High Immune Cell Infiltration Research Articles

Siglec-7 and Siglec-9 expression in primary triple negative and oestrogen receptor positive breast cancer and in vitro signalling.

PD-1/PD-L1 immune checkpoint blockade can be an effective treatment for advanced breast cancer patients. However, patients with oestrogen receptor positive (ER+) tumors often display only low lymphocyte infiltration, while a large part of triple negative (TN) breast tumors does not generate an effective immunotherapy response. Therefore, new treatment strategies have to be developed. Here, we investigate Siglec-7 and Siglec-9 as novel ITIM-bearing inhibitory immune checkpoint receptors similar to PD-1, but expressed on a broader range of immune cells. We assessed Siglec-7 and Siglec-9 (ligand) expression in TN and ER+ breast cancer tumors and their breast cancer cell line-induced signalling. We report that Siglec-7 and Siglec-9 are highly expressed in TN tumors, and to a low extent in ER+ tumors. Siglec-7 was observed on myeloid cells, Tcells, and NKcells and Siglec-9 preferentially on myeloid cells. Expression of sialoglycans, including Siglec-7 and Siglec-9 ligands, was observed in both TN and ER+ breast cancer tissue sections. Expression levels of Siglec-7 and Siglec-9 ligands were higher on in vitro cultured TN cell lines than ER+ cell lines. Importantly, by applying chimeric Siglec-7 reporter cells, we showed the induction of Siglec-7 signalling by multiple TN cell lines, but only by one ER+ cell line. Moreover, Siglec-7 signalling is directly related to Siglec-7 ligand expression levels of breast cancer cell lines. These data imply that immunotherapy targeting Siglec receptors may be particularly interesting for TN breast cancer patients not responding to current treatment strategies with tumors displaying high immune cell infiltration.

High infiltration of immune cells with lower immune activity mediated the heterogeneity of gastric adenocarcinoma and promoted metastasis

BackgroundGastric adenocarcinoma (GA) is a heterogeneous malignancy with high invasion and metastasis. We aimed to explore the metastatic characteristics of GA using single-cell RNA-sequencing (scRNA-seq) analysis. MethodsThe scRNA-seq dataset was downloaded from the GEO database and the “Seurat” package was used to perform the scRNA-seq analysis. The CellMarker2.0 database provided gene markers. Subsequently, differentially expressed genes (DEGs) were identified using the FindMarkers function and subjected to enrichment analysis with the “ClusterProlifer”. “GseaVis” package was used for visualizing the gene levels. Finally, the SCENIC analysis was performed for identifying key regulons. The expression level and functionality of the key genes were verified by quantitative real-time PCR (qRT-PCR), wound healing and transwell assays. ResultsA total of 7697 cells were divided into 8 cell subsets, in which the Cytotoxic NK/T cells, Myeloid cells and Myofibroblasts had higher proportion in the metastatic tissues. Further screening of DEGs and enrichment analysis revealed that in the metastatic tissues, NK cells, monocytes and inflammatory fibroblasts with low immune levels contributed to GA metastasis. In addition, this study identified a series of key immune-related regulons that mediated the lower immune activity of immune cells. Further in vitro experiment verified that CXCL8 was a key factor mediating the proliferation and migration of GA cells. ConclusionThe scRNA-seq analysis showed that high infiltration of immune cells with lower immune activity mediated heterogeneity to contribute to GA metastasis.

Natural killer cell-associated prognosis model characterizes immune landscape and treatment efficacy of diffuse large B cell lymphoma

PurposeNK cells are essential for the detection, identification and prediction of cancer. However, so far, there is no prognostic risk model based on NK cell-related genes to predict the prognosis and treatment outcome of DLBCL patients. This study aimed to explore a risk assessment model that could accurately predict the prognosis and treatment efficacy of DLBCL. MethodsBioinformatics analysis of the expression profiles of DLBCL samples in the GEO database was performed. Cox regression and LASSO regression analysis were used to determine NK cell-related genes associated with patient’s prognosis. Based on these genes, a risk assessment model was constructed to predict the prognosis of patients and the effectiveness of treatment. Finally, qRT-PCR was used to verify the expression of gene tags in clinical samples. ResultsWe identified seven prognosis-related NK cell-related genes (MAP2K1, PRKCB, TNFRSF10B, IL18, LAMP1, RASGRP1, and SP110), and DLBCL patients were divided into low- and high-risk groups based on these genes. Survival analysis showed that the prognosis of patients with low-risk group was better. Pathway enrichment analysis showed that the differentially expressed genes between the two risk groups were related to immune response pathways. Compared with the high-risk group, the low-risk group had higher infiltration of immune cells in tumor tissues. Besides, compared with high-risk group, low-risk patients by immunotherapy or other commonly used anti-tumor drugs might have better efficacy after treatment. In addition, qRT-PCR showed that the expression of risk genes including TNFRSF10B, IL18 and LAMP1 were significantly increased in most DLBCL samples compared to control samples, while the expression of protective genes including MAP2K1, PRKCB, RASGRP1 and SP110 were significantly decreased. ConclusionThe NK cell-related gene signatures were proved to be a reliable indicator of the success of immunotherapy in patients with DLBCL, thus providing a unique evaluation method.

