High Lymphocyte Infiltration Research Articles

Clinical prognosticators and targets in the immune microenvironment of intrahepatic cholangiocarcinoma

ABSTRACT Background Intrahepatic cholangiocarcinoma (ICC) is a disease with poor prognosis and limited therapeutic options. We investigated the tumor immune microenvironment (TIME) to identify predictors of disease outcome and to explore targets for therapeutic modulation. Methods Liver tissue samples were collected during 2008–2019 from patients (n = 139) diagnosed with ICC who underwent curative intent surgery without neoadjuvant chemotherapy. Samples from the discovery cohort (n = 86) were immunohistochemically analyzed on tissue microarrays (TMAs) for the expression of CD68, CD3, CD4, CD8, Foxp3, PD-L1, STAT1, and p-STAT1 in tumor core and stroma areas. Results were digitally analyzed using QuPath software and correlated with clinicopathological characteristics. For validation of TIME-related biomarkers, we performed multiplex imaging mass cytometry (IMC) in a validation cohort (n = 53). Results CD68+ cells were the predominant immune cell type in the TIME of ICC. CD4+high T cell density correlated with better overall survival (OS). Prediction modeling together with validation cohort confirmed relevance of CD4+ cells, PD-L1 expression by immune cells in the stroma and N-stage on overall disease outcome. In turn, IMC analyses revealed that silent CD3+CD4+ clusters inversely impacted survival. Among annotated immune cell clusters, PD-L1 was most relevantly expressed by CD4+FoxP3+ cells. A subset of tumors with high density of immune cells (“hot” cluster) correlated with PD-L1 expression and could identify a group of candidates for immune checkpoint inhibition (ICI). Ultimately, higher levels of STAT1 expression were associated with higher lymphocyte infiltration and PD-L1 expression. Conclusions These results highlight the importance of CD4+ T cells in immune response against ICC. Secondly, a subset of tumors with “hot” TIME represents potential candidates for ICI, while stimulation of STAT1 pathway could be a potential target to turn “cold” into “hot” TIME in ICC.

Predictors of response to immunotherapy in colorectal cancer.

Colorectal cancer (CRC) is a major cause of cancer-related deaths globally. While treatment advancements have improved survival rates, primarily through targeted therapies based on KRAS, NRAS, and BRAF mutations, personalized treatment strategies for CRC remain limited. Immunotherapy, mainly immune checkpoint blockade, has shown efficacy in various cancers but is effective in only a small subset of patients with CRC with deficient mismatch repair (dMMR) proteins or high microsatellite instability (MSI). Recent research has challenged the notion that CRC is immunologically inert, revealing subsets with high immunogenicity and diverse lymphocytic infiltration. Identifying precise biomarkers beyond dMMR and MSI is crucial to expanding immunotherapy benefits. Hence, exploration has extended to various biomarker sources, such as the tumor microenvironment, genomic markers, and gut microbiota. Recent studies have introduced a novel classification system, consensus molecular subtypes, that aids in identifying patients with CRC with an immunogenic profile. These findings underscore the necessity of moving beyond single biomarkers and toward a comprehensive understanding of the immunological landscape in CRC, facilitating the development of more effective, personalized therapies.

Differential analysis of histopathological and genetic markers of cancer aggressiveness, and survival difference in EBV-positive and EBV-negative prostate carcinoma

Several studies have shown an association between prostate carcinoma (PCa) and Epstein-Barr virus (EBV); however, none of the studies so far have identified the histopathological and genetic markers of cancer aggressiveness associated with EBV in PCa tissues. In this study, we used previously characterized EBV-PCR-positive (n = 39) and EBV-negative (n = 60) PCa tissues to perform an IHC-based assessment of key histopathological and molecular markers of PCa aggressiveness (EMT markers, AR expression, perineural invasion, and lymphocytic infiltration characterization). Additionally, we investigated the differential expression of key oncogenes, EMT-associated genes, and PCa-specific oncomiRs, in EBV-positive and -negative tissues, using the qPCR array. Finally, survival benefit analysis was also performed in EBV-positive and EBV-negative PCa patients. The EBV-positive PCa exhibited a higher percentage (80%) of perineural invasion (PNI) compared to EBV-negative PCa (67.3%) samples. Similarly, a higher lymphocytic infiltration was observed in EBV-LMP1-positive PCa samples. The subset characterization of T and B cell lymphocytic infiltration showed a trend of higher intratumoral and tumor stromal lymphocytic infiltration in EBV-negative tissues compared with EBV-positive tissues. The logistic regression analysis showed that EBV-positive status was associated with decreased odds (OR = 0.07; p-value < 0.019) of CD3 intratumoral lymphocytic infiltration in PCa tissues. The analysis of IHC-based expression patterns of EMT markers showed comparable expression of all EMT markers, except vimentin, which showed higher expression in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Furthermore, gene expression analysis showed a statistically significant difference (p < 0.05) in the expression of CDH1, AR, CHEK-2, CDKN-1B, and CDC-20 and oncomiRs miR-126, miR-152-3p, miR-452, miR-145-3p, miR-196a, miR-183-3p, and miR-146b in EBV-positive PCa tissues compared to EBV-negative PCa tissues. Overall, the survival proportion was comparable in both groups. The presence of EBV in the PCa tissues results in an increased expression of certain oncogenes, oncomiRs, and EMT marker (vimentin) and a decrease in CD3 ITL, which may be associated with the aggressive forms of PCa.

