Presence Of Immune Infiltrates Research Articles

FBLN5 as One Presumably Prognostic Gene Potentially Modulating Tumor Immune Microenvironment for Renal Clear Cell Carcinoma in Children and Young Adults.

To investigate the role of FBLN5in renal clear cell carcinoma (KIRC), in particular on the tumor's immune microenvironment, including children and young adults. FBLN5 expression in tumor and normal samples was explored using SangerBox, TIMER2.0, GEPIA, UALCAN, HPA databases. The Linkedomics database was used to obtain FBLN5 co-expressed genes in KIRC tissue. SangerBox was also used to estimate immune infiltration of FBLN5 in KIRC. The Kaplan-Meier plotter was used to investigate the survival effects of FBLN5 expression in the presence of immune infiltration. We then collected 48 cases from 7 hospitals over a-20 year period to calculate the impact of FBLN5 on the prognosis of children and young adults with KIRC. FBLN5 expression was significantly reduced in KIRC tissue compared to normal adjacent tissue. FBLN5 was potentially involved in the immune-related biological processes. In addition, FBLN5 expression has been linked to a number of immune checkpoints, cytokines, chemokines and chemokine receptors in KIRC. At the same time, the expression of FBLN5 affected the survival rates differently in KIRC patients with high or low levels of immune infiltration. High expression of FBLN5 in children and young adults with KIRC was associated with a favorable prognosis. This study shed light on the potential of FBLN5 as a prognostic marker in children and young adults with KIRC and as an immune-related target for clinical treatment.

The utilization of single-cell sequencing technology in investigating the immune microenvironment of ccRCC.

The growth and advancement of ccRCC are strongly associated with the presence of immune infiltration and the tumor microenvironment, comprising tumor cells, immune cells, stromal cells, vascular cells, myeloid-derived cells, and extracellular matrix (ECM). Nevertheless, as a result of the diverse and constantly evolving characteristics of the tumor microenvironment, prior advanced sequencing methods have frequently disregarded specific less prevalent cellular traits at varying intervals, thereby concealing their significance. The advancement and widespread use of single-cell sequencing technology enable us to comprehend the source of individual tumor cells and the characteristics of a greater number of individual cells. This, in turn, minimizes the impact of intercellular heterogeneity and temporal heterogeneity of the same cell on experimental outcomes. This review examines the attributes of the tumor microenvironment in ccRCC and provides an overview of the progress made in single-cell sequencing technology and its particular uses in the current focus of immune infiltration in ccRCC.

Identification of copper death-associated molecular clusters and immunological profiles in rheumatoid arthritis.

An analysis of the relationship between rheumatoid arthritis (RA) and copper death-related genes (CRG) was explored based on the GEO dataset. Based on the differential gene expression profiles in the GSE93272 dataset, their relationship to CRG and immune signature were analysed. Using 232 RA samples, molecular clusters with CRG were delineated and analysed for expression and immune infiltration. Genes specific to the CRGcluster were identified by the WGCNA algorithm. Four machine learning models were then built and validated after selecting the optimal model to obtain the significant predicted genes, and validated by constructing RA rat models. The location of the 13 CRGs on the chromosome was determined and, except for GCSH. LIPT1, FDX1, DLD, DBT, LIAS and ATP7A were expressed at significantly higher levels in RA samples than in non-RA, and DLST was significantly lower. RA samples were significantly expressed in immune cells such as B cells memory and differentially expressed genes such as LIPT1 were also strongly associated with the presence of immune infiltration. Two copper death-related molecular clusters were identified in RA samples. A higher level of immune infiltration and expression of CRGcluster C2 was found in the RA population. There were 314 crossover genes between the 2 molecular clusters, which were further divided into two molecular clusters. A significant difference in immune infiltration and expression levels was found between the two. Based on the five genes obtained from the RF model (AUC = 0.843), the Nomogram model, calibration curve and DCA also demonstrated their accuracy in predicting RA subtypes. The expression levels of the five genes were significantly higher in RA samples than in non-RA, and the ROC curves demonstrated their better predictive effect. Identification of predictive genes by RA animal model experiments was also confirmed. This study provides some insight into the correlation between rheumatoid arthritis and copper mortality, as well as a predictive model that is expected to support the development of targeted treatment options in the future.