A diagnostic signature developed based on the necroptosis-related genes and its association with immune infiltration in osteosarcoma

IntroductionOsteosarcoma is a bone-derived malignancy that often leads to lung metastasis and death. Material and methodsThe RNA-seq data of TARGET-osteosarcoma were collected from TARGET database. GSE16088 and GSE12865 datasets of osteosarcoma x from Gene Expression Database (GEO) were donwloaded. ConsensusClusterPlus was used for molecular subtype classification. Univariate Cox and Lasso regression was employed to develop a risk model. To analyze the regulatory effects of model feature genes on the malignant phenotype of osteosarcoma cell lines, qRT-PCR, Transwell and wound healing assays were performed. The abundance of immune cell infiltration was assessed using MCP-Counter, Gene Set Enrichment Analysis (GSEA), and ESTIMATE. The Tumor Immune Dysfunction and Exclusion (TIDE) software was employed to evaluate immunotherapy and response to conventional chemotherapy drugs. ResultsThree clusters (C1, C2 and C3) were classified using 39 necroptosis score-associated genes. In general, C1 and C2 showed better prognosis outcome and lower death rate than C3. Specifically, C2 could benefit more from immunotherapy, while C3 was more sensitive to traditional medicines, and C1 had higher immune cell infiltration. Next, an 8-gene signature and a risk score model were developed, with a low risk score indicating better survival and immune cell infiltration. ROC analysis showed that 1-, 3-, and 5-year overall survival of osteosarcoma could be correctly predicted by the risk score model. Cellular experiments revealed that the model feature gene IFITM3 promoted the osteosarcoma cell migration and invasion. Furthermore, the overall survival of osteosarcoma patients from TARGET and validation datasets can be accurately evaluated using the nomogram model. ConclusionsOur prognostic model developed using necroptosis genes could facilitate the prognostic prediction for patients suffering from osteosarcoma, offering potential osteosarcoma targets.

A Mature Tertiary Lymphoid Structure with a Ki-67-Positive Proliferating Germinal Center Is Associated with a Good Prognosis and High Intratumoral Immune Cell Infiltration in Advanced Colorectal Cancer.

Tertiary lymphoid structures (TLSs) are complex lymphocyte clusters that arise in non-lymphoid tissues due to inflammation or cancer. A mature TLS with proliferating germinal centers is associated with a favorable prognosis in various cancers. However, the effect of TLS maturity on advanced colorectal cancer (CRC) remains unexplored. We analyzed the significance of TLS maturity and tumor Ki-67 expression in surgically resected tumors from 78 patients with pathological T4 CRC. Mature TLS was defined as the organized infiltration of T and B cells with Ki-67-positive proliferating germinal centers. We analyzed the relationship between TLS maturity and intratumoral immune cell infiltration. Mature TLS with germinal center Ki-67 expression was associated with microsatellite instability and improved survival; however, high tumor Ki-67 expression was associated with poor survival in the same cohort. Multivariate analysis identified the absence of mature TLS as an independent predictor of poor post-recurrence overall survival. Intratumoral infiltration of T lymphocytes and macrophages was significantly elevated in tumors with mature TLS compared to those lacking it. High Ki-67 levels and absent mature TLS were identified as poor prognostic factors in advanced CRC. Mature TLS could serve as a promising marker for patients at high-risk of CRC.

Multi-Omics analysis elucidates tumor microenvironment and intratumor microbes of angiogenesis subtypes in colon cancer.

Angiogenesis plays an important role in colon cancer (CC) progression. To investigate the tumor microenvironment (TME) and intratumor microbes of angiogenesis subtypes (AGSs) and explore potential targets for antiangiogenic therapy in CC. The data were obtained from The Cancer Genome Atlas database and Gene Expression Omnibus database. K-means clustering was used to construct the AGSs. The prognostic model was constructed based on the differential genes between two subtypes. Single-cell analysis was used to analyze the expression level of SLC2A3 on different cells in CC, which was validated by immunofluorescence. Its biological functions were further explored in HUVECs. CC samples were grouped into two AGSs (AGS-A and AGS-B) groups and patients in the AGS-B group had poor prognosis. Further analysis revealed that the AGS-B group had high infiltration of TME immune cells, but also exhibited high immune escape. The intratumor microbes were also different between the two subtypes. A convenient 6-gene angiogenesis-related signature (ARS), was established to identify AGSs and predict the prognosis in CC patients. SLC2A3 was selected as the representative gene of ARS, which was higher expressed in endothelial cells and promoted the migration of HUVECs. Our study identified two AGSs with distinct prognoses, TME, and intratumor microbial compositions, which could provide potential explanations for the impact on the prognosis of CC. The reliable ARS model was further constructed, which could guide the personalized treatment. The SLC2A3 might be a potential target for antiangiogenic therapy.

