Evaluation Of Immune Infiltration Research Articles

Integrating single-cell RNA-Seq and bulk RNA-Seq data to explore the key role of fatty acid metabolism in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a predominant cause of cancer-related mortality globally, noted for its propensity towards late-stage diagnosis and scarcity of effective treatment modalities. The process of metabolic reprogramming, with a specific emphasis on lipid metabolism, is instrumental in the progression of HCC. Nevertheless, the precise mechanisms through which lipid metabolism impacts HCC and its viability as a therapeutic target have yet to be fully elucidated. In the current investigation, single-cell RNA sequencing in conjunction with weighted gene co-expression network analysis (WGCNA) was utilized to delineate lipid metabolism-related genes correlated with the prognostic outcomes of hepatocellular carcinoma (HCC). Data procurement encompassed transcriptomic and clinical datasets from HCC patients, sourced from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) repositories. Subsequent to this, consensus clustering analysis was implemented to stratify patients into distinct subgroups, contingent upon the expression patterns of lipid metabolism genes. Further analytical procedures involved functional enrichment analysis, evaluation of immune infiltration, and examination of the mutation landscape.PTGES3 was identified as a pivotal gene associated with lipid metabolism. Subsequent to its identification, cellular communication analysis was employed to assess the immunological attributes of PTGES3 within the tumor microenvironment. The functional role of PTGES3 was further corroborated through molecular docking simulations and in vitro experimental assays. We identified 27 genes associated with lipid metabolism, 18 of which exhibited significant correlation with overall survival in HCC patients. PTGES3 emerged as a central gene, demonstrating a robust association with immune cell infiltration and unfavorable prognosis. Cellular communication analysis revealed that PTGES3 exhibits the highest communication intensity with T cells, modulating the tumor microenvironment by potentiating the FN1/CD44 + MDK/NCL signaling pathway. Elevated expression of PTGES3 was linked to immunosuppressive cascades, diminished responsiveness to immunotherapy, and inferior overall survival outcomes. Molecular docking analysis indicated that etoposide, methotrexate, and doxorubicin could effectively bind to PTGES3. In vitro experiments confirmed that PTGES3 knockdown significantly impaired the proliferation, invasion, and migration of HCC cells. This study highlights the pivotal role of lipid metabolism in HCC progression and identifies PTGES3 as a potential prognostic biomarker and therapeutic target. These findings offer new insights into the development of targeted therapies for HCC, particularly in patients with high PTGES3 expression.

Exploring the role of aggrephagy-related signatures in immune microenvironment, prognosis, and therapeutic strategies of breast cancer.

Breast cancer (BC) is 1 of the most common malignant tumors among women globally. This study aimed to develop a prognostic signature based on aggrephagy-related genes (ARGs). Transcriptomic and clinical data for BC patients were downloaded from the cancer genome atlas and GEO databases. Differential expression analysis, univariate Cox proportional hazards regression and least absolute shrinkage and selection operator Cox regression were employed to construct a prognostic signature. Consensus clustering, evaluation of immune infiltration and drug sensitivity, and gene set enrichment analysis, and development of nomogram were performed. The expression of ARGs was validated using data from the Cancer Cell Line Encyclopedia and clinical samples. Eleven ARGs were abnormally expressed in BC, with 5 showing significant correlations with BC prognosis. Consensus clustering identified 2 molecular subtypes with distinct prognoses. A prognostic signature including 5 ARGs (VIM, TUBB1, TUBA3E, TUBA3D, TUBA1C) was developed, which showed high performance in predicting BC prognosis. The low-risk group showed enrichment in extracellular matrix organization and cell migration processes while chromosome separation was suppressed. Additionally, patients in this group also show activation in several signaling pathways including MAPK, PI3K-AKT, and cAMP pathways, whereas cell cycle and neutrophil extracellular trap formation were significantly inhibited. The signature was also associated with immune infiltration and drug sensitivity. A nomogram incorporating the risk signature, clinical stage and chemotherapy was constructed, demonstrating excellent performance in predicting prognosis. The expression of signature-related genes were validated in patients with BC. This study successfully constructed molecular subtypes and a prognostic signature based on ARGs in BC, and developed a nomogram.

