLUSION: CD66b had a relatively high positive expression rate and special clinicopathological features in patients with LUAD. CD66b + TINs were related to the expression of ICs and associated with poor prognoses in LUAD. A combination of CD66b and ICs, especially LAG-3 could further stratify patients into different groups with distinct prognoses.

Neutrophil depletion enhances the therapeutic effect of PD-1 antibody on glioma.

Tumor-infiltrating neutrophils (TINs), the predominant leukocytes in the tumor microenvironment, are important for cancer-related immunosuppression. Combinations of multiple immune checkpoint inhibitors can significantly improve outcomes in murine glioma models. Here, we investigated TIN levels in human glioma samples and tested the antitumor efficacy of neutrophil depletion alone or in combination with an anti-programmed death 1 (PD-1) antibody. To investigate the clinical relevance, we determined the correlation between tumor grade or survival and TIN levels in 202 resected glioma specimens. TCGA and CGGA data were used to validate the results and analyze the biological functions of TINs in gliomas. An orthotopic xenograft glioma mouse model was used to study the therapeutic effect of anti-PD-1 and/or anti-ly6G. Decreased TIN levels correlated with lower grades, mutant isocitrate dehydrogenase, and favorable prognosis, which was validated by CGGA and TCGA dataset results. Bioinformatics analysis revealed that TINs are mainly involved in angiogenic, inflammatory, and interferon-γ responses in gliomas. TINs were positively correlated with programmed death ligand-1 expression. In xenograft models, combined anti-PD-1 and neutrophil depletion therapy significantly inhibited tumor growth and promoted survival. This study demonstrates that TINs were related to glioma tumorigenesis. Targeting neutrophils could thus enhance the therapeutic effect of PD-1 blockade for gliomas.

Intratumoral CCR5+ neutrophils identify immunogenic subtype muscle-invasive bladder cancer with favorable prognosis and therapeutic responses.

Our previous studies revealed tumor-infiltrating neutrophils (TINs) played dichotomous roles in different cancers, indicating diverse TINs subtypes might orchestrate anti-tumor immunity or immune evasion, respectively. This study aimed to investigate the clinical significance and immune characteristics of CCR5+TINs in muscle-invasive bladder cancer (MIBC). Two hundred and fifty-seven MIBC patients from two clinical centers and 95 fresh MIBC samples were included. CCR5+TINs were stained by immunohistochemistry, and the relationship between patients' clinic-pathological features and prognosis was evaluated, respectively. Immunohistochemistry and flow cytometry were applied to assess the immune features of CCR5+TINs and their correlations with other immune cells. In vitro study was conducted to estimate immune characteristics of CCR5+TINs and their predictive potential for pembrolizumab therapeutic response. In the two MIBC cohorts, we found that high CCR5+TINs infiltration could predict better overall survival (OS, P= .032, 0.039) and recurrence-free survival (RFS, P= .001, 0.006) and be associated with survival benefit from adjuvant chemotherapy (ACT, P< .001 for OS and P= .022 for RFS, respectively) in merely pT2N0 MIBC. Maraviroc could partly reduce IFN-γ secretion by CCR5+TINs (P< .001). CCR5+TINs correlated with higher expression of effector molecules within CD8+T cells. Notably, pembrolizumab treatment could only elevate the apoptosis status of tumor cells in the CCR5+TINs high subgroup (P < .001), other than CCR5+TINs low subgroup (P= .481). Our results indicate that CCR5+TINs could prime anti-tumor immune response through autonomous IFN-γ release, thus leading to favorable prognosis and superior therapeutic response to ACT and immunotherapy in MIBC.

Tumor-Infiltrating Neutrophils Predict Poor Survival of Non-Functional Pancreatic Neuroendocrine Tumor.