50P Transcriptomic profiles of CD32b in breast cancer predict outcome and are associated with immune activation

50P Transcriptomic profiles of CD32b in breast cancer predict outcome and are associated with immune activation

Presence of immune infiltrates, increased expression of transposable elements, and viral response pathways in sarcoma associate with response to checkpoint inhibition.

11510 Background: Response to checkpoint inhibition (CPI) in sarcoma is overall low and varies between and within subtypes. Understanding tumor intrinsic determinants of this response may improve efficacy and patient selection. The de-repression of transposable elements (TEs), which are epigenetically silenced repetitive DNA elements of viral origin, is linked to anti-tumor immunity through an antiviral inflammatory response. We hypothesize that baseline expression of TEs and epigenetic regulators correlates with overall response rate (ORR) in sarcoma CPI clinical trials. Methods: This is a retrospective analysis of bulk RNA-sequencing data from pre-treatment biopsies of patients on CPI trials in sarcoma (pembrolizumab plus talimogene laherparepvec, nivolumab plus bempegaldesleukin, and pembrolizumab plus epacadostat). Sixty-seven samples from unique patients representing 12 subtypes were analyzed. The MCP counter deconvolution method and unsupervised clustering were used to group samples by immune phenotypes resulting in immune ‘hot’ and ‘cold’ clusters. ORR was defined by RECIST. To determine if baseline expression of TEs and epigenetic regulators significantly predicted immune types, we implemented a lasso penalized logistic regression. Results: Immune ‘hot’ tumors were characterized by increased immune infiltrates including CD8+ T-cells, B-cells, and NK cells vs ‘cold’ tumors. Patients with ‘hot’ vs ‘cold’ tumors had an ORR of 30.5% (11/36) vs. 3.2% (1/31) (p = 0.003; chi-squared). The best predictors of ‘hot vs ‘cold’ was the increased expression of multiple TE families including MER45A, MER57F, and LTR21B (respective lasso coefficients, 0.27, 0.07, and 0.07). Expression of IKZF1, a chromatin-interacting transcription factor, was also predictive (lasso coefficient, 0.35) and increased expression correlated with improved ORR (p = 0.003; unpaired t-test). TE and IKFZ1 expression was significantly correlated with CD8+ T-cell signaling and antiviral response pathways such as cGAS-STING (MER57F, r2= 0.43, padj = 1.75E-4; IKZF1, r2= 0.63, padj = 6.28E-9) and type II interferon (MER57F, r2= 0.67, padj = 2.51E-10; IKZF1, r2= 0.60, padj = 7.19E-8). Increased expression of cGAS-STING (p = 3.9E-4; unpaired t-test) and type II interferon pathways (p = 1.89E-10; unpaired t-test) was significant in ‘hot’ tumors. Conclusions: Immune ‘hot’ baseline immune profiles of sarcoma are associated with improved ORR to CPI and with increased expression of TEs and IKZF1. These differences in gene expression correlate with increased inflammatory signaling, which suggests a response to TE-encoded viral-like sequences that are typically epigenetically silenced. Induction of TE de-repression and IKZF1 expression through epigenetic targeting warrants pre-clinical investigation as a strategy to promote CPI response in sarcomas.

Non-invasive Detection of Immunotherapy-Induced Adverse Events.

Cancer immunotherapy has markedly improved the prognosis of patients with a broad variety of malignancies. However, benefits are weighed against unique toxicities, with immune-related adverse events (irAE) that are frequent and potentially life-threatening. The diagnosis and management of these events are challenging due to heterogeneity of timing onset, multiplicity of affected organs, and lack of non-invasive monitoring techniques. We demonstrate the use of a granzyme B-targeted PET imaging agent (GZP) for irAE identification in a murine model. We generated a model of immunotherapy-induced adverse events in Foxp3-DTR-GFP mice bearing MC38 tumors. GZP PET imaging was performed to evaluate organs non-invasively. We validated imaging with ex vivo analysis, correlating the establishment of these events with the presence of immune infiltrates and granzyme B upregulation in tissue. To demonstrate the clinical relevance of our findings, the presence of granzyme B was identified through immunofluorescence staining in tissue samples of patients with confirmed checkpoint inhibitor-associated adverse events. GZP PET imaging revealed differential uptake in organs affected by irAEs, such as colon, spleen, and kidney, which significantly diminished after administration of the immunosuppressor dexamethasone. The presence of granzyme B and immune infiltrates were confirmed histologically and correlated with significantly higher uptake in PET imaging. The presence of granzyme B was also confirmed in samples from patients that presented with clinical irAEs. We demonstrate an interconnection between the establishment of irAEs and granzyme B presence and, for the first time, the visualization of those events through PET imaging.

