Analysis Of Tumor Immune Microenvironment Research Articles

Abstract C013: Analysis of tumor immune microenvironment and clinical outcomes in undifferentiated pancreatic carcinomas with and without osteoclast-like giant cells

Abstract Background: Undifferentiated carcinoma (UC) is a rare (<1%) subtype of pancreatic cancer that was revised in 2010 per WHO classification as UC with or without osteoclast-like giant cells (OGC), affecting interpretation of prior literature comparing UC to pancreatic ductal adenocarcinomas (PDAC). Limited retrospective data suggest UC-OGC occurs at younger age with larger tumor size but the impact on prognosis is unclear, and there no data on the tumor immune microenvironment (TIME). Methods: The aim of the study was to contrast the differences in targeted genomics, TIME and survival between UC and UC-OGC. TIME was assessed via H&E, bulk RNA sequencing (RNA-seq), and multiplex immunofluorescence (mIF). Results: We identified 47 adult consecutive patients with UC (n=32) and UC-OGC (n=15) treated at Michigan Medicine between 2005 and 2021. The demographics, presenting symptoms, tumor size, and CA 19-9 were similar (p>0.05) between the two cohorts. In patients with advanced stage, median overall survival (OS) was 2.1 months (n=19) for UC vs 9.0 months (n=2) for UC-OGC (p=0.42). Thirteen patients from each cohort underwent resection, with median OS 1.84 years vs 11.92 years in UC vs UC-OGC, respectively (p=0.08). Median OS for all patients with UC (n=32) was 0.40 years compared to 10.82 years in UC-OGC (n=15) (p=0.003). The genomic landscape was similar in UC (n=9), UC-OGC (n=5) and PDAC (TCGA; n=159). KRAS and TP53 mutations were the most common alterations identified along with CDKN2A and SMAD4. RNA-seq demonstrated few differentially expressed genes between UC (n=7) and UC-OGC (n=3). Deconvolution with CIBERTSORT-X using the LM22 dataset demonstrated significantly increased antigen-presenting cells (APCs), specifically M2 macrophages, decreased cytotoxic T-cells, and decreased regulatory T cells in UC and UC-OGC tumors compared to PDAC. These findings were confirmed as significant (p<0.5) by multiplex mIF. Though UC (n=13) and UC-OGC (n=5) tumors had many similarities in their TIME, notable differences included decreased regulatory T cells (p=0.36) and a trend toward decreased M2 macrophages in UC-OGC tumors compared to UC (p=0.19). Conclusions: In this institutional series of pancreas UC with and without OGC, median OS in patients with UC-OGC was longer as compared to UC in both advanced and resected patients. Interestingly, there were significant differences in the TIME but not in the genomic profiles. Increased pro-tumorigenic M2 macrophage infiltration and decreased Tregs in the TIME of UC/UC-OGC compared to PDAC suggests a potential role of macrophage inhibitors and other immune-modulatory therapies in combination with chemotherapy for these rare variants of pancreatic cancer. Citation Format: Jamie N. Mills, Valerie Gunchick, Jiaqi Shi, Aaron Udager, Timothy Frankel, Mark M. Zalupski, Vaibhav Sahai. Analysis of tumor immune microenvironment and clinical outcomes in undifferentiated pancreatic carcinomas with and without osteoclast-like giant cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C013.

Detailed characterization of PD-1/PD-L1 and CTLA4 expression and tumor-infiltrating lymphocytes in yolk sac tumors