OBJECTIVE: This study retrospectively characterized the immune infiltrating profile in nonfunctional pancreatic neuroendocrine tumors (NF-PanNETs). METHODS: Tumor tissues from the 109-patient Fudan cohort and a 73-patient external validation set were evaluated by immunohistochemistry for 9 immune cell types: tumor-infiltrating neutrophils (TINs), tumor-associated macrophages (TAMs), CD11c+ dendritic cells, anti-NCR1+ natural killer (NK) cells, CD4+ and CD8+ T cells, CD45RO+ memory T cells, FOXP3+ regulatory T cells (Tregs), and CD20+ B cells. RESULTS: TINs were primarily distributed in the intratumoral area, dendritic cells and NK cells were scattered evenly in intratumoral and stromal areas, and Tregs were rarely detected. The remaining 5 cell types were primarily present in peritumoral stroma. Total TINs (P < .001) and TAMs (P = .002) increased as NF-PanNET grade rose. Kaplan-Meier analyses showed that high intratumoral TINs, total TAMs, and stromal CD4+ T-cell infiltration correlated with shorter recurrence-free survival (RFS, P = .010, P = .027, and P = .035, respectively) and overall survival (OS, P = .017, P = .029, and P = .045, respectively). Additionally, high intratumoral CD8+ T cell infiltration correlated with prolonged RFS (P = .039). Multivariate Cox regression demonstrated that intratumoral TINs, World Health Organization (WHO) classification, and eighth edition of the American Joint Committee on Cancer tumor-node-metastasis staging system (AJCC8th TNM) were independent factors for RFS (P = .043, P = .023, and P = .029, respectively), whereas intratumoral TINs and WHO classification were independent factors for OS (P = .010 and P = .007, respectively). Furthermore, the combination of TINs, WHO classification, and AJCC8th TNM remarkably improved prognostic accuracy for RFS. These results have been verified in the external validation set. CONCLUSION: Intratumoral TINs are an independent and unfavorable predictor of postoperative NF-PanNETs. A combination of TINs, WHO classification, and AJCC8th TNM could improve prognostic accuracy for RFS.

High density of CD66b in primary high-grade ovarian cancer independently predicts response to chemotherapy.

PURPOSE: Ovarian carcinoma (OC) is the most lethal female genital cancer. After a primary curative surgical approach followed by chemotherapy, a fraction of the patients recur with chemoresistant disease. Data indicate a favorable therapeutic effect of tumor-infiltrating neutrophils (TIN) in OC. Our aim was to investigate the prognostic role of CD66b expression, corresponding to neutrophilic infiltration for recurrence-free survival (RFS) and overall survival (OS) in patients with OC. METHODS: A collective of 47 primary serous ovarian carcinoma and their matching recurrences were processed and stained with CD66b using immunohistochemistry. Tumors from patients with RFS of more than 6 months were defined as chemosensitive. Statistical analysis of CD66b expression was performed to assess the clinical endpoints. RESULTS: High density of CD66b expressing neutrophils in primary carcinoma was associated with chemosensitivity (p = 0.014) and longer RFS (p = 0.001). Univariate analysis identified high density of CD66b expressing neutrophils as a predictor for favorable RFS (HR 0.41, 95% CI 0.22-0.76, p < 0.005). Residual disease > 2 cm (HR 3.67, 95% CI 1.62-8.31, p < 0.002) and higher number of chemotherapy cycles (HR 1.28, 95% CI 1.05-1.55, p < 0.013) were associated with worse RFS. Multivariate analysis showed that high density of CD66b expressing neutrophils (HR 0.22, 95% CI 0.10-0.48, p < 0.001) and residual disease > 2 cm (HR 3.69, 95% CI 1.43-9.53, p < 0.007) were independent predictors of RFS but had no impact on OS. CONCLUSION: High CD66b neutrophil density in primary high-grade OC predicts good response to initial chemotherapy and longer recurrence-free survival independent of known risk factors.

Cowpea Mosaic Virus Promotes Anti-Tumor Activity and Immune Memory in a Mouse Ovarian Tumor Model.