Abstract 389: The role of endoplasmic reticulum stress in immune surveillance and effectiveness of immunotherapy in renal and ovarian carcinoma

Abstract In complex tumor-host interactions, the immune response has a dual role, with the capacity to both promote and suppress tumor growth, requiring complex interactions of regulatory molecules within the cellular network of the tumor-immune microenvironment (TIME). Various factors in TIME, such as low pH and hypoxia, cause Endoplasmic Reticulum Stress (ERS). Immune cells exposed to ERS lack antigen presentation, exhibit a suppressor phenotype, and can initiate apoptosis. Therefore, relieving ERS can benefit immune surveillance and response to immunotherapy. Our research is focused on tumor-immune cells interaction, specifically in ovarian and renal cancers. We conducted in vitro co-cultures of these cancer cells with peripheral blood mononuclear cells (PBMC) and manipulated ERS using a well-established ERS inducer tunicamycin and a chemical chaperone tauroursodeoxycholic acid (TUDCA). We then employed various techniques such as MTT, RT-qPCR, western blotting, immunofluorescence staining, scratch wound assay, and 3D co-cultures to analyze cellular viability, gene expression, protein levels of ERS markers (e.g., BiP, CHOP, PERK), immune cell activity (e.g., IL-2, INFγ, FoxP3, PD-1), migration, invasiveness, interaction, spheroid formation, and immune cell infiltration. Furthermore, we examined ERS and immune profiles in tumor samples from patients with ovarian and renal cancer who underwent conventional therapy or immunotherapy.Relieving ERS increased the infiltration of PBMC into tumor spheroids and reduced the expression of BiP and PERK in PBMC. In vitro, the viability of ovarian and renal cancer cells increased when cultured alone after ERS alleviation. However, when co-cultured with PBMC, their viability decreased, indicating enhanced immune cell response. Additionally, the growth and migration of ovarian and renal cancer cells were limited in these co-cultures, along with increased expression of BiP and CHOP and an elevated CHOP/BiP ratio. We connected these findings to the molecular background of ERS in patient samples and observed a positive correlation between a favorable clinical outcome, higher lymphocyte infiltration at the tumor site, and increased levels of BiP and CHOP in cancer cells. We further applied these results to OC and RCC in vivo syngeneic mouse models, currently with very promising outcomes.Based on these results we expect ERS alleviation as a suitable approach to improving the survival and effectiveness of immune cell populations within hostile tumor-induced microenvironment with positive effects on anticancer immunotherapy. Supported by Ministry of Health of the Czech Republic, grant nr. NU21-03-00539, and by the Johannes Amos Comenius Programme, project SALVAGE nr. CZ.02.01.01/00/22_008/0004644. All rights reserved. Citation Format: Marek Svoboda, Lukas Moran, Barbora Vavrusakova, Lenka Krejci, Kamila Souckova, Katerina Vasickova, Milos Holanek. The role of endoplasmic reticulum stress in immune surveillance and effectiveness of immunotherapy in renal and ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 389.