Evaluation of Immune Infiltrates in Ovarian Endometriosis and Endometriosis-Associated Ovarian Cancer: Relationship with Histological and Clinical Features.

the association between ovarian endometriosis (OE) and endometriosis-associated ovarian cancer (EAOC) is extensively documented, and misfunction of the immune system might be involved. The primary objective of this study was to identify and compare the spatial distribution of tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs) in OE and EAOC. Secondary objectives included the analysis of the relationship between immunosuppressive populations and T-cell exhaustion markers in both groups. TILs (CD3, CD4, and CD8) and macrophages (CD163) were assessed by immunochemistry. Exhaustion markers (PD-1, TIM3, CD39, and FOXP3) and their relationship with tumour-associated macrophages (CD163) were assessed by immunofluorescence on paraffin-embedded samples from n = 43 OE and n = 54 EAOC patients. we observed a predominantly intraepithelial CD3+ distribution in OE but both an intraepithelial and stromal pattern in EAOC (p < 0.001). TILs were more abundant in OE (p < 0.001), but higher TILs significantly correlated with a longer overall survival and disease-free survival in EAOC (p < 0.05). CD39 and FOXP3 significantly correlated with each other and CD163 (p < 0.05) at the epithelial level in moderate/intense CD4 EAOC, whereas in moderate/intense CD8+, PD-1+ and TIM3+ significantly correlated (p = 0.009). Finally, T-cell exhaustion markers FOXP3-CD39 were decreased and PD-1-TIM3 were significantly increased in EAOC (p < 0.05). the dysregulation of TILs, TAMs, and T-cell exhaustion might play a role in the malignization of OE to EAOC.

Comparison of T cell infiltration in hepatocellular carcinoma between whole exome and transcriptome sequencing data.

e16226 Background: Immunotherapy is one of the most promising therapeutics for hepatocellular carcinoma (HCC), and the immune infiltration status in tumors could be used as a biomarker for immunotherapy. However, evaluation of immune infiltration is usually based on RNA-Seq (transcriptome sequencing), which is limited in clinical practice due to the difficulty of handling RNA. On the other hand, WES (whole exome sequencing) is frequently performed to detect tumor mutations in cancer clinic. Therefore, we try to compare T cell infiltration in hepatocellular carcinoma obtained from WES data and RNA-Seq data, evaluate the potential of T cell infiltration from WES data as a biomarker for immunotherapy. Besides, we also evaluated the association of T cell fraction obtained from WES data and other clinical information. Methods: Seventy pairs of HCC tumor and matched normal tissue samples underwent both WES and RNA-Seq. T cell ExTRECT score and mcpCounter score were used to represent the immune infiltration status, and they were generated from WES data and RNA-Seq data respectively. Tumor mutational burden (TMB) score and some characteristics (tumor size, blood pressure, etc.) were also calculated for correlation analysis. Results: T cell ExTRECT score was positively correlated with mcpCounter score (Spearman correlation coefficient: 0.54). While TMB score showed no correlation with T cell ExTRECT score (Spearman correlation coefficient: -0.06) and mcpCounter score (Spearman correlation coefficient: -0.09). Besides, T cell ExTRECT score was negatively associated with main tumor size (Spearman correlation coefficient: -0.38) and High Blood Pressure (Spearman correlation coefficient: -0.37). Conclusions: These results suggest that T cell ExTRECT score obtained from WES and mcpCounter score generated by RNA-seq data is highly correlated, and neither of them is associated with TMB score. T cell ExTRECT score could be used to evaluate immune infiltration in hepatocellular carcinoma, and may serve as a biomarker for immunotherapy regardless of TMB score. Further investigation is still needed to evaluate the clinical outcome of immunotherapy for hepatocellular carcinoma with T cell ExTRECT score.