Transcriptomic mapping on Notch signaling in A luminal phenotype breast cancer.

e12539 Background: The Notch signaling pathway plays an important role in cell-differentiation, survival, proliferation, stem-cell renewal, and in determining cell fate during development and morphogenesis. The dysregulation of the Notch pathways contributes to carcinogenesis, cancer stem cell renewal, angiogenesis, and chemo-resistance. Elevated levels of Notch receptors and ligands have been associated with cancer-progression and poor survival. A less explored function of Notch pathways in cancer is their role in leukocyte homing and activation. Understanding their role and relationship with immune infiltrates is an area of interest in cancer research. Methods: The expression of four different Notch genes (Notch1, Notch2, Notch3 and Notch4) was explored in relation with A luminal breast cancer patient outcome using transcriptomic data (Affymetrix dataset, exploratory cohort) and the METABRIC study (validation cohort). The TIMER online tool was used to explore the association of the identified notch and immune infiltration, and the TCGA and METABRIC studies to analyze the correlation between notch1 - 4 expression and genomic signatures of immune activation. Results: We identified 2 individual genes called Notch1 and Notch2, which predict favorable prognosis in luminal A breast cancer. Their expression positively correlated with the presence of immune infiltrates within the tumor (dendritic cells, CD4+ T cells, neutrophils, CD8+ T cells and B cells), with markers of T cell activation and antigen presentation, and with gene signatures of immune surveillance (cytotoxic T lymphocyte activation and IFN gamma signature). By contrast Notch3 and Notch4 which predicted for detrimental outcome were not associated with any of these parameters. Conclusions: Our analysis identifies a Notch signature composed of 2 genes with potential to recognize immune infiltrated and activated A luminal phenotype breast cancers with favorable prognosis.

Chaperonin-Containing TCP1 Subunit 6A Is a Prognostic Potential Biomarker That Correlates With the Presence of Immune Infiltrates in Colorectal Cancer.

AimsChaperonin-containing TCP1 subunit (CCT) 6A is an oncogenic 6th subunit of the CCT family. Nevertheless, not much is documented regarding its function in colorectal cancer (COAD). This investigation seeks to explore the role of CCT6A in the prognosis of COAD.Main MethodsSequencing data from the Gene Expression Omnibus (GEO) and Cancer Genome Atlas database (TCGA) were employed to analyze the expression of CCT6A and its involvement in various regulatory networks behind COAD. Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA) analyzed Levels of expression and survival rates, while GEPIA was used to uncover further the functional networks that involved CCT6A. Database for Annotation, Visualization, and Integrated Discovery (DAVID) tools were used to interpret Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. Evaluation of the expression levels of CCT6A in COAD samples was also verified via immunohistochemistry.Key FindingsWe found that the expression of CCT6A is up-regulated in COAD. CCT6A correlated with poor prognosis and decreased immune infiltrates such as CD4+ T cells, B cells, and dendritic cells. CCT6A is increased in COAD patients. CCT6A is associated with several gene networks related to the DDX family and mismatch repair pathways.SignificanceOur data showed that data mining was able to uncover data regarding levels of CCT6A and its involvement in genetic regulating pathways in COAD.

Abstract PO-068: Intratumoral heterogeneity of prognostic multigene signatures for breast cancer