Neutralizing IL-16 enhances the efficacy of targeting Aurora-A therapy in colorectal cancer with high lymphocyte infiltration through restoring anti-tumor immunity

Cancer cells can evade immune elimination by activating immunosuppressive signaling pathways in the tumor microenvironment (TME). Targeting immunosuppressive signaling pathways to promote antitumor immunity has become an attractive strategy for cancer therapy. Aurora-A is a well-known oncoprotein that plays a critical role in tumor progression, and its inhibition is considered a promising strategy for treating cancers. However, targeting Aurora-A has not yet got a breakthrough in clinical trials. Recent reports have indicated that inhibition of oncoproteins may reduce antitumor immunity, but the role of tumor-intrinsic Aurora-A in regulating antitumor immunity remains unclear. In this study, we demonstrated that in tumors with high lymphocyte infiltration (hot tumors), higher tumor-intrinsic Aurora-A expression is associated with a better prognosis in CRC patients. Mechanically, tumor-intrinsic Aurora-A promotes the cytotoxic activity of CD8+ T cells in immune hot CRC via negatively regulating interleukin-16 (IL-16), and the upregulation of IL-16 may impair the therapeutic effect of Aurora-A inhibition. Consequently, combination treatment with IL-16 neutralization improves the therapeutic response to Aurora-A inhibitors in immune hot CRC tumors. Our study provides evidence that tumor-intrinsic Aurora-A contributes to anti-tumor immunity depending on the status of lymphocyte infiltration, highlighting the importance of considering this aspect in cancer therapy targeting Aurora-A. Importantly, our results suggest that combining Aurora-A inhibitors with IL-16-neutralizing antibodies may represent a novel and effective approach for cancer therapy, particularly in tumors with high levels of lymphocyte infiltration.

Effects of tumour budding on adjuvant chemotherapy in colorectal cancer.

High tumour budding has been indicated as a risk factor of poor survival in colorectal cancer. This study aimed to investigate the impact of tumour budding grades and the use of adjuvant chemotherapy on prognosis in patients with colorectal cancer. This study included consecutive colorectal cancer patients who underwent radical surgery for primary colorectal adenocarcinoma at The Sixth Hospital of Sun Yat-sen University between 2009 and 2019. Tumour budding was assessed based on the recommendations of the International Tumor Budding Consensus Conference using haematoxylin and eosin (H&E)-stained slides with tumour samples. The primary outcome of interest was to correlate tumour budding with disease-free survival and overall survival; the secondary outcome was investigation of the impact of adjuvant therapy on different tumour budding grades. In addition, a subgroup analysis was performed for the effects of lymphocytic infiltration on adjuvant chemotherapy in patients with Bd3. Of 709 eligible patients, 412 with colorectal cancer were included. According to the International Tumor Budding Consensus Conference, 210 (50.9 per cent), 127 (30.8 per cent) and 75 (18.2 per cent) were classified as low budding (Bd1), intermediate budding (Bd2) and high budding (Bd3) respectively. Patients with Bd1, Bd2 and Bd3 had 5-year disease-free survival rates of 82.9 per cent, 70.1 per cent and 49.3 per cent respectively, and 5-year overall survival rates of 90 per cent, 79.5 per cent and 62.7 per cent respectively (P <0.001). Adjuvant chemotherapy yielded a significant survival benefit in patients with Bd3 (5-year disease-free survival, 65 per cent versus 31.4 per cent, P <0.001; 5-year overall survival, 84.4 per cent versus 63.1 per cent, P <0.001), but not in those with Bd1 or Bd2. In patients with Bd3, the benefit of adjuvant chemotherapy was maintained in those with low, but not high lymphocytic infiltration. High grade of tumour budding was strongly correlated with poorer survival outcomes in colorectal cancer. Patients with Bd3 benefited from adjuvant chemotherapy, with the exclusion of patients with high lymphocytic infiltration.

Low CDKN1B Expression Associated with Reduced CD8+ T Lymphocytes Predicts Poor Outcome in Breast Cancer in a Machine Learning Analysis.

The cyclin-dependent kinase inhibitor 1B (CDKN1B) gene, which encodes the p27Kip1 protein, is important in regulating the cell cycle process and cell proliferation. Its role in breast cancer prognosis is controversial. We evaluated the significance and predictive role of CDKN1B expression in breast cancer prognosis. We investigated the clinicopathologic factors, survival rates, immune cells, gene sets, and prognostic models according to CDKN1B expression in 3794 breast cancer patients. We performed gene set enrichment analysis (GSEA), in silico cytometry, pathway network analyses, gradient boosting machine (GBM) learning, and in vitro drug screening. High CDKN1B expression levels in breast cancer correlated with high lymphocyte infiltration signature scores and increased CD8+ T cells, both of which were associated with improved prognosis in breast cancer. which were associated with a better prognosis. CDKN1B expression was associated with gene sets for the upregulation of T-cell receptor signaling pathways and downregulation of CD8+ T cells. Pathway network analysis revealed a direct link between CDKN1B and the pathway involved in the positive regulation of the protein catabolic process pathway. In addition, an indirect link was identified between CDKN1B and the T-cell receptor signaling pathway. In in vitro drug screening, BMS-345541 demonstrated efficacy as a therapeutic targeting of CDKN1B, effectively impeding the growth of breast cancer cells characterized by low CDKN1B expression. The inclusion of CDKN1B expression in GBM models increased the accuracy of survival predictions. CDKN1B expression plays a significant role in breast cancer progression, implying that targeting CDKN1B might be a promising strategy for treating breast cancer.