BackgroundImmune checkpoint blockade (ICB) is considered as a promising approach for cancer treatment. However, the potency of ICB therapy in yolk sac tumors (YSTs) has not been confirmed, and the comprehensive analysis of tumor immune microenvironment and the expression of PD-1/PD-L1 and CTLA4 were also not thoroughly evaluated. MethodsImmunohistochemistry was performed in formalin-fixed, paraffin-embedded tumor specimens from 23 YSTs patients to detect the density and distribution of tumor-infiltrating T cells, tertiary lymphoid structures (TLSs), as well as the expression of PD-1/PD-L1 and CTLA4. ResultsOverall, more than half (61 %) of all patients exhibited an immune-desert phenotype based on CD3+ T cells. PD-1 expression was identified in five tumor samples (21.7 %), and PD-L1 expression exhibited a different positive rate in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) (39.1 % and 17.4 %). Noteworthily, the rate of positive CTLA4 expression in both TCs and TILs was markedly higher (69.6 % and 56.5 %) than those of PD-1 and PD-L1 expression. Furthermore, TLSs were observed in 21.74 % of all tissues, and samples with TLSs exhibited significantly higher densities of TILs and higher expression of immune checkpoint molecules, particularly PD-1/PD-L1. In addition, tumors located in testes also exhibited a higher density of TILs and higher expression of immune checkpoint molecules. ConclusionGenerally a high frequency of CTLA4 expression was found, PD-1/PD-L1 expression, the immune-inflamed phenotype, and TLSs were low frequency in YSTs, however, YSTs in testes showed a higher density of TILs and higher expression of immune checkpoint molecules.

Tumor immune microenvironment (TIME) analysis of stage I lung adenocarcinoma with micropapillary pattern.

e20510 Background: Micropapillary is one of the most aggressive histologic subtypes of lung adenocarcinoma, which is considered a poor prognostic marker. Comprehensive analysis of tumor immune microenvironment (TIME) features of micropapillary of early invasive lung adenocarcinoma may provide better insight and facilitate the development of novel strategies for early detection and intervention. Methods: A total of 15 patients who underwent anatomical resection and were pathologically diagnosed with stage I micropapillary adenocarcinomas were included in this study. Gene expression profiles (GEPs) of tumor immune-related 289 genes were analyzed using NanoString nCounter. Differentially expressed genes(DEGs), estimation of TIME cell infiltration, and TIME signatures between micropapillary and non-micropapillary were assessed. Results: The number of micropapillary and non-micropapillary patients was 5 and 10. The comparison revealed CD69, IL6, CXCL8, CXCL2, POLR2A and so on were significantly upregulated in the micropapillary group compared with the non-micropapillary group, while HLA-DPA1, IFIT2, and other seven genes were down-regulated in the micropapillary group. These DEGs were mostly enriched in “positive regulation of cytokine production”， “T cell activation”， “receptor ligand activity，” and “signaling receptor activator activity” pathways according to GO enrichment analysis. The results of the KEGG analysis revealed that genes were significantly enriched in “IL-17 signaling pathway”, “TNF signaling pathway”, “cytokine-cytokine receptor interaction”, and so on. Meanwhile, different immune infiltration of the two groups was analyzed. Totally, there was a better TIME cell infiltration in the micropapillary group compared with the non-micropapillary group. Especially, the score of DCs and mast cells was significantly higher in micropapillary group. However, TIME signature scores were similar in both groups. Conclusions: Our study demonstrated that the TIME between micropapillary and non-micropapillary of stage I lung adenocarcinoma was different. Increased mast cells explained poor prognosis in lung adenocarcinoma with micropapillary pattern. A high level of DCs infiltration indicated that micropapillary adenocarcinoma might benefit from immunotherapy.

The changes of HER3 expression in head and neck cancer patients treated with induction chemotherapy.