Abstract Background Multigene assays for breast cancer are becoming more commonly used for research, diagnostic, and treatment-selection purposes. These assays are commonly based on a single tumor sample, so this study assesses whether clinically-relevant multigene scores are subject to misclassification due to technical variation or intratumoral heterogeneity. Methods Using FFPE blocks, tumors from 37 patients were sampled at different pathologist-selected spatial locations, targeting regions with distinct histological appearance. A second pathologist assessed mitotic activity and presence of immune infiltration for each specimen. Gene expression was quantified using the PAM50 assay. Samples were classified with respect to intrinsic subtype (Luminal A, Luminal B, HER2-enriched, or Basal-like) and risk of recurrence with proliferation score (ROR-P, high vs. med/low). Euclidean distances between samples were calculated across the 50 genes, and the distribution of Euclidean distances were compared for samples that were concordant vs. discordant for each multigene classifier for PAM50 or ROR-P. Results Of the 37 tumors, 11 had samples with discordant PAM50 subtype and 7 had samples with discordant ROR-P group (75% and 83% agreement, respectively). The Euclidean distance between paired samples with discordant PAM50 intrinsic subtype or ROR-P score was significantly greater than that among concordant tumors. Samples discordant for ROR-P had more heterogeneity in histological characteristics, while discordance of PAM50 subtype call was generally not predicted by heterogeneity of these histological characteristics. Conclusion The concordance of PAM50 subtype calls and ROR-P, despite oversampling of heterogenous-appearing tumor regions, demonstrates reproducibility of the PAM50 classifier for breast cancer tumor intrinsic subtyping. However, assay sampling strategies for histologically heterogeneous tumors merit further consideration. Citation Format: Amber N. Hurson, Alina Hamilton, Linnea T. Olsson, Erin L. Kirk, Benjamin C. Calhoun, Joseph Geradts, Mark E. Sherman, Melissa A. Troester. Intratumoral heterogeneity of prognostic multigene signatures for breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-068.

The immune microenvironment of uterine adenosarcomas

BackgroundUterine adenosarcoma (UA) is an extremely rare sarcoma subtype. There has been limited evaluation of the immune microenvironment in these tumors. The objective of this study is to examine and describe the immune infiltrate and PD-1/PD-L1 expression in UA and to correlate these changes in the tumor micro-environment with the overall survival status or the disease-free survival status (DFSS), respectively.MethodsPatients (pts) treated at our center from 1982 to 2014 with UA were identified. Fifteen cases had tumor paraffin-embedded blocks available. Immunohistochemistry studies for CD3, CD8, FOXP3, CD163, PD-1 and PD-L1 (clone 22C3) were performed. Image analysis was used to assess the density (cells/mm2), except in PD-L1, where the percentage of membranous staining on tumor cells was noted.ResultsImmune infiltrate analysis median (range) density in cells/mm2 varied broadly: CD3 178 (15–802); CD8 117 (11–661); FoxP3 4.8 (0.2–82); CD163 791 (264–1861); and PD1 5 (1–65). 3 cases had rare (1%) PD-L1 tumor membranous labeling. The reports yielded that ten pts were alive, and 5 were dead. Pts who were alive had significant higher CD3 and CD8 median densities in tumors than those who were dead (p = 0.040). There was no correlation between DFSS and CD3 or CD8 median densities. Patients who had no local recurrence had significantly higher CD3 and CD8 median densities in tumors than those who had local recurrence (p = 0.040).ConclusionsIn conclusion, this is the first report characterizing the presence of immune infiltrate and PD-1/PD-L1 expression in UA. CD3+ CD8+ T-cells density may be prognostic. The immune-responsiveness of UA needs to be further investigated in a larger study.

The role of immune infiltrates as prognostic biomarkers in patients with breast cancer.

The presence of immune infiltrates in the tumor microenvironment has been documented in many types of cancer. Moreover, the preexistent or endogenous immunity which consists of interactions between intratumoral lymphocytes and tumor cells is mostly relevant for the successful application of various anticancer therapies, including standard chemotherapy, immune checkpoint inhibition-based immunotherapy and targeted therapies. The immunoscore defines densities of intratumoral immune infiltrates which determine poor or favorable prognosis depending on their quantity and quality in the tumor compartments. Results from large clinical studies have demonstrated an association between high densities of cytotoxic and memory TILs in the tumor compartments with improved prognosis. Importantly, we have demonstrated that differential combined densities of immune infiltrates jointly analyzed in the tumor center (TC) and the invasive margin (IM) have a significant prognostic value in breast cancer patients with poor clinicopathological parameters.