Abstract CT109: Radiomic features in tumor assessment, preliminary results from a phase 2 study of intratumoral administration of BO-112 with pembrolizumab in patients with anti PD1 refractory melanoma

Abstract Background: There is a lack of predictive biomarkers for immunotherapy in melanoma. Immunohistochemistry and gene sequencing are frequently assessed as part of clinical research. Radiomic signatures may also add valuable information, based on parameters which can be related to immune infiltration, therefore defining an imaging biomarkers panel for this clinical scenario. BO-112 is a double stranded synthetic RNA formulated with polyethyleneimine (PEI) that, by mimicking the effect of a viral infection, mobilizes the immune system. The role of imaging biomarkers is being explored in the present phase II clinical trial. Study design: Single arm study with BO-112 plus pembrolizumab (NCT04570332) in patients with advanced melanoma in progression to anti-PD1 therapy. As part of exploratory objectives, a radiomics analysis was performed to detect changes in lesion texture features. Quantitative features were obtained by using Quibim Precision 2.9 platform (Quibim, Valencia, Spain) after the manual delineation of lesions on the CT study of each subject at each timepoint to obtain information about injected/non-injected lesions. Images were normalized by taking into account Hounsfield Units (HU) bias across scanners in a cross-calibration phantom and the Z-score. The difference (Delta) in the features between baseline and week 8 was calculated. Results: Studies were assessed based on event (progression) and based on whether lesions had been injected. Patients with only cutaneous disease were not included in this analysis. Out of 23 patients who had at least two imaging assessments, 6 developed progressive disease by week 8, and 17 subjects had no event by that time. Due to the small sample size, the radiomic analysis was based on hypothesis testing. With regards to volume, 50% of the non-progressing lesions reduced their value from that of the baseline. Regarding injected versus non-injected changes, up to 50% of injected lesions decreased their volume after 8 weeks of injected treatment whereas in non-injected lesions volume decreased in less than 25% of lesions. From the independent sample t-test of delta radiomics features against the injected/non-injected lesions variable, there were 4 features with a statistically significant difference between groups; all of them related to the Low Grey-Level Zone Emphasis. Specifically, Delta original GLRLM Low Grey-Level Run Emphasis showed the highest significant difference between injected and non-injected lesions (p=0.004), with higher and positive delta values in the injected group (75% injected lesions were above 0). Conclusions: Imaging biomarkers provide a large number of quantitative image features with a wide span of information, from size to heterogeneity of the tissue which may be indicator of tumor progression and immune infiltrate. In the analysis of radiomics features, delta GLRLM low grey-level zone emphasis was sensitive to the tumoral changes happening in injected lesions at week 8. This might add insights into the imaging-based evaluation of immune infiltration in intratumoral immunotherapy and the creation of associated imaging biomarkers panels. Citation Format: Paula Moreno, Philippe Saiag, Luis de la Cruz Merino, Caroline Dutriaux, Eduardo Castanon Álvarez, Caroline Robert, Juan F. Rodríguez-Moreno, Pablo Cerezuela-Fuentes, Ana M. Arance, Henry Montaudié, Miguel F. Sanmamed, María González Cao, María Pilar López Criado, Julie Charles, Alfonso Berrocal, Enrique de Miguel, Elisa Funk-Brentano, Pablo Sau Llanas, Sorilla Prey, Eva Muñoz-Couselo, Delvys Rodríguez Abreu, Juan Martin-Liberal, Ángel Alberich-Bayarri, Javier Sánchez López, Sonia Macia, Marisol Quintero, Marya F. Chaney, Stéphane Dalle, Iván Márquez-Rodas. Radiomic features in tumor assessment, preliminary results from a phase 2 study of intratumoral administration of BO-112 with pembrolizumab in patients with anti PD1 refractory melanoma [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 CT109.