e18004 Background: Erb-b2 receptor tyrosine kinase 3 (HER3) is broadly expressed in various types of cancer and is associated with poor prognosis. Recent evidence supports HER3 plays a role in acquired resistance to chemotherapy. The change of HER3 expression by chemotherapy in head and neck squamous cell carcinoma (HNSCC) remains unclear. Methods: We evaluated 95 patients with locally advanced HNSCC who had received induction chemotherapy (ICT) between 2009 and 2019. Immunohistochemical staining for HER3 was performed using formalin-fixed paraffin-embedded surgical specimens and biopsy samples. H-score of membrane staining was evaluated. Results: Median age of patients was 64 years, and 85% were male. Primary locations included oropharynx (54.8%), hypopharynx (21.5%), oral cavity (17.2%), and salivary gland (6.5%). Mean H-score of HER3 in tumor tissues was 77.6 ± 7.9. H-score of HER3 in HPV-positive tumors was higher than HPV-negative tumors (p = 0.019), which was more pronounced in oropharynx and hypopharynx tumors. While, H-score of HER3 in adjacent normal tissues of these tumors did not significantly differ. Compared with the non-smokers, tumor tissues of current or ex-smokers demonstrated higher H-score of HER3 (p = 0.033), independent of tumor location. In contrast, in adjacent normal tissues, no significant differences of H-score of HER3 were observed depending on the smoking status. The H-score higher than 100 was defined as HER3-high group. Among oropharynx and hypopharynx tumors with positive correlation between H-score of HER3 and HPV status (n = 56), HER3-high group was significantly correlated with HPV positivity (p = 0.009) and good response to ICT (p = 0.035). Good clinical response was also strongly related to HPV-positivity (p = 0.001). In addition, there was a tendency for longer overall survival in HER3-high group than low HER3 expression group (93 vs. 70 months, p = 0.053). The paired tumor samples collected at pre- and post-ICT (n = 21) were analyzed. HER3 expressions in adjacent normal tissue were higher at post-ICT compared to pre-ICT (p = 0.003), and these results were more pronounced in the non-smoking group, but no differences were found according to HPV-status. Conclusions: Taken together, our results showed that H-score of HER3 was significantly higher in both HPV-positive and smoking group. Interestingly, high HER3 expression in oropharynx and hypopharynx tumors was associated with HPV positivity and good clinical response to ICT. Additional analysis of tumor immune microenvironment in tumor tissues are underway.

The predictive value of prognosis and therapeutic response for STAT family in pancreatic cancer

BackgroundSignal transducers and activators of transcription (STAT) proteins, well-known cytoplasmic transcription factors, were found to be abnormally expressed in various cancers and play essential parts in the initiation, progression and therapy resistance of cancer. Nevertheless, the functions of different STATs in pancreatic cancer (PC) and their relationship to the prognosis and immune infiltration as well as drug efficacy in PC patients have not been systematically elucidated. MethodsExpression, prognosis, genetic alterations and pathway enrichment analyses of the STAT family were investigated via Oncomine, GEPIA, Kaplan Meier-plotter, cBioPortal, Metascape and GSEA. Analysis of tumor immune microenvironment was conducted by ESTIMATE and TIMER. “pRRophetic” packages were used for analysis of chemotherapeutic response. Finally, the diagnostic and prognostic value of key STATs were further validated through public datasets and immunohistochemistry. ResultsIn this study, only STAT1 mRNA level was significantly increased in tumor tissues and highly expressed in PC cell lines via multiple datasets. PC patients with higher STAT1/4/6 expression had a worse overall survival (OS) and progression-free survival (PFS), while higher STAT5B expression was correlated with better prognosis in the TCGA cohort. The STATs-associated genes were enriched in pathways about the remodeling of tumor immune microenvironment. The STATs levels were significantly correlated with immune infiltration, except STAT6. The STAT1 was identified as a potential biomarker and its diagnostic and prognostic value were further validated at mRNA and protein levels. GSEA showed that STAT1 may be involved in the progression and immune regulations of PC. Moreover, STAT1 expression was significantly related to the level of immune checkpoint, and predicted immunotherapy and chemotherapy responses. ConclusionSTAT family members were comprehensively analyzed and STAT1 was identified as an effective biomarker for predicting the survival and therapeutic response, which could be beneficial to develop better treatment strategies.

Comparative analysis of tumor immune microenvironment in primary tumors vs metastatic tissue in microsatellite stable metastatic colorectal cancer.