A hot and cold tumor‑related prognostic signature for stage II colorectal cancer.

Globally, colorectal cancer (CRC) is one of the most lethal and prevalent malignancies. Based on the presence of immune cell infiltration in the tumor microenvironment, CRC can be divided into immunologically 'hot' or 'cold' tumors, which in turn leads to the differential efficacy of immunotherapy. However, the immune characteristics of hot and cold CRC tumors remain largely elusive, prompting further investigation of their properties regarding the tumor microenvironment. In the present study, a predictive model was developed based on the differential expression of proteins between cold and hot CRC tumors. First, the differentially expressed proteins (DEPs) were identified using digital spatial profiling and mass spectrometry-based proteomics analysis, and the pathway features of the DEPs were analyzed using functional enrichment analysis. A novel eight-gene signature prognostic risk model was developed (IDO1, MAT1A, NPEPL1, NT5C, PTGR2, RPL29, TMEM126A and TUBB4B), which was validated using data obtained from The Cancer Genome Atlas. The results revealed that the risk score of the eight-gene signature acted as an independent prognostic indicator in patients with stage II CRC (T3-4N0M0). It was also found that a high-risk score in the eight-gene signature was associated with high immune cell infiltration in patients with CRC. Taken together, these findings revealed some of the differential immune characteristics of hot and cold CRC tumors, and an eight-gene signature prognostic risk model was developed, which may serve as an independent prognostic indicator for patients with stage II CRC (T3-4N0M0).

Signature identification based on immunogenic cell death-related lncRNAs to predict the prognosis and immune activity of patients with endometrial carcinoma.

Endometrial carcinoma (EC) is one of the most prevalent gynecologic malignancies and requires further classification for treatment and prognosis. Long non-coding RNAs (lncRNAs) and immunogenic cell death (ICD) play a critical role in tumor progression. Nevertheless, the role of lncRNAs in ICD in EC remains unclear. This study aimed to explore the role of ICD related-lncRNAs in EC via bioinformatics and establish a prognostic risk model based on the ICD-related lncRNAs. We also explored immune infiltration and immune cell function across prognostic groups and made treatment recommendations. A total of 552 EC samples and clinical data of 548 EC patients were extracted from The Cancer Genome Atlas (TCGA) database and University of California Santa Cruz (UCSC) Xena, respectively. A prognostic-related feature and risk model was developed using the least absolute shrinkage and selection operator (LASSO). Subtypes were classified with consensus cluster analysis and validated with t-Distributed Stochastic Neighbor Embedding (tSNE). Kaplan-Meier analysis was conducted to assess differences in survival. Infiltration by immune cells was estimated by single sample gene set enrichment analysis (ssGSEA), Tumor IMmune Estimation Resource (TIMER) algorithm. Quantitative polymerase chain reaction (qPCR) was used to detect lncRNAs expression in clinical samples and cell lines. A series of studies was conducted in vitro and in vivo to examine the effects of knockdown or overexpression of lncRNAs on ICD. In total, 16 ICD-related lncRNAs with prognostic values were identified. Using SCARNA9, FAM198B-AS1, FKBP14-AS1, FBXO30-DT, LINC01943, and AL161431.1 as risk model, their predictive accuracy and discrimination were assessed. We divided EC patients into high-risk and low-risk groups. The analysis showed that the risk model was an independent prognostic factor. The prognosis of the high- and low-risk groups was different, and the overall survival (OS) of the high-risk group was lower. The low-risk group had higher immune cell infiltration and immune scores. Consensus clustering analysis divided the samples into four subtypes, of which cluster 4 had higher immune cell infiltration and immune scores. A prognostic signature composed of six ICD related-lncRNAs in EC was established, and a risk model based on this signature can be used to predict the prognosis of patients with EC.

Comprehensive Analysis of the Function and Prognostic Value of TAS2Rs Family-Related Genes in Colon Cancer.