Development and validation of a novel mitophagy-related gene prognostic signature for glioblastoma multiforme

BackgroundGlioblastoma multiforme (GBM) is one of the most malignant tumors in brain with high morbidity and mortality. Mitophagy plays a significant role in carcinogenesis, metastasis, and invasion. In our study, we aim to construct a mitophagy-related risk model to predict prognosis in GBM.MethodsRNA-seq data combined with clinical information were downloaded from TCGA. The 4-gene risk model and nomograph was then constructed and validated in external cohort. Evaluation of immune infiltration, functional enrichment and tumor microenvironment (TME) were then performed.ResultA mitophagy-related risk model was established and patients in TCGA and CGGA were classified into low-risk and high-risk groups. In both cohorts, patients in low-risk group had improved survival, while high-risk group had poor prognosis. Also, the risk model was identified as an independent factor for predicting overall survival via Cox regression. Furthermore, a prognostic nomogram including mitophagy signatures was established with excellent predictive performance. In addition, the risk model was closely associated with regulation of immune infiltration as well as TME.ConclusionIn conclusion, our study constructed a mitophagy-related risk model, which can be utilized for the clinical prognostic prediction in GBM.

Prognostic value of p16, p53, and pcna in sarcoma and an evaluation of immune infiltration

Backgroundp16, p53, and proliferating cell nuclear antigen (pcna) genes play significant roles in many chromatin modifications and have been found to be highly expressed in a variety of tumor tissues. Therefore, they have been used as target genes for some tumor therapies. However, the differential expressions of the p16, p53, and pcna genes in human sarcomas and their effects on prognosis have not been widely reported.MethodsThe Oncomine dataset was used to analyze the transcription levels of p16, p53, and pcna genes, and the gene expression profile interactive analysis (GEPIA) dataset was used to analyze the differential expressions of p16, p53, and pcna. The expression levels of p16, p53, and pcna were further analyzed by Western Blotting. GEPIA and Kaplan–Meier analyses were used to analyze the prognostic value of p16, p53, and pcna. Furthermore, p16, p53, and pcna gene mutations and their association with overall survival (OS) and disease-free survival (DFS) were analyzed using cBioPortal datasets. In addition, genes co-expressed with p16, p53, and pcna were analyzed using Oncomine. The DAVID dataset was used to analyze the functional enrichment of p16, p53, pcna, and their co-expressed genes by Gene Ontology (GO) and Metascape were used to construct a network map. Finally, the immune cell infiltration of p16, p53, and pcna in patients with sarcoma was reported by Tumor Immune Estimation Resource (TIMER).Resultsp16, p53, and pcna were up-regulated in human sarcoma tissues and almost all sarcoma cell lines. Western Blotting showed that the expression of p16, p53, and pcna was elevated in osteosarcoma cell lines. The expression of pcna was correlated with OS, the expression of p16, p53, and pcna was correlated with relapse-free survival, and the genetic mutation of p16 was negatively correlated with OS and DFS. We also found that p16, p53, and pcna genes were positively/negatively correlated with immune cell infiltration in sarcoma.ConclusionsThe results of this study showed that p16, p53, and pcna can significantly affect the survival and immune status of sarcoma patients. Therefore, p16, p53, and pcna could be used as potential biomarkers of prognosis and immune infiltration in human sarcoma and provide a possible therapeutic target for sarcoma.

Construction of an Immune-Autophagy Prognostic Model Based on ssGSEA Immune Scoring Algorithm Analysis and Prognostic Value Exploration of the Immune-Autophagy Gene in Endometrial Carcinoma (EC) Based on Bioinformatics.