187 Background: We previously demonstrated differences in responses to checkpoint inhibitors between liver and non-liver metastases (mets) in microsatellite stable (MSS) metastatic colorectal cancer (mCRC). Here, we investigate differences in tumor microenvironment (TME) and mutational landscape of primary and mCRC sites. Methods: We used the Tempus clinicogenomic database to select de-identified cases of MSS mCRC which underwent Tempus xT testing. Tempus xT is a 648-gene NGS panel that detects somatic alterations and includes RNA sequencing. Gene expression patterns of different immune cells were used to predict their relative abundance within the tumor. Analyzed tumors were divided into 4 categories based: primary tumor (colon), liver, lung, and peritoneal mets. Demographics, genomic alterations, tumor mutational burden (TMB), PD-L1 expression, and proportions of B, T (CD4+, CD8+), NK cells, and macrophages were compared between all 4 categories. Chi-squared/Fisher’s exact tests or Kruskal-Wallis tests were used to assess statistical significance. Results: Genomic profiles from 4,345 unique mCRC patients were analyzed (Primary CRC = 1734; Liver = 1825; Lung = 443; Peritoneal = 343). Median TMB and PD-L1 expressions had small differences across sites that were statistically significant. KRAS mutations were more frequent and SMAD4 mutations were less prevalent in lung mets. RNA-Seq revealed significant differences in immune cell composition. Lung mets exhibited a higher percentage of B cells and lower percentage of macrophages than liver and peritoneum (p< 0.001), similar to primary tumors. CD4+ T cells were highest in primary tumors, whereas CD8+ T cells were highest in lung mets (p < 0.001). Conclusions: The TME of lung mets has a more favorable immune composition (increased CD8+ T cells and B cells, decreased macrophages) than that of liver and peritoneal mets, suggesting that MSS mCRC to the lung may potentially be more responsive to immunotherapy.[Table: see text]

Replication stress identifies novel molecular classification associated with treatment outcomes in pancreatic cancer

BackgroundReplication stress is a prominent hallmark of tumor cells, which is crucial for maintaining genomic integrity. However, it remains poorly understood whether replication stress can serve as a surrogate biomarker to indicate prognosis and treatment response of pancreatic cancer. MethodsTranscriptomic and clinical data were obtained from The Cancer Genome Atlas and literature. An integrated signature of 18 replication-stress associated genes (termed as REST18) was established using the cox proportional hazards regression analysis. Tumors were sorted into REST18-low and REST18-high groups. Survival analysis, gene set enrichment analysis and composition of immune cells were compared between these tumors. ResultsPatients with REST18-high tumors showed worse prognoses than those with REST18-low tumors in the TCGA database and the finding is validated in an independent cohort of pancreatic cancer. Comparison of REST18 model and other molecular classifications showed that REST18-high tumors are positively correlated to basal-like or squamous phenotypes, which have higher metastasis potential. DNA repair pathway is enriched in the REST18-high tumors. Analysis of tumor immune microenvironment found that REST18-high tumors are characterized with “immune-cold” features. Univariate and multivariate analysis show that REST18 is an independent risk factor for overall survival and predicts outcomes of chemotherapy in pancreatic cancer. ConclusionREST18 is a novel biomarker to indicate prognosis and treatment response of chemotherapy in pancreatic cancer.

Comprehensive analysis of tumor immune microenvironment and prognosis of m6A-related lncRNAs in gastric cancer