In the realm of colon carcinoma, significant genetic and epigenetic diversity is observed, underscoring the necessity for tailored prognostic features that can guide personalized therapeutic strategies. In this study, we explored the association between the type 2 bitter taste receptor (TAS2Rs) family-related genes and colon cancer using RNA-sequencing and clinical datasets from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). Our preliminary analysis identified seven TAS2Rs genes associated with survival using univariate Cox regression analysis, all of which were observed to be overexpressed in colon cancer. Subsequently, based on these seven TAS2Rs prognostic genes, two colon cancer molecular subtypes (Cluster A and Cluster B) were defined. These subtypes exhibited distinct prognostic and immune characteristics, with Cluster A characterized by low immune cell infiltration and less favorable outcomes, while Cluster B was associated with high immune cell infiltration and better prognosis. Finally, we developed a robust scoring system using a gradient boosting machine (GBM) approach, integrated with the gene-pairing method, to predict the prognosis of colon cancer patients. This machine learning model could improve our predictive accuracy for colon cancer outcomes, underscoring its value in the precision oncology framework.

The roles of IRF8 in nonspecific orbital inflammation: an integrated analysis by bioinformatics and machine learning

BackgroundNonspecific Orbital Inflammation (NSOI) represents a persistent and idiopathic proliferative inflammatory disorder, characterized by polymorphous lymphoid infiltration within the orbit. The transcription factor Interferon Regulatory Factor 8 (IRF8), integral to the IRF protein family, was initially identified as a pivotal element for the commitment and differentiation of myeloid cell lineage. Serving as a central regulator of innate immune receptor signaling, IRF8 orchestrates a myriad of functions in hematopoietic cell development. However, the intricate mechanisms underlying IRF8 production remain to be elucidated, and its potential role as a biomarker for NSOI is yet to be resolved.MethodsIRF8 was extracted from the intersection analysis of common DEGs of GSE58331 and GSE105149 from the GEO and immune- related gene lists in the ImmPort database using The Lasso regression and SVM-RFE analysis. We performed GSEA and GSVA with gene sets coexpressed with IRF8, and observed that gene sets positively related to IRF8 were enriched in immune-related pathways. To further explore the correlation between IRF8 and immune-related biological process, the CIBERSORT algorithm and ESTIMATE method were employed to evaluate TME characteristics of each sample and confirmed that high IRF8 expression might give rise to high immune cell infiltration. Finally, the GSE58331 was utilized to confirm the levels of expression of IRF8.ResultsAmong the 314 differentially expressed genes (DEGs), some DEGs were found to be significantly different. With LASSO and SVM-RFE algorithms, we obtained 15 hub genes. For biological function analysis in IRF8, leukocyte mediated immunity, leukocyte cell-cell adhesion, negative regulation of immune system process were emphasized. B cells naive, Macrophages M0, Macrophages M1, T cells CD4 memory activated, T cells CD4 memory resting, T cells CD4 naive, and T cells gamma delta were shown to be positively associated with IRF8. While, Mast cells resting, Monocytes, NK cells activated, Plasma cells, T cells CD8, and T cells regulatory (Tregs) were shown to be negatively linked with IRF8. The diagnostic ability of the IRF8 in differentiating NSOI exhibited a good value.ConclusionsThis study discovered IRF8 that are linked to NSOI. IRF8 shed light on potential new biomarkers for NSOI and tracking its progression.

Unraveling Differences in Molecular Mechanisms and Immunological Contrasts between Squamous Cell Carcinoma and Adenocarcinoma of the Cervix.

This study aims to refine our understanding of the inherent heterogeneity in cervical cancer by exploring differential gene expression profiles, immune cell infiltration dynamics, and implicated signaling pathways in the two predominant histological types of cervix carcinoma, Squamous Cell Carcinoma (SCC) and Adenocarcinoma (ADC). Targeted gene expression data that were previously generated from samples of primary cervical cancer were re-analyzed. The samples were grouped based on their histopathology, comparing SCC to ADC. Each tumor in the study was confirmed to be high risk human papilloma virus (hrHPV) positive. A total of 21 cervical cancer samples were included, with 11 cases of SCC and 10 of ADC. Data analysis revealed a total of 26 differentially expressed genes, with 19 genes being overexpressed in SCC compared to ADC (Benjamini-Hochberg (BH)-adjusted p-value < 0.05). Importantly, the immune checkpoint markers CD274 and CTLA4 demonstrated significantly higher expression in SCC compared to ADC. In addition, SCC showed a higher infiltration of immune cells, including B and T cells, and cytotoxic cells. Higher activation of a variety of pathways was found in SCC samples including cytotoxicity, interferon signaling, metabolic stress, lymphoid compartment, hypoxia, PI3k-AKT, hedgehog signaling and Notch signaling pathways. Our findings show distinctive gene expression patterns, signaling pathway activations, and trends in immune cell infiltration between SCC and ADC in cervical cancer. This study underscores the heterogeneity within primary cervical cancer, emphasizing the potential benefits of subdividing these tumours based on histological and molecular differences.