Clinicopathological analysis of CD47 and signal regulatory protein alpha expression in myeloid sarcoma patients: CD47 expression is a favourable prognostic factor

Myeloid sarcoma is a rare extramedullary haematopoietic malignancy. Interaction between CD47 and signal regulatory protein α (SIRPα) inhibits phagocytosis. CD47-positive tumours confer poor prognoses in various malignant tumours, including acute myeloid leukaemia. This study aimed to investigate the clinicopathological effects of CD47 and SIRPα expression in myeloid sarcoma. Immunohistochemistry (IHC) of CD47 and SIRPα was performed in 84 biopsy samples obtained from patients with myeloid sarcoma, some of which were CD47-positive. Patients were categorised into the following two groups based on IHC of SIRPα: those with SIRPα-positive neoplastic cells (nSIRPα) and, SIRPα expression on non-neoplastic stromal cells in tumour microenvironment (miSIRPα). In addition, patients with CD47 positivity had higher lymphocytic infiltration into the tumour microenvironment. Overall, these patients had significantly higher overall survival, however, no significant difference was observed in progression-free survival. No significant prognostic differences were observed between the nSIRPα and miSIRPα groups. This is the first study to demonstrate an association between CD47 expression and improved prognosis in myeloid sarcoma. Nonetheless, it will be necessary to conduct additional research on gene expression and genomic abnormalities to elucidate the corresponding pathogenesis of myeloid sarcoma.

Diagnosis and prognosis of pancreatic cancer with immunoglobulin heavy constant delta blood marker.

Pancreatic cancer (PC) is highly malignant and difficult to detect, while few blood markers are currently available for diagnosing PC. We obtained differential expression genes (DEGs) from GEO (gene expression omnibus) database and assessed by quantitative real-time polymerase chain reaction (qRT-PCR), receiver operating characteristic (ROC), univariate and multifactorial regression analysis, and survival analysis in our clinic center. Through the TCGA (the cancer genome atlas) database, we analyzed functional enrichment, different risk groups with survival analysis, immunological features, and the risk score established by the Cox regression model and constructed a nomogram. Immunoglobulin heavy constant delta (IGHD) was remarkably upregulated in peripheral blood from PC patients, and IGHD was a potential independent biomarker for PC diagnosis (ROC sensitivity, 76.0%; specificity, 74.2%; area under the curve (AUC) = 0.817; univariate logistic regression analysis: odds ratio (OR) 1.488; 95% confidence interval (CI) 1.182-1.872; P < 0.001; multiple logistic: OR 2.097; 95% CI 1.276-3.389, P = 0.003). In addition, the IGHD expression was remarkably reduced after resectioning the primary tumor. High IGHD expression indicated higher lymphocyte infiltration and increased activities of immunological pathways in PC patients. KRAS and SMAD were observed with a prominent difference among top mutated genes between the two groups. The risk score predicted reliable clinical prognosis and drug responses. Furthermore, a nomogram with the risk score and clinical characteristics was constructed, showing a better predictive performance. IGHD is a valuable PC diagnosis, prognosis, and therapeutic response marker.

Regorafenib induces damage-associated molecular patterns, cancer cell death and immune modulatory effects in a murine triple negative breast cancer model

Damage associated molecular patterns (DAMPs), including calreticulin (CRT) exposure, high-mobility group box 1 protein (HMGB1) elevation, and ATP release, characterize immunogenic cell death (ICD) and may play a role in cancer immunotherapy. Triple negative breast cancer (TNBC) is an immunogenic subtype of breast cancer with higher lymphocyte infiltration. Here, we found that regorafenib, a multi-target angiokinase inhibitor previously known to suppress STAT3 signaling, induced DAMPs and cell death in TNBC cells. Regorafenib induced the expression of HMGB1 and CRT, and the release of ATP. Regorafenib-induced HMGB1 and CRT were attenuated following STAT3 overexpression. In a 4T1 syngeneic murine model, regorafenib treatment increased HMGB1 and CRT expression in xenografts, and effectively suppressed 4T1 tumor growth. Immunohistochemical staining revealed increased CD4+ and CD8+ tumor-infiltrating T cells in 4T1 xenografts following regorafenib treatment. Regorafenib treatment or programmed death-1 (PD-1) blockade using anti-PD-1 monoclonal antibody reduced lung metastasis of 4T1 cells in immunocompetent mice. While regorafenib increases the proportion of MHC II high expression on dendritic cells in mice with smaller tumors, the combination of regorafenib and PD-1 blockade did not show a synergistic effect on anti-tumor activity. These results suggest that regorafenib induces ICD and suppresses tumor progression in TNBC. It should be carefully evaluated when developing a combination therapy with an anti-PD-1 antibody and a STAT3 inhibitor.