Atezolizumab for the treatment of breast cancer

ABSTRACTIntroduction: Breast cancer (BC) is the most common cancer diagnosed among women. The development of new personalized therapeutic strategies has reshaped the landscape in this field. However, BC is still the first cause of death among women. Interestingly, several preclinical studies and some clinical evidences are focused their attention on the role of immune system and immunotherapy on cancer control, also in BC.Areas covered: Usually, BC has been considered a not immunogenic tumor for its low mutational load. However, recent studies have evidenced that some subtypes, triple negative and HER-2 positive BC, are ‘hot’ tumors, thus more immunogenic. Moreover, the presence of immune infiltrate is positively associated with favorable prognosis. Therefore, the use of immune-checkpoint inhibitors seems to be an encouraging treatment option also in BC. Among these drugs, atezolizumab is an anti-PD-L1 monoclonal antibody with a particular structure that reduce antibody-dependent cellular cytotoxicity against T cells, increasing quantitatively and qualitatively the effective response.Expert opinion: The use of immunotherapy is a promising option for BC. However, at the same time it still raises many doubts. Surely, the research and the validation of immune biomarkers can permit to identify patients who more benefit from these drugs.

Epididymal cysteine-rich secretory proteins are required for epididymal sperm maturation and optimal sperm function.

What is the role of epididymal cysteine-rich secretory proteins (CRISPs) in male fertility? While epididymal CRISPs are not absolutely required for male fertility, they are required for optimal sperm function. CRISPs are members of the CRISP, Antigen 5 and Pathogenesis related protein 1 (CAP) superfamily and are characterized by the presence of an N-terminal CAP domain and a C-terminal CRISP domain. CRISPs are highly enriched in the male reproductive tract of mammals, including in the epididymis. Within humans there is one epididymal CRISP, CRISP1, whereas in mice there are two, CRISP1 and CRISP4. In order to define the role of CRISPs within the epididymis, Crisp1 and Crisp4 knockout mouse lines were produced then interbred to produce Crisp1 and 4 double knockout (DKO) mice, wherein the expression of all epididymal CRISPs was ablated. Individual and DKO models were then assessed, relative to their own strain-specific wild type littermates for fertility, and sperm output and functional competence at young (10-12 weeks of age) and older ages (22-24 weeks). Crisp1 and 4 DKO and control mice were also compared for their ability to bind to the zona pellucida and achieve fertilization. Knockout mouse production was achieved using modified embryonic stem cells and standard methods. The knockout of individual genes was confirmed at a mRNA (quantitative PCR) and protein (immunochemistry) level. Fertility was assessed using breeding experiments and a histological assessment of testes and epididymal tissue. Sperm functional competence was assessed using a computer assisted sperm analyser, induction of the acrosome reaction using progesterone followed by staining for acrosome contents, using immunochemical and western blotting to assess the ability of sperm to manifest tyrosine phosphorylation under capacitating conditions and using sperm-zona pellucida binding assays and IVF methods. A minimum of three biological replicates were used per assay and per genotype. While epididymal CRISPs are not absolutely required for male fertility, their production results in enhanced sperm function and, depending on context, CRISP1 and CRISP4 act redundantly or autonomously. Specifically, CRISP1 is the most important CRISP in the establishment of normally motile sperm, whereas CRISP4 acts to enhance capacitation-associated tyrosine phosphorylation, and CRISP1 and CRISP4 act together to establish normal acrosome function. Both are required to achieve optimal sperm-egg interaction. The presence of immune infiltrates into the epididymis of older, but not younger, DKO animals also suggests epididymal CRISPs function to produce an immune privileged environment for maturing sperm within the epididymis. Caution should be displayed in the translation of mouse-derived data into the human wherein the histology of the epididymis is someone what different. The mice used in the study were housed in a specific pathogen-free environment and were thus not exposed to the full range of environmental challenges experienced by wild mice or humans. As such, the role of CRISPs in the maintenance of an immune privileged environment, for example, may be understated. The combined deletion of Crisp1 and Crisp4 in mice is equivalent to the removal of all CRISP expression in humans. As such, these data suggest that mammalian CRISPs, including that in humans, function to enhance sperm function and thus male fertility. These data also suggest that in the presence of an environmental challenge, CRISPs help to maintain an immune privileged environment and thus, protect against immune-mediated male infertility. Not applicable. This study was funded by the National Health and Medical Research Council, the Victorian Cancer Agency and a scholarship from the Chinese Scholarship Council. The authors have no conflicts of interest to declare.