Comprehensive analysis of the relationship between ubiquitin-specific protease 21 (USP21) and prognosis, tumor microenvironment infiltration, and therapy response in colorectal cancer

BackgroundUbiquitin-specific proteases family is crucial to host immunity against pathogens. However, the correlations between USP21 and immunosurveillance and immunotherapy for colorectal cancer (CRC) have not been reported.MethodsThe differential expression of USP21 between CRC tissues and normal tissues was analyzed using multiple public databases. Validation was carried out in clinical samples through qRT-PCR and IHC. The correlation between USP21 and the prognosis, as well as clinical pathological characteristics of CRC patients, was investigated. Moreover, cell models were established to assess the influence of USP21 on CRC growth and progression, employing CCK-8 assays, colony formation assays, and wound-healing assays. Subsequently, gene set variation analysis (GSVA) was used to explore the potential biological functions of USP21 in CRC. The study also examined the impact of USP21 on cytokine levels and immune cell infiltration in the tumor microenvironment (TME). Finally, the effect of USP21 on the response to immunotherapy and chemotherapy in CRC was analyzed.ResultsThe expression of USP21 was significantly upregulated in CRC. High USP21 is correlated with poor prognosis in CRC patients and facilitates the proliferation and migration capacities of CRC cells. GSVA indicated an association between low USP21 and immune activation. Moreover, low USP21 was linked to an immune-activated TME, characterized by high immune cell infiltration. Importantly, CRC with low USP21 exhibited higher tumor mutational burden, high PD-L1 expression, and better responsiveness to immunotherapy and chemotherapeutic drugs.ConclusionThis study revealed the role of USP21 in TME, response to therapy, and clinical prognosis in CRC, which provided novel insights for the therapeutic application in CRC.

Death receptors 4/5 mediate tumour sensitivity to natural killer cell-mediated cytotoxicity in mismatch repair deficient colorectal cancer.

Identifying the target of natural killer (NK) cells in colorectal cancer (CRC) is critical for optimising the clinical use of NK cell-mediated immunotherapy. Mismatch repair deficiency (dMMR) is associated with high immune cell infiltration and MHC Class I defects. Whether dMMR CRC responses to NK cell therapy remains unclear. MLH1, DR4, and DR5 knockout cell lines were established using CRISPR-Cas9 system. NK92-MI or NK cell isolated from BABL/C mice were used as effector cells against tumour cells. Inflammatory cytokines secretion by CRC cells was assessed via cytokine analysis. NK-cell-deficient/proficient animal models were used to validate the NK cell sensitivity. We observed that dMMR CRC cells were more sensitive to NK cell-mediated cytotoxicity than were mismatch-repair-proficient (pMMR) CRC cells. In dMMR CRC, Death receptor (DR)4/5 was upregulated and mediated sensitivity to NK cell-mediated cytotoxicity. DR4/5-mediated secretion of interleukin -12 sustained NK cell viability in dMMR CRC. NK cell depletion induced dMMR CRC tumour growth, and NK cell transfer inhibited lung metastasis of dMMR CRC with DR4/5 expression in vivo. TP53 upregulated DR4/DR5 expression in dMMR CRC. dMMR associated with increased sensitivity to NK cell-mediated cytotoxicity in CRC. DR4/DR5 sensitise dMMR CRC to NK cell-mediated cytotoxicity.

Resistance to immune checkpoint inhibitors in colorectal cancer with deficient mismatch repair/microsatellite instability: misdiagnosis, pseudoprogression and/or tumor heterogeneity?

Colorectal carcinoma (CRC) with deficiency of the deficient mismatch repair (dMMR) pathway/microsatellite instability (MSI) is characterized by a high mutation load and infiltration of immune cells in the tumor microenvironment. In agreement with these findings, clinical trials have demonstrated a significant activity of immune checkpoint inhibitors (ICIs) in dMMR/MSI metastatic CRC (mCRC) patients and, more recently, in CRC patients with early disease undergoing neoadjuvant therapy. However, despite high response rates and durable clinical benefits, a fraction of mCRC patients, up to 30%, showed progressive disease when treated with single agent anti-programmed cell death 1 (PD-1) antibody. This article discusses the three main causes that have been associated with early progression of dMMR/MSI mCRC patients while on treatment with ICIs, i.e., misdiagnosis, pseudoprogression and tumor heterogeneity. While pseudoprogression probably does not play a relevant role, data from clinical studies demonstrate that some dMMR/MSI CRC cases with rapid progression on ICIs may be misdiagnosed, underlining the importance of correct diagnostics. More importantly, evidence suggests that dMMR/MSI mCRC is a heterogeneous group of tumors with different sensitivity to ICIs. Therefore, we propose novel diagnostic and therapeutic strategies to improve the outcome of dMMR/MSI CRC patients.

High immune cell infiltration predicts improved survival in cholangiocarcinoma.