Cowpea mosaic virus (CPMV) is a promising platform nanotechnology with applications as a cancer therapeutic. To understand the therapeutic potential of CPMV in more detail, its antitumor mechanisms are investigated using a syngeneic immunocompetent murine orthotopic ovarian cancer model (ID8-Defb29/Vegf-A). CPMV treatment in situ promotes tumor regression and prevents tumor recurrence. Although CPMV does not kill tumor cells directly, it promotes an intra-tumoral cytokine response which induces pre-existing myeloid cells to break immunotolerance and initiate antitumor responses. The upregulation of interleukin-6 and interferon-γ as well as the downregulation of IL-10 and transforming growth factor ß are observed, associated with activation and repolarization of tumor-associated macrophages and neutrophils to an anti-tumor phenotype. Furthermore, the in situ administration of CPMV recruits dendritic cells and natural killer cells to the tumor site, and induces the expression of costimulatory molecules on CD11b- myeloid cells. By converting immunosuppressive myeloid cells into potent antigen-presenting cells, in situ CPMV treatment significantly improves effector and memory CD4+ and CD8+ T cell responses and promoted systemic tumor-specific cytotoxic CD8+ T cell activity. CPMV in situ immunotherapy induces significant tumor control in an aggressive ovarian tumor model by coordinating innate and adaptive immune responses involving neutrophils, macrophages, and T cells.

Tumor-infiltrating neutrophils predict therapeutic benefit of tyrosine kinase inhibitors in metastatic renal cell carcinoma.

Tumor-infiltrating neutrophils (TINs) show diverse predictive effects in the context of different cancer types and therapeutic regimens. In this study we investigated their relevance with therapeutic effect of tyrosine kinase inhibitors (TKIs) in metastatic renal cell carcinoma (mRCC). Two independent datasets including 271 mRCC patients treated by TKIs or IL-2/IFN-α based immunotherapy were retrospective included, and TINs were detected by immunohistochemistry. The presence of TINs was observed in 50 (45.0%) samples of the TKI cohort and in 73 (45.6%) samples of the immunotherapy cohort. TINs were associated with shorter overall survival (HR, 1.776; 95%CI, 1.191-2.650; p = 0.004) in the TKI cohort, but not in the immunotherapy cohort (HR, 1.074; 95%CI, 0.767-1.505; p = 0.672). Multivariate Cox analysis confirmed the independent prognostic value of TINs for TKI-treated patients (HR, 2.078, 95%CI, 1.352-3.195; p = 0.001), apart from other parameters. Moreover, survival benefit of TKI therapy was superior to IL-2/IFN-α immunotherapy only among TINs-absent patients (HR, 1.561; 95%CI, 0.927-2.629; p = 0.094). Data mining in the TCGA cohort of renal cell carcinoma revealed the predominant immunosuppressive function of TINs in renal cell carcinoma. The negative correlation between TINs and intratumoral CD8+ T cells was further confirmed in the TKI cohort (p = 0.019), the immunotherapy cohort (p = 0.001) and the TCGA cohort (p < 0.001). In conclusion, the presence of TINs was an independent, unfavorable prognostic factor in TKI-treated mRCC patients. TINs could also predict therapeutic benefit of TKIs over IL-2/IFN-α immunotherapy. These findings should be further confirmed within datasets of clinical trials or prospective observational studies.

The prognostic values of tumor-infiltrating neutrophils, lymphocytes and neutrophil/lymphocyte rates in bladder urothelial cancer.

Tumor-infiltrating neutrophils (TINs) and lymphocytes (TILs) are found to play essential roles in many tumors and associate with the prognosis of patients. But, the prognostic values of TINs, TILs and NLR (neutrophils-lymphocytes ratio) in bladder cancer (BC) are still undefined. The object of our study was to systematically interrogate the associations of these immune cells with clinical outcomes of BC patients. In our study, a total of 102 patients pathologically diagnosed with BC were included. CD66b+ and CD8+ antibodies were used to mark neutrophils and CD8+ lymphocytes by immunohistochemistry. The results found that TINs and NLR were significantly associated with pathological T-stages of tumors (P < 0.01), but TILs were not. And TINs were also related to pathological tumor grades (P = 0.012). Regarding the prognostic values, TINs was related to the high risk of recurrence in non-muscle invasive BC (NMIBC) patients. Elevated TINs and NLR were associated with poor overall survivals of BC patients, whereas higher TILs were related to longer survivals (P < 0.01). Multivariate analysis showed that both of TINs (HR 2.427, 1.024-5.752, P = 0.044) and NLR (HR 3.529, 1.147-10.864, P = 0.028) were independent unfavorable prognosis markers. In conclusion, Tumor infiltrating immune cells, including TINs, TILs and NLR were important markers in predicting the prognosis of bladder cancer patients. TINs and NLR were more likely to be negative predictors, but TILs were favorable in patients with BC.