Abstract 2936: Molecular characterization of the immune subclass of hepatocellualr carcinoma

Abstract Background: Immune checkpoint inhibitors have emerged as a promising therapeutic approach in different solid tumors, including hepatocellular carcinoma (HCC). Nonetheless, little is known about the immune-component of HCC or potential biomarkers of response to these therapies. Aims: To perform comprehensive characterization of the HCC immunological profile and to identify biomarkers to select immunotherapy candidates. Methods: We performed gene expression array deconvolution through non-negative matrix factorization in 228 resected HCCs. Characterization of the transcriptional landscape was conducted using >1,000 signatures representing distinct immune cells by gene set enrichment and nearest template prediction analyses. Presence of immune infiltration, tertiary lymphoid structure (TLS), PD-1 and PD-L1 immunostainings was investigated using immunohistochemistry. DNA methylation profile of 450K CpG sites was analyzed to identify those with significant differences for each group. Extensive validation of the immune classifier was performed in 728 independent HCC samples. Results: Overall, an immune-related subclass of HCC was identified in ~27% of patients. The immune subclass was characterized by gene signatures identifying immune cells (i.e. T cells, TLS, cytotox, p<0.001), signatures of response to immune checkpoint therapy (p<0.001), presence of high immune infiltration (p=0.01), TLS (≥5 foci, 19/51 vs 33/168, p=0.01) and PD-1 and PD-L1 protein expression (p<0.05). The methylation levels of 363 CpG sites in 192 immune response gene promoters were able to capture the Immune class (ANOVA, p<0.05, Δβ>0.2 Tukey test). Integration with HCC molecular classifications revealed significant enrichment of the Immune subclass with IFN and S1 (p<0.001) and exclusion of the CTNNB1 and S2 (p<0.001) subclasses. The immune class contains two distinct microenvironment-based types: A) Exhausted immune response type (~35%) characterized by stromal activation, T cell exhaustion signatures, and presence of immunosuppressive components such as TGFB, LGALS1, M2 macrophages and pathways able to recruit myeloid-derived-suppressor cells (FDR<0.01); and B) Active immune response type (~65%) characterized by overexpression of adaptive immune response genes and IFN signaling (p<0.001). Tumors within the active immune response type showed a trend towards better survival vs rest (p=0.07). Conclusions: Around 27% of HCC patients belong to the Immune class, characterized by activation of immune cells and signatures of response to immunotherapies. Within this subclass, two distinct types have been characterized by presenting active or exhausted immune responses, a feature that provides the rationale for precision medicine-based therapies. Note: This abstract was not presented at the meeting. Citation Format: Daniela Sia, Yang Jiao, Iris Martinez, Olga Kuchuk, Carlos Villacorta Martin, Manuel Castro de Moura, Juan Putra, Genis Camprecios, Swan Thung, Samuel Waxman, Vincenzo Mazzaferro, Manel Esteller, Augusto Villanueva, Josep Maria Llovet. Molecular characterization of the immune subclass of hepatocellualr carcinoma [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 2936. doi:10.1158/1538-7445.AM2017-2936

Abstract 1845: Mutant p53 promotes progression and metastasis of mouse oral tumors induced by 4NQO, associated with specific immune infiltrates