Background Endometrial carcinoma (EC) is a malignant cancer spreading worldwide and in the fourth position among all other types of cancer in women. The purpose of this paper is to explore the prognostic value of the immune-autophagy gene in endometrial carcinoma (EC) based on bioinformatics, construct an immune-autophagy prognostic model of endometrial carcinoma, search for independent prognostic markers, and finally predict the potential therapeutic drugs of TCGA subgroup. Methods The Cancer Genome Atlas (TCGA) database was used to extract transcriptome sequencing data of patients suffering from EC; 28 kinds of immune cells were scored by ssGSEA, and the immune subtypes were grouped by consistency cluster analysis. The accuracy and effectiveness of the grouping were verified by the analysis of differential gene expression and survival rate of immune checkpoints in the two groups to provide the premise and basis for the establishment of independent prognostic factors. The expression of different genes in high and low immune groups was analyzed. The analysis of various genes' expression in immune groups (high and low) has been performed. Go function annotation and KEGG pathway enrichment analysis were used to evaluate the difference of immune infiltration between high and low immune groups. The immune and autophagy genes were crossed, the key (hub) genes were selected, the risk was scored, the prognosis model was constructed, and the independent prognostic markers were established. CAMP and CTRP 2.0 were used to test the drug sensitivity. Results According to the level of immune cell enrichment, the results have been subcategorized into two immune subtypes: high immunity group_ H and low immunity group_ L. Two immune subtypes, CD274, PDCD1, and CTLA4, were detected in the immune system_ H and immunity_L. A significant difference was detected between these two groups in the expression and survival rate. Few more differences were also detected between the two groups through the evaluation of immune infiltration, which proved the grouping's accuracy and effectiveness. Differential gene expression analysis showed that there were 721 DEGs and 3 hub genes. DEGs are mainly involved in lymphocyte activation, proliferation, differentiation, leukocyte proliferation, and other biological processes, mediate chemokines' activities, chemokine receptor binding, and other molecular functions, and are enriched in the outer plasma membrane, endoplasmic reticulum, and T cell receptor complex. The enriched pathways are allograft, complex, inflammatory, interferon-alpha, interferon-gamma, E2F, G2M, mitotic, etc. Conclusion Through bioinformatics analysis, we successfully constructed the immuno-autophagy prognosis model of endometrial cancer and identified three high-risk immunoautophagy genes, including VEGFA, CCL2, and Ifng. Four potential therapeutic drugs were predicted as sildenafil, sunitinib, TPCA-1, and etoposide.

Hyperglycemia Enhances Immunosuppression and Aerobic Glycolysis of Pancreatic Cancer Through Upregulating Bmi1-UPF1-HK2 Pathway.

Background & AimsAccumulating evidence strongly suggests that hyperglycemia promotes the progression of pancreatic cancer (PC). Approximately 80% of patients with PC are intolerant to hyperglycemic conditions. In this study, we define the role of Bmi1, a stemness-related oncogene, in controlling the Warburg effect, and immune suppression under hyperglycemia conditions.MethodsThe diabetes mellitus model was established by intraperitoneal injection of streptozotocin. The role of the hyperglycemia-Bmi1-HK2 axis in glycolysis-related immunosuppression was examined in both orthotopic and xenograft in vivo models. Evaluation of immune infiltrates was carried out by flow cytometry. Human PC cell lines, SW1990, BxPC-3, and CFPAC-1, were used for mechanistic in vitro studies.ResultsThrough bioinformatics analysis, we found that hyperglycemia was strongly related to aerobic glycolysis, immunosuppression, and cancer cell stemness. High glucose condition in the tumor microenvironment promotes immune suppression by upregulating glycolysis in PC cells, which can be rescued via knockdown Bmi1 expression or after 2-deoxy-D-glucose treatment. Through gain-/loss-of-function assessments, we found that Bmi1 upregulated the expression of UPF1, which enhanced the stability of HK2 mRNA and thereby increased the expression of HK2. The role of the hyperglycemia-Bmi-HK2 pathway in the inhibition of antitumor immunity was further verified via the immune-competent and immunodeficient mice model. We also demonstrated that hyperglycemia promotes the expression of Bmi1 by elevating the intracellular acetyl-CoA levels and histone H4 acetylation levels.ConclusionsOur results suggest that the previously unreported Bmi1-UPF1-HK2 pathway contributes to PC progression and immunosuppression, which may bring in new targets for developing effective therapies to treat patients with PC.

Identification and integrative analysis of ACLY and related gene panels associated with immune microenvironment reveal prognostic significance in hepatocellular carcinoma