ALOX5AP is an Indicator for High CD8 Lymphocyte Infiltration and "Hot" Tumor Microenvironment in Osteosarcoma: A Bioinformatic Study.

Little progress has been made in the treatment and prognosis of osteosarcoma in the past 40years. Tumor microenvironment (TME) plays a critical role in the progression of osteosarcoma. This study aims to determine immune-associated prognostic biomarkers for osteosarcoma patients. With the help of analytical tools including ESTIMATE, differential gene expression, LASSO, and univariate cox and multivariate cox regression analysis, osteosarcoma gene expression data from Gene Expression Omnibus (GEO) databases were investigated. Following the establishment of a prognostic risk score model, internal and external validations using the GEO and TARGET databases were carried out. A total of 44 and 55 samples respectively in the GSE21257 and the TARGET databases were included. Our analysis found 93 differentially expressed genes (DEGs) between the high and low-ImmuneScore groups. Through univariate cox and LASSO analysis, ALOX5AP was identified as an indicator of TME in osteosarcomas. ALOX5AP was then used to construct a prognostic risk model. Internal and external verification revealed that higher expression of ALOX5AP was correlated with lower risk. Through the CIBERSORT algorithm, the level of CD8 T cells was found to negatively correlate with the risk score. This study revealed that ALOX5AP is an indicator for predicting high CD8 lymphocyte infiltration and "hot" tumor microenvironment in osteosarcomas. Thus, ALOX5AP has the potential to act as a biomarker for effective immunotherapies in osteosarcoma patients.

Abstract 5169: Aurora-A kinase promotes anti-tumor immunity in high lymphocyte-infiltrated colorectal cancer

Abstract Introduction: The tumor microenvironment (TME) is composed of different cell types and is involved in tumor progression. Several immunosuppressive signaling pathways can promote tumor growth by inhibiting anti-tumor immunity in TEM. Therefore, targeting the immunosuppressive signaling pathways is a potential cancer treatment. Recent studies reported that inhibition of oncoproteins could lead to reduced anti-tumor immunity in several cancers; therefore, it’s critical to identify the potential oncoproteins which can lead to enhanced anti-tumor immunity. Aurora-A is a serine/threonine kinase that plays an important role in the centrosome, cell mitosis, and chromosome stability. Previous studies indicated that cancer patients with poor prognoses are associated with a high Aurora-A level in tumor cells. Inhibition of Aurora-A is considered as a promising strategy for cancer treatment. However, Aurora-A inhibitors have not had a breakthrough in phase II or phase III clinical trials. The role of Aurora-A in tumor immunity is still unclear. Here, we investigated the role of Aurora-A in tumor immunity. Materials and Methods: shRNA was used to knock down Aurora-A, and Alisertib was added to inhibit Aurora-A. RT-qPCR and western blot were performed to determine the IL-16 expression. The secretion of cytokines was measured by cytokine array and ELISA. BALB/c and NOD/SCID mice were subcutaneously injected with CT26 and then administered daily with Alisertib or treated with anti-IL-16 antibody. The tumor-infiltrating immune populations were analyzed by flow cytometry. IHC and H/E stain were performed to analyze Aurora-A expression and lymphocyte infiltration in human CRC specimens. Results: CRC patients with lower lymphocyte infiltration and higher Aurora-A are associated with poor prognosis and vice versa. Knockdown of Aurora-A in CT26 promotes tumor growth in immunocompetent mice by inhibiting the infiltration and cytotoxic activity of CD8+ T cells. Aurora-A negatively regulates IL-16 expression in a kinase-dependent manner in CRC cells. Blockade of IL-16 by anti-IL-16 antibody can reverse the tumor growth and CD8+ T cell cytotoxic activity in Aurora-A knocked-down CT26 tumors. Alisertib can inhibit CT26 tumor growth and promote IL-16 secretion in TME in mice. A combination of Alisertib and IL-16 antibody can promote the therapeutic effect of Alisertib in CT26 tumors. Conclusion: Tumor-intrinsic Aurora-A plays a diverse role in tumor progression depending on the status of lymphocyte infiltration in TME. Aurora-A can promote anti-tumor immunity via negatively regulating IL-16 in a kinase activity-dependent manner in hot CRC tumors. In tumors with high lymphocyte infiltration, Aurora-A inhibition can suppress cancer cell growth and reduce anti-tumor immunity; the combination of Aurora-A inhibitors and IL-16 antibodies may provide a novel and effective strategy for cancer therapy in hot tumors. Citation Format: Shiang-Jie Yang, Ming-Derg Lai, Liang-Yi Hung. Aurora-A kinase promotes anti-tumor immunity in high lymphocyte-infiltrated colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5169.