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. The vast majority of the human HNSCCs contain p53 mutations, some of which acquire oncogenic gain-of-function (GOF) activities. Previous mouse models in which either the p53R172H GOF mutation or deletion of the p53 gene were co-activated with oncogenic K-ras showed that p53R172H can predispose to oral tumor initiation, accelerate tumor growth and promote progression to SCC. However, the impact of p53 gain- and loss-of-function (LOF) mutations in metastasis could not be assessed because of the low rates of progression to SCC in that model. To overcome this limitation, we used the tobacco-surrogate 4NQO to induce oral lesions that may progress to advance carcinomas following stepwise changes that resemble the gradual accumulation of histological and molecular abnormalities observed during human oral cancer progression. To examine the role of p53 mutations during SCC malignant progression we applied 4NQO to mice in which the p53R172H mutation or homozygous deletion of p53 were activated in oral epithelial cells. We observed that oral tumors appeared faster in the presence of p53 mutations than in mice with wtp53. No significant difference was observed between the p53R172H mutation and p53 deletion. However, survival was shorter in mice expressing p53R172H than in mice with loss of p53. Notably, regional and distant metastasis were only observed in mice with oral tumors expressing p53R172H. Therefore, these mice are excellent tools for pre-clinical studies designed to target early stages of tumor development or latest stages of progression and metastasis. As immunotherapy is becoming adopted as part of the standard treatment for head and neck cancer patients, we conducted a detailed characterization of the immune infiltrates of the lesions that developed in these mice in order to assess their immunogenicity. We observed a progressive increase in T-cell infiltration from normal oral mucosa to oral premalignant lesions (OPL) to SCC, in the presence or absence of p53 mutations. Similarly, the expression of immune checkpoint proteins, including PD1, PDL1 and CTLA4 increased during oral tumor progression, regardless of the p53 status. Interestingly, we observed a sharp increase in the presence of immune infiltrates expressing the co-stimulatory OX40 in SCCs relative to premalignant lesions, with different extent depending on the nature of the p53 mutation. The significance of these observations is currently being analyzed. In summary, p53R172H promotes metastasis in oral tumors induced by 4NQO. In addition, T-cell infiltration and activation of immune checkpoints during oral tumor progression suggest that this is an excellent model to test immunotherapy-based strategies. Furthermore, our findings suggest that OX40-based therapies may be tailored to patients according to their p53 status. Citation Format: Jin Wang, Yuanyuan Zhang, Bingbing Wang, Adel K. El-Naggar, Jeffrey N. Myers, Carlos Caulin. Mutant p53 promotes progression and metastasis of mouse oral tumors induced by 4NQO, associated with specific immune infiltrates [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 1845. doi:10.1158/1538-7445.AM2017-1845

Association of mesenchymal subtype with the presence of immune infiltrates in squamous cancer classifier.

e23073 Background: Lehmann and colleagues (Lehmann 2011) devised a classification system for triple negative breast cancer (TNBC) consisting of seven subtypes—IM, BL1, BL2, LAR, M, MSL, and UNS. Recent work has shown the IM group (characterized by the presence of immunomodulatory genes) is due to tumor infiltrating lymphocytes (TILs), and should be measured independently of the other subtypes. Furthermore, the m subtype (characterized by genes associated with epithelial-mesenchymal transition (EMT)) was shown to have a highly inverse relationship with the IM group (Lehmann 2016). We (Ring 2016) modified the original algorithm into a 101-gene algorithm that also confirmed this inverse relationship between IM and m signatures (Grigoriadis SABCS 2016). While this is a novel and interesting finding, we wanted to know whether this inverse relationship applies to other epithelial cell origin cancers such as squamous lung cancer. Methods: We downloaded gene expression data from 548 squamous cell lung cancers from the TCGA database, and applied the 101-gene TNBC algorithm to the lung cohort without optimization or adjustment. Results: Of the 548 samples, 389 (71%) were able to be classified by the 101-gene algorithm. Of these, 82 (21%) had an m phenotype. 128 patients (33%) were positive for the IM signature. The expected overlap of patients positive for both IM and m was 27 patients (7%). The observed overlap was 0 patients (chi-squared = 29; p < 0.001). Conclusions: The unoptimized 101-gene TNBC algorithm validated the observation that gene expression signatures for IM and m are inversely correlated. While any attempt to apply a TNBC algorithm to squamous lung cancer would need careful optimization and validation, these data support that the inverse relationship noted in TNBC may transcend the tissue of origin.

The Inhibitory G Protein α-Subunit, Gαz, Promotes Type 1 Diabetes-Like Pathophysiology in NOD Mice.

The α-subunit of the heterotrimeric Gz protein, Gαz, promotes β-cell death and inhibits β-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gαz protein would preserve functional β-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in Gαz knockout mice. By 17 weeks of age, Gαz-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive β-cells in Gαz-null islets despite similar immune infiltration in control mice. Islets from Gαz-null mice also exhibited a higher percentage of Ki-67-positive β-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, β-cell-specific Gαz-null mice phenocopy whole-body Gαz-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a β-cell-centric role for Gαz in diabetes pathophysiology. We propose that Gαz plays a key role in β-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of β-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response.