Antitumoral immune response has a crucial role in constraining cancer. However, previous studies on cholangiocarcinoma (CCA), a rare and aggressive cancer, have reported contradictory findings on the prognostic impact of tumor-infiltrating T-lymphocytes. We aimed to clarify the effect of tumor-infiltrating CD3+ and CD8+ lymphocytes and PD-1/PD-L1 expression on CCA prognosis. CD3+, CD8+, and PD-1+ lymphocyte densities, as well as PD-L1 expression rate were analyzed from stained tissue microarray samples from the tumor center and invasive margin of 47 cholangiocarcinomas. The association of CD3+ and CD8+ based Immune cell score (ICS) and its components with overall survival was evaluated, adjusting for age, sex, TNM stage, radicality of surgery, tumor location, and PD-L1 expression on immune cells. Low ICS was a strong independent prognostic factor for worse overall survival (Hazard ratio 9.27, 95% confidence interval 2.72-31.64, P<0.001). Among the ICS components, high CD8+ lymphocyte infiltration at the tumor center had the most evident impact on patient outcome. PD-1 and PD-L1 expression on immune cells did not have a significant impact on overall survival alone; however, PD-L1 positivity seemed to impair survival for ICSlow subgroup. Identifying patient subgroups that could benefit from immunotherapy with PD-1/PD-L1 pathway blockade may help improve treatment strategies for this aggressive cancer. Our findings highlight the importance of evaluating the immune contexture in cholangiocarcinoma, as ICS serves as a strong independent prognostic and selective factor for patients who might benefit from immunotherapy.

Triglyceride-inflammation score established on account of random survival forest for predicting survival in patients with nasopharyngeal carcinoma: a retrospective study.

This study aimed to establish an effective prognostic model based on triglyceride and inflammatory markers, including neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR), and platelet-to-lymphocyte ratio (PLR), to predict overall survival (OS) in patients with nasopharyngeal carcinoma (NPC). Additionally, we aimed to explore the interaction and mediation between these biomarkers in their association with OS. A retrospective review was conducted on 259 NPC patients who had blood lipid markers, including triglyceride and total cholesterol, as well as parameters of peripheral blood cells measured before treatment. These patients were followed up for over 5 years, and randomly divided into a training set (n=155) and a validation set (n=104). The triglyceride-inflammation (TI) score was developed using the random survival forest (RSF) algorithm. Subsequently, a nomogram was created. The performance of the prognostic model was measured by the concordance index (C-index), time-dependent receiver operating characteristic (ROC) curve, and decision curve analysis (DCA). The interaction and mediation between the biomarkers were further analyzed. Bioinformatics analysis based on the GEO dataset was used to investigate the association between triglyceride metabolism and immune cell infiltration. The C-index of the TI score was 0.806 in the training set, 0.759 in the validation set, and 0.808 in the entire set. The area under the curve of time-dependent ROC of TI score in predicting survival at 1, 3, and 5 years were 0.741, 0.847, and 0.871 respectively in the training set, and 0.811, 0.837, and 0.758 in the validation set, then 0.771, 0.848, and 0.862 in the entire set, suggesting that TI score had excellent performance in predicting OS in NPC patients. Patients with stage T1-T2 or M0 had significantly lower TI scores, NLR, and PLR, and higher LMR compared to those with stage T3-T3 or M1, respectively. The nomogram, which integrated age, sex, clinical stage, and TI score, demonstrated good clinical usefulness and predictive ability, as evaluated by the DCA. Significant interactions were found between triglyceride and NLR and platelet, but triglyceride did not exhibit any medicating effects in the inflammatory markers. Additionally, NPC tissues with active triglyceride synthesis exhibited high immune cell infiltration. The TI score based on RSF represents a potential prognostic factor for NPC patients, offering convenience and economic advantages. The interaction between triglyceride and NLR may be attributed to the effect of triglyceride metabolism on immune response.

Single cell analysis unveils B cell-dominated immune subtypes in HNSCC for enhanced prognostic and therapeutic stratification

Head and neck squamous cell carcinoma (HNSCC) is characterized by high recurrence or distant metastases rate and the prognosis is challenging. There is mounting evidence that tumor-infiltrating B cells (TIL-Bs) have a crucial, synergistic role in tumor control. However, little is known about the role TIL-Bs play in immune microenvironment and the way TIL-Bs affect the outcome of immune checkpoint blockade. Using single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database, the study identified distinct gene expression patterns in TIL-Bs. HNSCC samples were categorized into TIL-Bs inhibition and TIL-Bs activation groups using unsupervised clustering. This classification was further validated with TCGA HNSCC data, correlating with patient prognosis, immune cell infiltration, and response to immunotherapy. We found that the B cells activation group exhibited a better prognosis, higher immune cell infiltration, and distinct immune checkpoint levels, including elevated PD-L1. A prognostic model was also developed and validated, highlighting four genes as potential biomarkers for predicting survival outcomes in HNSCC patients. Overall, this study provides a foundational approach for B cells-based tumor classification in HNSCC, offering insights into targeted treatment and immunotherapy strategies.