EHBP1L1 Drives Immune Evasion in Renal Cell Carcinoma through Binding and Stabilizing JAK1.

High lymphocyte infiltration and immunosuppression characterize the tumor microenvironment (TME) in renal cell carcinoma (RCC). There is an urgent need to elucidate how tumor cells escape the immune attack and to develop novel therapeutic targets to enhance the efficacy of immune checkpoint blockade (ICB) in RCC. Overactivated IFN-γ-induced JAK/STAT signaling involves in such TME, but the underlying mechanisms remain elusive. Here, EH domain-binding protein 1-like protein 1 (EHBP1L1) is identified as a crucial mediator of IFN-γ/JAK1/STAT1/PD-L1 signaling in RCC. EHBP1L1 is highly expressed in RCC, and high EHBP1L1 expression levels are correlated with poor prognosis and resistance to ICB. EHBP1L1 depletion significantly inhibits tumor growth, which is attributed to enhanced CD8+ T cell-mediated antitumor immunity. Mechanistically, EHBP1L1 interacts with and stabilizes JAK1. By competing with SOCS1, EHBP1L1 protects JAK1 from proteasomal degradation, which leads to elevated JAK1 protein levels and JAK1/STAT1/PD-L1 signaling activity, thereby forming an immunosuppressive TME. Furthermore, the combination of EHBP1L1 inhibition and ICB reprograms the immunosuppressive TME and prevents tumor immune evasion, thus significantly reinforcing the therapeutic efficacy of ICB in RCC patient-derived xenograft (PDX) models. These findings reveal the vital role of EHBP1L1 in immune evasion in RCC, which may be a potential complement for ICB therapy.

IL-2/GM-CSF enhances CXCR3 expression in CAR-T cells via the PI3K/AKT and ERK1/2 pathways.

To investigate the effects of cytokines IL-2 and GM-CSF on CXCR3 expression and chemotaxis of CAR-T cells. High lymphocyte infiltration within the tumor is a basic requirement for good results in tumor immunotherapy; C-X-C motif chemokine receptor 3 (CXCR3) is an important factor for the chemotaxis of lymphocytes to tumor tissues. The tumor microenvironment can exhibit diverse cytokine suppression or promote antitumor immunity. Both interleukin (IL)-2 and granulocyte macrophage colony-stimulating factor (GM-CSF) contribute to the regulation of immunosuppression in the tumor microenvironment. However, the effects of IL-2 and GM-CSF on CXCR3 expression on the T cell surface and its mechanisms are not well understood. Here, we explored the effects of polycytokines on CXCR3 expression in chimeric antigen receptor T cells (CAR-T cells) and on HuH-7 in situ hepatocellular carcinoma. Peripheral blood mononuclear cells (PBMCs) were isolated, followed by purifying using CD3 immunomagnetic beads. Cells were divided into three groups. After 24h of activation using CD3/CD28 antibody, T cells were transfected using lentiviral vector, pGC-SV40-EGFP-GPC3-CAR. Three culture methods were used to amplify the transfected T cells. Method 'A' was to incubate T cells with CD3/CD28 antibody; method 'B' was with CD3/CD28 antibody and IL-2 at a final concentration of 1000 U/ml; method 'C' was with method B in addition of GM-CSF at a final concentration of 1000 U/ml. The phosphorylation of MAPK and PI3K/AKT was determined by western blot. The chemotaxis effect of CAR-T cells on Huh-7 HCCIA in situ was assayed by immunofluorescence and immunohistochemistry. The CD3/CD28/IL-2/GM-CSF combination is the most potent for stimulating activated CAR-T cell proliferation and CXCR3 expression in vitro; CD3/CD28/IL-2 induces CAR-T cell expression of CXCR3 through the activation of the PI3K/APK pathway and GM-CSF induces CXCR3 expression in CAR-T cells through the activation of ERK1/2 rather than the p38 MAPK signaling pathway. CAR-GPC3-T cells with high CXCR3 expression showed increased chemotaxis ability to HuH in situ hepatocellular carcinoma, and considerably inhibited the growth of in situ tumors in nude mouse livers. A multi-factorial amplification protocol can effectively improve CXCR3 expression on the surface of activated CAR-T cells in vitro, as well as enhance the chemotaxis ability of CAR-T cells in vivo, which significantly inhibit the growth of liver cancer.