BackgroundN6-methyladenosine (m6A) modification and long non-coding RNAs (lncRNAs) play pivotal roles in gastric cancer (GC) progression. The emergence of immunotherapy in GC has created a paradigm shift in the approaches of treatment, whereas there is significant heterogeneity with regard to degree of treatment responses, which results from the variability of tumor immune microenvironment (TIME). How the interplay between m6A and lncRNAs enrolling in the shaping of TIME remains unclear.MethodsThe RNA sequencing and clinical data of GC patients were collected from TCGA database. Pearson correlation test and univariate Cox analysis were used to screen out m6A-related lncRNAs. Consensus clustering method was implemented to classify GC patients into two clusters. Survival analysis, the infiltration level of immune cells, Gene set enrichment analysis (GSEA) and the mutation profiles were analyzed and compared between two clusters. A competing endogenous RNA (ceRNA) network and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were applied for the identification of pathways in which m6A-related lncRNAs enriched. Then least absolute shrinkage and selection operator (LASSO) COX regression was implemented to select pivotal lncRNAs, and risk model was constructed accordingly. The prognosis value of the risk model was explored. In addition, the response to immune checkpoint inhibitors (ICIs) therapy were compared between different risk groups. Finally, we performed qRT-PCR to detect expression patterns of the selected lncRNAs in the 35 tumor tissues and their paired adjacent normal tissues, and validated the prognostic value of risk model in our cohort (N = 35).ResultsThe expression profiles of 15 lncRNAs were included to cluster patients into 2 subtypes. Cluster1 with worse prognosis harbored higher immune score, stromal score, ESTIMATE score and lower mutation rates of the genes. Different immune cell infiltration patterns were also displayed between the two clusters. GSEA showed that cluster1 preferentially enriched in tumor hallmarks and tumor-related biological pathways. KEGG pathway analysis found that the target mRNAs which m6A-related lncRNAs regulated by sponging miRNAs mainly enriched in vascular smooth muscle contraction, cAMP signaling pathway and cGMP-PKG signaling pathway. Next, eight lncRNAs were selected by LASSO regression algorithm to construct risk model. Patients in the high-risk group had poor prognoses, which were consistent in our cohort. As for predicting responses to ICIs therapy, patients from high-risk group were found to have lower tumor mutation burden (TMB) scores and account for large proportion in the Microsatellite Instability-Low (MSI-L) subtype. Moreover, patients had distinct immunophenoscores in different risk groups.ConclusionOur study revealed that the interplay between m6A modification and lncRNAs might have critical role in predicting GC prognosis, sculpting TIME landscape and predicting the responses to ICIs therapy.

The analysis of tumor immune microenvironment in muscle invasive bladder cancer treated with neoadjuvant chemotherapy.

The analysis of tumor immune microenvironment in muscle invasive bladder cancer treated with neoadjuvant chemotherapy.

Comprehensive Analysis of Tumor Immune Microenvironment and Prognosis of m6A-Related IncRNAs in Lung Adenocarcinoma.

Lung cancer is a fatal disease with high morbidity and mortality. As a major type of non-small cell lung cancer (NSCLC), lung adenocarcinoma (LUAD) has come into the focus of the biological and clinical research. Nevertheless, potential functions of N6-methyladenosine (m6A) methylation-related long non-coding RNAs (lncRNAs) in the tumor immune microenvironment (TIME) remain unclear. Here, we analyzed RNA-seq data of 522 samples from the TCGA-LUAD project. Based on integrative analyses of lncRNA, immune, and clinical profiles, we developed a computational method to determine the characteristics of lncRNAs as an indicators of immune cell infiltration in LUAD patients. Subsequently, we investigated the influence of m6A-related lncRNAs on prognosis and immunotherapy in patients with LUAD. We identified 24 m6A-related lncRNAs that were related to LUAD prognosis and clustered in two molecular subtypes (clusters 1 and 2). Our results suggest that cluster 1 was significantly related to downregulation of PD-L1, enhancement of immune cell infiltration, and good prognosis. Furthermore, p53 and mTOR pathways were enriched in cluster 1, whereas in the cluster 2, PPAR was uncommonly enriched. Patients with LUAD who had lower risk scores exhibited higher immunoscores and lower expression levels of programmed cell death-ligand 1, compared with patients who had higher risk scores. Finally, m6A-related lncRNAs were implicated in the TIME; our findings indicate that these lncRNAs are critical within the TIME in LUAD. These signatures are promising as potential targets for improved LUAD immunotherapy.

A three-phase trans-ethnic study reveals B7-H3 expression is a significant and independent biomarker associated with colon cancer overall survival.