Abstract P1-07-03: Mesenchymal subtype negatively associates with the presence of immune infiltrates within a triple negative breast cancer classifier

Abstract Introduction: Lehmann and colleagues (Lehmann et al., 2011) devised a gene expression classification system for triple negative breast cancer (TNBC) consisting of seven subtypes—IM, BL1, BL2, LAR, M, MSL, and UNS (unselected). We (Ring et al., 2016) recently modified this original algorithm of 2188 gene subtyping into a 101-gene algorithm. In addition to a reduction of genes, the 101-gene algorithm has two methodological differences: first, the immunomodulatory (IM) signature was treated not as a subtype but rather as a binary feature of one of the other subtypes (e.g. BL1/IM+, LAR/IM-); second, when tumors—by a predefined correlation coefficient—showed traits of more than one subtype, both subtypes were reported as “dual subtypes.” Aim: Our aim was to apply the 101-gene algorithm for TNBC subtyping and to establish the relation of TNBC subtypes with their IM-status across several independent data sets. Methods: 951 patients from four independent TNBC cohorts with available gene expression data were analyzed by the 101-gene algorithm. Of these 848 were classified with at least one subtype. Results: The distribution of the 5 TNBC subtypes in both single and dual subtypes was 47%,10%,15%,18%,11%, for BL1, BL2, LAR, M, and MSL respectively. The majority of cases gave only one subtype (572, 67%) with M (Mesenchymal) being 9% (n=54) of these. Given this frequency of 9% of M as a baseline, in the remaining 276 (33%) cases with dual subtypes, the expectation that M would be one of the two is 11% (64 subtype calls). However, M is one of the two of the dual subtypes at a much higher frequency of 40% (222 subtype calls, Chi-Squared, P<0.0001). IM+ is a common feature across these cohorts (n=310 or 37%). When examining the IM feature within the patients exhibiting the M subtype as either a single subtype or one of the two dual subtypes (n=276, 33%), IM positive tumors are never of the M phenotype (Chi-Squared, p<0.0001). Conclusions: We further have confirmed with the 101-gene algorithm that the IM signature inversely associates with the M subtype as it has been observed with the 2188 gene algorithm (Lehmann et al., 2016). Moreover, the M signature is occasionally a confounder of other subtypes however still identifies those tumors negative for immune infiltrates. This raises important opportunities to understand the relationships between intrinsic tumor biology reflected in TNBC subtypes and their interaction with variable immune cell stroma which are the subject of ongoing analyses. Citation Format: Grigoriadis A, Quist J, Mirza H, Cheang MC, Ring BZ, Hout DR, Bailey DB, Seitz RS, Tutt AN. Mesenchymal subtype negatively associates with the presence of immune infiltrates within a triple negative breast cancer classifier [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-03.

155 - Presence of immune infiltrates in early phases of prostate cancer: Establishment of a preclinical efficacy model to promote immunotherapy development

155 - Presence of immune infiltrates in early phases of prostate cancer: Establishment of a preclinical efficacy model to promote immunotherapy development

CD8+ tumor infiltrating lymphocytes strongly correlate with molecular subtype and clinico-pathological characteristics in breast cancer patients from Sudan

Breast Cancer in the Middle East and North Africa region (MENA) occurs in a younger age group of patients and is more aggressive than in Europe and North America. Little is known about the presence of immune infiltration and clinico-pathologic correlates in patients from the MENA region. Immune infiltration of most solid tumors carries a prognostic role. Adaptive immune cells and cytotoxic T-cells, in particular, play a positive prognostic role in patients undergoing treatment for breast cancer. Therefore, our preliminary study was aimed to identify and quantify the presence of tumor infiltrating lymphocytes (TILs) (CD8+, CD45RO+, and CD3+) in invasive breast cancer patients from Sudan. Additionally, TIL was correlated with patients’ clinico-pathological characteristics. Forty-three archival Formalin Fixed Paraffin Embedded samples of patients diagnosed with invasive breast cancer were collected at The Radiation and Isotope Centre in Khartoum, Sudan. CD8, CD45RO and CD3 immunostaining of FFPE tissue sections was performed. The mean percentage of lymphocytes in the invasive margin (stromal TILs) was scored. Overall distribution of CD3+, CD8+ and CD45RO+ in the tumor invasive margin were 100 %, 100 %, and 92.9 %, respectively. CD8+ TILs were significantly correlated with HER2/neu positive breast cancers and HER2/neu (+) tumors. Additionally, CD3+ TILs were significantly associated with lymph node metastasis. None of the other clinico-pathological variables had any significant association with CD3+ and CD45RO+. Our limited and preliminary study identified a strong correlation between CD8+ TILs and HER2/neu positive invasive breast cancers and HER2/neu (+) tumors.