CEBPB is associated with active tumor immune environment and favorable prognosis of metastatic skin cutaneous melanoma.

Metastatic skin cutaneous melanoma (SKCM) is a common malignancy that accounts for low morbidity but high mortality of skin cancer. SKCM is characterized by high lymphocytic infiltration, whereas the states of infiltrated cells are variable in patients leading to a heterogeneous prognosis and hindering appropriate clinical decisions. It is therefore urgent to identify markers associated with lymphocytic infiltration, cellular conditions, and the prognosis of SKCM. In this study, we report that CEBPB, a transcriptional factor, is mainly expressed in macrophages in metastatic SKCM and associated with an active tumor immune environment and a favorable prognosis through integrated analysis of single-cell and bulk RNA-seq datasets. High CEBPB expression is significantly associated with active inflammation and immune response pathways in both macrophages and bulk SKCM tumor tissues. A signature based on CEBPB-associated genes that are specifically expressed in macrophages could robustly and prognostically separate different metastatic SKCM patients. In addition, the associations between the metastatic SKCM tumor signature and microenvironment with respect to T-cell recruitment and state, inflammation response, angiogenesis, and so on were also determined. In conclusion, we present here the first report on CEBPB in tumor immune environment and prognosis regulation in metastatic SKCM and construct a reliable signature, which should provide a useful biomarker for stratification of the patient's prognosis and therapeutic selection.

Transcriptional analysis highlights three distinct immune profiles of high-risk oral epithelial dysplasia.

Oral potentially malignant disorders (OPMD) are precursors of oral squamous cell carcinoma (OSCC), and the presence of oral epithelial dysplasia (OED) in OPMD confers an increased risk of malignant transformation. Emerging evidence has indicated a role for the immune system in OPMD disease progression; however, the underlying immune mechanisms remain elusive. In this study, we used immune signatures established from cancer to delineate the immune profiles of moderate and severe OED, which are considered high-risk OPMD. We demonstrated that moderate and severe OEDs exhibit high lymphocyte infiltration and upregulation of genes involved in both immune surveillance (major histocompatibility complex-I, T cells, B cells and cytolytic activity) and immune suppression (immune checkpoints, T regulatory cells, and tumor-associated macrophages). Notably, we identified three distinct subtypes of moderate and severe OED: immune cytotoxic, non-cytotoxic and non-immune reactive. Active immune surveillance is present in the immune cytotoxic subtype, whereas the non-cytotoxic subtype lacks CD8 immune cytotoxic response. The non-immune reactive subtype showed upregulation of genes involved in the stromal microenvironment and cell cycle. The lack of T cell infiltration and activation in the non-immune reactive subtype is due to the dysregulation of CTNNB1, PTEN and JAK2. This work suggests that moderate and severe OED that harbor the non-cytotoxic or non-immune reactive subtype are likely to progress to cancer. Overall, we showed that distinct immune responses are present in high-risk OPMD, and revealed targetable pathways that could lead to potential new approaches for non-surgical management of OED.

Immunotherapeutic Strategies for Head and Neck Squamous Cell Carcinoma (HNSCC): Current Perspectives and Future Prospects.

Neoantigens are abnormal proteins produced by genetic mutations in somatic cells. Because tumour neoantigens are expressed only in tumour cells and have immunogenicity, they may represent specific targets for precision immunotherapy. With the reduction in sequencing cost, continuous advances in artificial intelligence technology and an increased understanding of tumour immunity, neoantigen vaccines and adoptive cell therapy (ACT) targeting neoantigens have become research hotspots. Approximately 900,000 patients worldwide are diagnosed with head and neck squamous cell carcinoma (HNSCC) each year. Due to its high mutagenicity and abundant lymphocyte infiltration, HNSCC naturally generates a variety of potential new antigen targets that may be used for HNSCC immunotherapies. Currently, the main immunotherapy for HNSCC is use of immune checkpoint inhibitors(ICIs). Neoantigen vaccines and adoptive cell therapy targeting neoantigens are extensions of immunotherapy for HNSCC, and a large number of early clinical trials are underway in combination with immune checkpoint inhibitors for the treatment of recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC). In this paper, we review recent neoantigen vaccine trials related to the treatment of HNSCC, introduce adoptive cell therapy targeting neoantigens, and propose a potential treatment for HNSCC. The clinical application of immune checkpoint inhibitor therapy and its combination with neoantigen vaccines in the treatment of HNSCC are summarized, and the prospect of using neoantigen to treat HNSCC is discussed and proposed.