There have been inconsistent results and conflicting conclusions among the existing prognostic studies of B7-H3 expression in colon cancer patients. Therefore, the association between B7-H3 expression and colon cancer survival has remained largely unclear. We performed a three-phase and trans-ethnic prognostic study of B7-H3 expression in colon cancer patients involving perhaps the largest population to date. In the discovery phase, we utilized a Cox proportional hazards model adjusted for covariates to test the association between B7-H3 expression and colon cancer overall survival (OS) time in a European population from The Cancer Genome Atlas (TCGA) cohort (n=433). In the validation phase I, the association was replicated in a European population from Gene Expression Omnibus (GEO) cohort (n=811). In the validation phase II, we again confirmed the significant association in an Asian population from Fujian Medical University Union Hospital (UNION) cohort (n=179). Furthermore, a series of Kaplan-Meier analysis, bioinformatics analysis of tumor immune microenvironment (TIME), and immune checkpoint prognostic prediction analysis, as well as sensitivity analysis, were also conducted. Highly expressed B7-H3 was a significant and robust biomarker to colon cancer survival, with a large hazard ratio (HR) [HRTCGA =4.60, 95% confidence interval (CI): 2.15 to 9.83, P=8.37×10-05; HRGEO =1.47, 95% CI: 1.12 to 1.94, P=0.0056; HRUNION =1.63, 95% CI: 1.36 to 1.95, P=7.91×10-08]. We detected an involvement of B7-H3 in the tumor immune microenvironment (TIME). Meanwhile, B7-H3 was significantly and weakly correlated with 6 out of 27 well-recognized immune checkpoint genes. Even after adjusting for effects of other immune checkpoint genes, B7-H3 still exhibited a harmful effect on colon cancer survival using samples from TCGA and GEO cohorts (HR =1.47, 95% CI: 1.07 to 2.02, P=0.0184), indicating that it was an independent prognostic factor of colon cancer. We also proposed an immune checkpoint prognostic risk score which possessed the capability to identify colon cancers with high risk of mortality. The expression of B7-H3 is a significant, robust, and independent prognostic factor to colon cancer OS.

CD200 Blockade Modulates Tumor Immune Microenvironment but Fails to Show Efficacy in Inhibiting Tumor Growth in a Murine Model of Melanoma.

CD200-CD200R pathway regulates immune responses and has been implicated in the pathogenesis of a number of cancer types. CD200 blockade is considered a strategy for immunotherapy of CD200-positive cancers such as melanoma. Thus, it is critical to understand the potential impacts of CD200 blockade in a more human relevant tumor model. In this study, we evaluated these issues using the CD200+ Yumm1.7 mouse melanoma model. Yumm1.7 cells bear Braf/Pten mutations resembling human melanoma. We found that Yumm1.7 tumors grow significantly faster in CD200R–/– mice compared to wild type mice. Analysis of tumor immune microenvironment (TIME) revealed that tumors from CD200R–/– or anti-CD200 treated mice had downregulated immune cell contents and reduced TCR clonality compared to tumors from untreated wild type mice. T cells also showed impaired effector functions, as reflected by reduced numbers of IFN-γ+ and TNF-α+ T cells. Mechanistically, we found upregulation of the CCL8 gene in CD200R–/– tumors. In vitro co-culture experiments using Yumm1.7 tumor cells with bone marrow derived macrophages (BMDM) from WT and CD200R–/– mice confirmed upregulation of macrophage CCL8 in the absence of CD200-CD200R interaction. Finally, we found that anti-CD200 therapy failed to show efficacy either alone or in combination with checkpoint inhibitors such as anti-PD-1 or anti-CTLA4 in inhibiting Yumm1.7 tumor growth. Given that CD200R-deficiency or anti-CD200 treatment leads to reduced T cell responses in TME, using blockade of CD200 as an immunotherapy for cancers such as melanoma should be practiced with caution.

High Dimensional Imaging Mass Cytometry Panel to Visualize the Tumor Immune Microenvironment Contexture.

The integrative analysis of tumor immune microenvironment (TiME) components, their interactions and their microanatomical distribution is mandatory to better understand tumor progression. Imaging Mass Cytometry (IMC) is a high dimensional tissue imaging system which allows the comprehensive and multiparametric in situ exploration of tumor microenvironments at a single cell level. We describe here the design of a 39-antibody IMC panel for the staining of formalin-fixed paraffin-embedded human tumor sections. We also provide an optimized staining procedure and details of the experimental workflow. This panel deciphers the nature of immune cells, their functions and their interactions with tumor cells and cancer-associated fibroblasts as well as with other TiME structural components known to be associated with tumor progression like nerve fibers and tumor extracellular matrix proteins. This panel represents a valuable innovative and powerful tool for fundamental and clinical studies that could be used for the identification of prognostic biomarkers and mechanisms of resistance to current immunotherapies.