Abstract LB312: Subtype- and race-specific differences in immune landscape of breast cancer

Abstract Tumor mass comprises of not just the tumor cells but a variety of stromal cells. Immune cells residing within the tumor microenvironment modulate the growth and behavior of the tumor cells through a bi-directional crosstalk and affect the outcomes of cancer therapies. Breast cancer (BC) is a highly heterogeneous disease having four major molecular subtypes. Triple-negative breast cancer (TNBC) is the most aggressive subtype and has limited available therapeutic options. Further, it disproportionately affects African American (AA) women, with nearly two times higher incidence as compared to Caucasian American (CA) women. It is associated with an earlier onset and overall higher mortality of BC. Here, we investigated the differences in the immune landscape between TNBC and non-TNBC subtypes and examined if there existed any race-specific patterns. A total of 81 breast cancer samples with a distribution of 55 non-TNBC (28 CA and 27 AA) and 26 TNBC (12 CA and 14 AA) were selected after histopathologic analysis. Tissues were analysis using the NanoString nCounter® pancancer immune panel to determine tissue-specific immune cell populations. We observed a significantly higher presence of tumor-infiltrating lymphocytes in TNBC compared to non-TNBC, with a higher immune score for CD45+ (total lymphocytes), T-cells, B-cells and tumor-associated macrophages. Specifically, among the T-cell population, Treg cells, Th1 cells, and CD8+ cells were higher in TNBC samples. The pathway prediction indicated 'primary immunodeficiency' and 'cancer immunotherapy by PD-1 blockade' pathways being significantly up-regulated in TNBC samples. Overexpression of CTLA4, IDO1, PDL1 and PDL2 as well as Treg marker genes, such as FOXP3 and LAG3, which are involved in immunosuppressive pathways, was observed in TNBC. In race-wise comparisons of non-TNBC and TNBC sample between CA and AA patients, we found that AA non-TNBC tumors had higher Treg cell scores. On the other hand, CA TNBC samples had higher Th1 and CD8+ T-cell scores compared to AA TNBC. The pathway prediction from differentially expressed genes between CA and AA non-TNBC samples showed complement activation pathway to be down-regulated in AA patients, which could be associated with an increased presence of Treg cells. Further, when comparing CA vs AA TNBC samples, we found significant up-regulation of oncogene BMI1 and down-regulation of genes involved in T-cell activation such as CD47, CD44, FOS, and S100A8 in AA TNBC, supporting their more aggressive nature. In conclusion, our data show that TNBC patients have a higher infiltration of immune cells, which remain suppressed due to the presence of Treg cells and PD1 signaling pathway. The presence of higher Treg cells in AA non-TNBC and lower Th1 and CD8+ T-cells in AA TNBC samples suggests an overall immune suppressed microenvironment in AA patients compared to CA patients. Overall, our data suggest that TNBC could be good candidate for immune therapy. Citation Format: Amod Sharma, Kunwar Somesh Vikramdeo, Sarabjeet Kour Sudan, Mohammad Aslam Khan, Mohammad Tahir, James Elliot Carter, Cindy Nelson, Ajay P Singh, Seema Singh. Subtype- and race-specific differences in immune landscape of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB312.

Abstract 2549: Activity-regulated cytoskeleton-associated protein (ARC) gene expression is associated with high infiltration of stromal cells and immune cells, but with less cancer cell proliferation and better overall survival in ER-positive/HER2-negative breast cancers