BackgroundCumulating evidence reveals the key role of aberrant lipogenesis and immunogenomic features in hepatocellular carcinoma (HCC). However, there are still obstacles in our understanding of the complicated interaction between metabolic reprogramming and tumor immune microenvironment.MethodsWe compared metabolomic, transcriptomic and immunogenomic characteristics of portal vein tumor thrombosis (PVTT) and primary tumor to seek valuable markers. Human HCC samples with PVTT (n = 28) was analyzed through ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Transcript levels of mRNA in two cohorts from published database GEO (n = 60) and TCGA (n = 411) were downloaded to explore differentially expressed genes and functional enriched gene set. Evaluation of immune infiltration was estimated and validated from transcriptomic data in both cohorts through six immune deconvolution algorithms and in a high-resolution mode (CIBERSORTx). Survival analysis (Kaplan–Meier and multivariable Cox regression model) was performed to examine prognostic value of ACLY, related immune checkpoints and immune infiltration levels from TCGA cohort. LASSO regression was further conducted to determine a gene panel to further predict survival outcomes associated with ACLY.ResultsWe identified a novel signature, ATP citrate lyase, through transcriptomic and metabolomic approaches. We demonstrated that the metabolism adaptations in both fatty acid and cholesterol biosynthesis triggered by ACLY oncogenic activation. We illustrated the crucial function of ACLY in lipogenesis and its potential interaction with immune microenvironment. CD276, a promising target in immune checkpoint blockade, showed correlation to ACLY and differential expression in ACLY risk classification. Combination of ACLY, CD276 and immune infiltration level and a novel ACLY-associated panel from a predictive model retrieved from published database validated the prognostic value to risk stratification in patients with HCC.ACLY blockade to counteract metabolic activation and immunosuppressive status of the tumor microenvironment highlighted attractive prospect for translational application.ConclusionsWe investigated ACLY and its indispensable role in metabolism, immune function and a prognostic gene panel in HCC. We anticipate that the multifaced role of ACLY may reveal the potential value for mechanistic research and combinational therapy, suggesting that the combination blockade of ACLY and immune checkpoints may work as a promising strategy.

Repurposing the serotonin agonist Tegaserod as an anticancer agent in melanoma: molecular mechanisms and clinical implications

BackgroundNew therapies are urgently needed in melanoma particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors.MethodsDrug screening, IC50 determinations as well as synergy assays were detected by the MTT assay. Apoptosis using Annexin V and 7AAD staining was assessed using flow cytometry. TUNEL staining was performed using immunocytochemistry. Changes in phosphorylation of key molecules in PI3K/Akt/mTOR and other relevant pathways were detected by western blot as well as immunocytochemistry. To assess in vivo anti-tumor activity of Tegaserod, syngeneic intravenous and subcutaneous melanoma xenografts were used. Immunocytochemical staining was performed to detect expression of active Caspase-3, cleaved Caspase 8 and p-S6 in tumors. Evaluation of immune infiltrates was carried out by flow cytometry.ResultsUsing a screen of 770 pharmacologically active and/or FDA approved drugs, we identified Tegaserod (Zelnorm, Zelmac) as a compound with novel anti-cancer activity which induced apoptosis in murine and human malignant melanoma cell lines. Tegaserod (TM) is a serotonin receptor 4 agonist (HTR4) used in the treatment of irritable bowel syndrome (IBS). TM’s anti-melanoma apoptosis-inducing effects were uncoupled from serotonin signaling and attributed to PI3K/Akt/mTOR signaling inhibition. Specifically, TM blunted S6 phosphorylation in both BRAFV600E and BRAF wildtype (WT) melanoma cell lines. TM decreased tumor growth and metastases as well as increased survival in an in vivo syngeneic immune-competent model. In vivo, TM also caused tumor cell apoptosis, blunted PI3K/Akt/mTOR signaling and decreased S6 phosphorylation. Furthermore TM decreased the infiltration of immune suppressive regulatory CD4+CD25+ T cells and FOXP3 and ROR-γt positive CD4+ T cells. Importantly, TM synergized with Vemurafenib, the standard of care drug used in patients with late stage disease harboring the BRAFV600E mutation and could be additively or synergistically combined with Cobimetinib in both BRAFV600E and BRAF WT melanoma cell lines in inducing anti-cancer effects.ConclusionTaken together, we have identified a drug with anti-melanoma activity in vitro and in vivo that has the potential to be combined with the standard of care agent Vemurafenib and Cobimetinib in both BRAFV600E and BRAF WT melanoma.