Abstract PS16-09: High infiltration of adipocytes is associated with inflammation, metastasis as well as favorable tumor immune microenvironment whereas low infiltration is associated with highly proliferative tumor in ER positive breast cancer patients

Abstract Background: Cancer associated adipocytes are known to cause inflammation and leads to the cancer progression, and metastasis. In the current study, we defined intra-tumoral adipocytes transcriptionally utilizing a computational algorithm, xCell, which allowed us to quantify infiltrating adipocytes. We hypothesized that high amount of intra-tumoral adipocytes is associated with aggressive cancer characteristics and poor survival outcome. Material and Methods: The clinicopathological data and survival information of 1090 breast cancer patients were obtained from The Cancer Genome Atlas (TCGA), GSE25066 and study by Yau et al. Survival analysis, gene set enrichment analysis (GSEA) were conducted comparing the intra-tumoral adipocyte high and low groups. The association between the amount of intra-tumoral adipocytes and Nottingham pathological grade or AJCC stage were evaluated. CYT score and xCell were utilized to evaluate intratumoral immune cell composition within breast cancer. Results: Our transcriptomically defined intra-tumoral adipocyte appropriately reflected mature adipocytes in a bulk tumor. Surprisingly, intra-tumoral adipocytes high tumors did not demonstrate worse survival, neither OS or DFS, in the whole cohort or any of the subtypes. Less aggressive Stage 1 even trended toward increased intra-tumoral adipocytes although statistically insignificant. Further, less proliferative grade 1 was associated with increased intra-tumoral adipocytes compared with other grades in whole and ER positive/HER2 negative (ER+/HER2-) subtype of TCGA cohort (p < 0.001). This result was consistent in GSE25066 cohort (p < 0.001). An amount of intra-tumoral adipocytes was weakly inversely correlated with MKI67 expression in three cohorts; TCGA, GSE25066 and the study by Yau et al (r = -0.235, r = -2.09 and r = -0.355, respectively). In agreement with the previous in vitro studies, adipocyte high tumors enriched the gene sets associated with inflammation, such as TNF-α and Inflammatory response. Further, intra-tumoral adipocyte high tumors enriched the gene sets related to metastasis, such as EMT, TGF-β signaling, and Notch signaling. On the other hand, adipocyte high tumors enriched the gene sets related to immune response such as IL2 signaling and IFN-γ especially in ER+/HER2- subtype. The analysis of tumor immune microenvironment revealed that intra-tumoral adipocyte high tumors were associated with favorable tumor immune microenvironment in ER+/HER2- subtype, but not in triple negative (TN). Consistent with the association with pathological grade and MKi67, intra-tumoral adipocyte low tumors enriched the cell cycle and cell proliferation gene sets, such as G2M checkpoint, in both TCGA and GSE25066 cohorts. These results force us to speculate that intra-tumoral adipocytes does cause inflammation and evoke metastatic pathways as previously reported in in vitro studies, but also associate with immune response, whereas adipocyte low tumors are highly proliferative cancers, thus, there are no difference in clinical outcome between them. Conclusion: High infiltration of adipocyte was associated with inflammation, metastatic pathways and favorable tumor immune microenvironment, whereas low infiltration of adipocytes was associated with highly proliferative tumor in ER-positive breast cancer. These cancer biologies may explain the reason why adipocyte infiltration high tumors did not demonstrate worse survival. Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Eriko Katsuta, Nobuhisa Matsuhashi, Manabu Futamura, Kazuhiro Yoshida, Kazuaki Takabe. High infiltration of adipocytes is associated with inflammation, metastasis as well as favorable tumor immune microenvironment whereas low infiltration is associated with highly proliferative tumor in ER positive breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-09.

The Impact of Immunofunctional Phenotyping on the Malfunction of the Cancer Immunity Cycle in Breast Cancer.