Abstract Introduction: Recently peritumoral lidocaine infiltration prior to removal was reported to be associated with better survival in early-stage breast cancer (BC). This led us to hypothesize that innervation to the tumor affects its biology and patient survival. Activity-regulated cytoskeleton-associated protein (ARC) gene expression is known to be regulated by neuronal activity. Therefore, we studied the clinical relevance of ARC gene expression as a surrogate of neuronal activity in BC. Methods: Sweden Cancerome Analysis Network-Breast (SCAN-B (GSE96058), n=3273) cohort was analyzed, and the results were validated using The Cancer Genome Atlas (TCGA, n=1069). Results: ER+/HER2- and Luminal A type cancers expressed significantly higher ARC compared to the other subtypes in both cohorts (p&amp;lt;0.005). In the tumor microenvironment, the stromal cells (fibroblasts, endothelial cells and adipocytes) were all found to be significantly infiltrated in high ARC BC (p&amp;lt;0.01). Multiple immune cells were significantly infiltrated in high ARC BC, including CD8, CD4 memory cells, helper type II T cells, regulatory T cells, M1 and M2 macrophages, dendritic cells and B cells (all p&amp;lt;0.03 in both cohorts). In terms of cancer characteristics, there was no difference in silent or nonsilent mutations, single nucleotide variant or indel neoantigens between tumors with low or high ARC expression. However, high ARC BC was significantly associated with less homologous recombination deficiency, intratumor heterogeneity and fraction altered mutation rate compared to low ARC BC in TCGA (p&amp;lt;0.001). High ARC expression was significantly associated with smaller tumor size (p&amp;lt;0.001) and without lymph node metastasis in the SCAN-B cohort (p&amp;lt;0.02), and less Stage IV disease in the TCGA cohort (p&amp;lt;0.02); however, these results were not validated by the other. High ARC BC was significantly associated with lower Nottingham histologic grade and lower MKi67 expression in both cohorts (p&amp;lt;0.001). Cell proliferation-related gene sets in the Hallmark collection (E2F targets, G2M checkpoint, and MTORC1 signaling) were significantly less enriched to high ARC BC consistently in both cohorts. Overall survival (OS) was significantly better in high ARC BC in the ER+/HER2- subtype consistently in both cohorts (p&amp;lt;0.01) and when including all subtypes in the SCAN-B cohort (p&amp;lt;0.001); however this was not validated in TCGA. No significant difference in OS was found between low and high ARC gene expression in triple negative BC in either cohort. Conclusion: ARC gene expression as a surrogate of neuronal activity in BC was associated with high infiltration of stromal cells and immune cells but with less cancer cell proliferation and better overall survival, particularly in the ER+/HER2- subtype. Citation Format: Gabrielle Yee, Rongrong Wu, Takashi Ishikawa, Kazuaki Takabe. Activity-regulated cytoskeleton-associated protein (ARC) gene expression is associated with high infiltration of stromal cells and immune cells, but with less cancer cell proliferation and better overall survival in ER-positive/HER2-negative breast cancers [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 2549.

Abstract 7570: Molecular subtyping and immunohistochemistry validation identifies muscle Invasive bladder cancer subgroups with poorer overall survival

Abstract Introduction: Muscle-invasive bladder cancers (MIBC) are molecularly heterogeneous and have poor clinical outcomes, compared to non-muscle invasive disease. The molecular characterization of subtypes and immune status has shown prognostic value and the potential to guide precision intervention. However, a more comprehensive understanding of the association between subtypes and immune cells is still required. To this end, an integrative multi-omics analysis was performed on patients with MIBC. Methods: From a cohort of 244 MIBC patients who did not receive neoadjuvant chemotherapy, cystectomy tissue was collected to assess molecular features associated with poor outcomes. Macro-dissected FFPE slides were used to perform whole transcriptome RNA sequencing (N=208, passing quality control) and both the consensus single-sample classifier and TCGA classifier were applied to the RNAseq data to determine molecular subtypes. Immunohistochemistry (IHC) staining for canonical markers of basal (KRT5, 14 and CD44), luminal (KRT20, GATA3, FOXA1) and immune cells (CD3, CD8, PD-L1 (SP263)) was performed, and scoring was assessed by two independent pathologists. Tumor subtypes derived from either RNAseq or IHC were compared and correlated with disease specific survival. Results: Among the 244 MIBC patients 30.7% were T2, 47.6% were T3 and 21.7% were T4. The consensus molecular classification identified mRNA subtypes and showed agreement with the TCGA molecular classification. Correlations with disease specific survival revealed that luminal subtypes trended towards best outcome while stroma-rich subtype trended towards poorest outcomes compared to other subtypes. IHC markers associated with luminal-like and basal-like tumors recapitulated the molecularly defined luminal/basal subtype assignment. Stroma-rich subtypes were enriched with fibroblast signatures, implying high desmoplastic stromal cell infiltration and low immune cell infiltration. By integrating molecular classification and IHC immune markers (PD-L1, CD3, CD8 - serving as a proxy for ‘immune signature’), we found that the immune signatures themselves stratified the survival benefit, as previously shown. Interestingly, we observed heterogeneity of the IHC-immune signature within mRNA subtypes. Conclusions: Integrating MIBC subtyping, IHC of immune markers and patient outcomes data provides a biological framework that allows for potential biomarker identification for this lethal disease. The studies presented here underscore the existence of heterogeneity in immune phenotypes within tumor subtypes. A deeper understanding of the association between these tumor subtypes and their immunological states is crucial to guide treatment decisions, particularly for patients facing worse prognostic outcomes. Citation Format: Jiarui Zhang, Mingxiao Feng, Neil Beeharry, Shibu Thomas, Ralph Wirtz, Arndt Hartmann, Markus Eckstein, Woonyoung Choi. Molecular subtyping and immunohistochemistry validation identifies muscle Invasive bladder cancer subgroups with poorer overall survival [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 7570.