Abstract 5628: Trilaciclib (G1T28), a CDK4/6 inhibitor, enhances the efficacy of combination chemotherapy and immune checkpoint inhibitor treatment in preclinical models

Abstract While immune checkpoint inhibitors are efficacious and lead to durable responses in patients with various cancers, only a minority of patients respond. An approach to increase the response rate of immune checkpoint inhibitors is to combine them with chemotherapy in order to enhance immunogenic cell death and “prime” the immune system. However, chemotherapy itself can cause damage to various cell types of the immune system, potentially diminishing the activity of the chemotherapy/checkpoint inhibitor combination. Therefore, a targeted approach to preserve immune system function during chemotherapy would allow optimal activity of the checkpoint inhibitor therapy. Trilaciclib (G1T28), a potent and selective IV CDK4/6 inhibitor, preserves hematopoietic stem cells and enhances immune system function during chemotherapy. We and others have previously shown T lymphocytes are extremely sensitive to CDK4/6 inhibition causing a transient arrest of proliferation and protection from damage by chemotherapy. Post treatment with trilaciclib and chemotherapy, T lymphocytes are released from the transient arrest in the presence of chemotherapy-induced immunogenic cell death allowing better priming of an anti-tumor immune response. To evaluate the effect of trilaciclib combination treatments, MC38 tumor bearing mice were treated with trilaciclib, oxaliplatin, or anti-PD-L1 alone or in combination, and tumor size was measured during and post treatment for 100 days. Our results demonstrate that the addition of trilaciclib to an oxaliplatin/anti-PD-L1 combination (TOP) treatment significantly improves anti-tumor activity. Specifically, twice as many mice treated with TOP for 15 days had a complete response (CRs) when compared to oxaliplatin/anti-PD-L1 (OP); 6/10 CRs vs 3/10 CRs, respectively. In addition, the CRs were durable and without any evidence of recurrence at 100 days. Furthermore, TOP treatment caused a 60% increase in overall survival (OS) compared to mice treated with OP; median OS for TOP was 98 days compared to 61 days (HR, 0.53) for the OP treatment group. Assessment of ex-vivo stimulation of cytokine release from harvested splenocytes to measure T-cell activation, and evaluation of immune infiltrates and T-cell receptor repertoire within the tumors are ongoing. Taken together, we demonstrate that the short-acting IV CDK4/6 inhibitor, trilaciclib, which has been shown to preserve immune function during chemotherapy, enhances the anti-tumor activity of chemotherapy/anti-PD-L1 combination therapy. Citation Format: Jessica A. Sorrentino, Anne Y. Lai, Jay C. Strum, Patrick J. Roberts. Trilaciclib (G1T28), a CDK4/6 inhibitor, enhances the efficacy of combination chemotherapy and immune checkpoint inhibitor treatment in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5628. doi:10.1158/1538-7445.AM2017-5628

Evaluation of immune infiltration in the colonic mucosa of patients with ipilimumab-related colitis

ABSTRACTApproximately 30% of patients treated with ipilimumab will develop gastrointestinal toxicity. The immunological drivers that underpin the clinical observations in human tissues are poorly understood. We report here on the immune consequences of ipilimumab treatment in the colorectal mucosa of patients with treatment-related colitis. Using immunohistochemistry, we evaluated the immune infiltrate by CD8+, FoxP3, and granzyme B (GzmB) in colonic biopsies from 20 patients with ipilimumab-related colitis. We assessed 10 cases with normal colon biopsies for comparison. In eight cases (four on steroids only, four on steroids and infliximab), we evaluated two sequential biopsies. We observed that CD8+, FoxP3+, and GzmB T cell counts were significantly higher in patients with ipilimumab-related colitis compared to normal colon (p < 0.0001). Patients who required infliximab for the resolution of their colitis had a significantly higher CD8+/FoxP3 ratio than those treated only with steroids and this correlated with clinical severity. The analysis of repeat samples revealed that resolution of the colitis was associated with a decrease in CD8+ and FoxP3+ cells both in patients treated with steroids and infliximab. Our data suggest that counts of cytotoxic T cells and Tregs in the colonic mucosa from patients with ipilimumab-related colitis correlate with clinical findings and may predict severity and guide management.