Tumor-infiltrating lymphocytes predict survival in ≥ pT2 urothelial bladder cancer

Tumor-infiltrating lymphocytes (TILs) are associated with improved survival in several types of cancers, including genitourinary cancers. However, multiple different scoring methods used to assess TILs complicate the comparison of different studies and are not always suitable for daily practice. In 2014, the International TILs Working Group (ITWG) proposed a simple and robust assessment method for a more standardized evaluation of TILs. Here, we validated this system in muscle-invasive urinary bladder cancer (MIBC). Patient history and histologic specimens from 203 patients with MIBC were retrospectively analyzed. The stromal TIL (sTIL) score was determined using the ITWG system and 3 groups were defined according to the degree of stromal lymphocytic infiltration: low (0–10%), intermediate (10–55%) and high (55–100%). Associations between sTIL score, clinicopathological variables, tumor-specific survival (TSS), overall survival (OS), and disease-free survival (DFS) were analyzed. High stromal lymphocytic infiltration was associated with significantly higher OS, TSS and DFS when compared to low grade sTILs. The survival benefit remained statistically significant in multivariate analyses, confirming that sTILs are a strong independent positive prognostic factor in patients with MIBC. In summary, the degree of sTILs as defined by the ITWG robustly predicts survival in MIBC patients. Prospective studies with larger case numbers are needed to determine whether sTILs should be included in staging guidelines and how they could aid in therapeutic decision making.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Abstract CT529: A Phase 1b Study of CG0070 combined with nivolumab in cisplatin ineligible patients with muscle invasive bladder cancer

Abstract Background: Up to 50% of patients with muscle invasive bladder cancer (MIBC) are unable to receive neoadjuvant chemotherapy, creating an unmet clinical need for alternative systemic treatment. Results from recent neoadjuvant immune checkpoint blockade (ICB) studies signal pCR rates between 31-42%. However, there was a clear dichotomy in response between patients with high vs. low pre-treatment lymphocytic infiltration. In tumors exhibiting immune excluded or immune desert phenotypes, therapeutic strategies to attract CD8+ T cells infiltration prior to ICB may be beneficial. CG0070 is an oncolytic adenovirus designed to preferentially replicate through a transcriptionally regulated promoter that is up-regulated in RB-pathway-defective cells (&amp;gt;70% of MIBC). In addition, CG0070 expresses GM-CSF to attract antigen presenting cells to the tumor microenvironment. We hypothesize that combination therapy using intravesical CG0070 and systemic Nivolumab is 1) safe and 2) can enhance local and systemic treatment response in the neoadjuvant setting against MIBC. Methods: This is a phase Ib trial evaluating 2 doses of nivolumab (4 weeks apart) with 6 weekly intravesical CG0070 followed by cystectomy. Dose-limiting toxicities (DLTs) will be assessed in 6 DLT-evaluable patients in a safety lead-in phase. The combination would be declared tolerable if the incidence of DLTs is &amp;lt;33%, and the study expanded to 30 patients. Key eligibility: cT2-T4aN0-1M0 stage, no autoimmume disease within 2 years, and no concurrent invasive upper tract carcinoma or other active cancer. The primary objective is to evaluate the safety of combination therapy according to CTCAE v5.0. The secondary objective is to assess the changes in the lymphocytic infiltration following combination therapy and to assess the antitumor efficacy of combination therapy. Exploratory endpoints include assessing the predictive value of E2F expression, tumor mutational burden, immune infiltration, and various peripheral blood for treatment response. Study Progress: Study is actively accruing. Clinical trial information: NCT04610671. An update on the study will be provided at the time of presentation. Citation Format: Roger Li, Wade Sexton, Phillipe Spiess, Scott Gilbert, Julio Pow-Sang, Jingsong Zhang, Gustavo Borjas, Paola Grandi, James Burke, Rohit Jain, Jose Conejo-Garcia. A Phase 1b Study of CG0070 combined with nivolumab in cisplatin ineligible patients with muscle invasive bladder cancer [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 CT529.