Simple SummaryThe cancer-immunity cycle (CIC) is a series of self-sustaining stepwise events to fight cancer growth by the immune system. We hypothesized that immunofunctional phenotyping that represent the malfunction of the CIC is clinically relevant in breast cancer (BC) utilizing total of 2979 BC cases; 1075 from TCGA cohort, 1904 from METABRIC cohort were analyzed. The immunofunctional phenotype was classified as follows: hot T-cell infiltrated, high immune cytolytic activity (CYT), cold T-cell infiltrated, high frequency of CD8+ T cells and low CYT, and non-inflamed, low frequency of CD8+ T cells and low CYT. We demonstrated that immunofunctional phenotyping not only indicated the degree of anti-cancer immune dysfunction, but also served as a prognostic biomarker and HTI was inversely related to estrogen response.The cancer-immunity cycle (CIC) is a series of self-sustaining stepwise events to fight cancer growth by the immune system. We hypothesized that immunofunctional phenotyping that represent the malfunction of the CIC is clinically relevant in breast cancer (BC). Total of 2979 BC cases; 1075 from TCGA cohort, 1904 from METABRIC cohort were analyzed. The immunofunctional phenotype was classified as follows: hot T-cell infiltrated (HTI), high immune cytolytic activity (CYT), Cold T-cell infiltrated (CTI), high frequency of CD8+ T cells and low CYT, and non-inflamed, low frequency of CD8+ T cells and low CYT. The analysis of tumor immune microenvironment in the immunofunctional phenotype revealed that not only immunostimulatory factors, but also immunosuppressive factors were significantly elevated and immunosuppressive cells were significantly decreased in HTI. Patients in HTI were significantly associated with better survival in whole cohort and patients in CTI were significantly associated with worse survival in triple negative. Furthers, HTI was inversely related to estrogen responsive signaling. We demonstrated that immunofunctional phenotype not only indicated the degree of anti-cancer immune dysfunction, but also served as a prognostic biomarker and HTI was inversely related to estrogen response.

Spatial analysis of tumor immune microenvironment (TIME) in patients treated with Bintrafusp alfa.

3070 Background: Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of TGF-βRII receptor (TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. In preclinical models, bintrafusp alfa treatment promoted CD8+ T cell and NK cell activation, and both immune cell (IC) populations were required for optimal bintrafusp alfa mediated tumor control. However, the effect of bintrafusp alfa on TIME in humans has not been reported. Methods: In this unplanned interim analysis of a biomarker expansion cohort (NCT 02517398), patients (pts) with advanced non-small cell lung cancer (NSCLC) underwent paired biopsies (bx) before and on treatment with bintrafusp alfa (~ 50 days apart). The objective was to evaluate frequency and localization of tumor infiltrated ICs by IHC. Out of 12 pts, 7 had matched (Pre vs Post) tumor-containing specimens sufficient for multiplex immunofluorescence (MxIF) analysis of TIME. Four pts were excluded as Post bx histology for 3/12 [2 PR (partial response), 1 SD (stable disease)] was negative for tumor (necrosis or fibrosis) and 1/12 did not have a Post bx performed. Results: TIME study shows CD8 T cell infiltrates were increased in Post compared to Pre bx (median 161 vs 62/mm²; interquartile range [IQR] 65–396/mm² vs 31–135/mm²; p = 0·04). While M2 macrophages were also increased (median 800 vs 367/mm²; IQR 776–1131/mm² vs 171–831/mm²; p = 0·04), the ratio of M1/M2 was reversed in pts with SD (↑) compared to pts with PD (↓). Other ICs such as CD4, T-regs, NK cells and M1 macrophages were not changed. On average compared to baseline, M2 macrophages were > 2 fold closer to every other IC in pts with PD, but > 2 fold further from any IC in pts with SD. Tregs were relatively closer to other IC in PD pts. Linear Discriminant Analysis was also performed and results indicate that differential IC densities (mainly M1 macrophages and CD4 T cells) do perform as classifiers between long ( > 5 months) and short ( < 5 months) term responses. Conclusions: This study suggests that bintrafusp alfa not only can enhance intratumoral effector IC infiltrates (CD8) but also has a modulating effect on the spatial distribution of both M1/M2 macrophages within the NSCLC TIME. The differential proximity of M2 macrophages to other IC infiltrates and changes in M1/M2 ratios in association with response suggests that an M1/M2 macrophage balance is directly involved in response and/or resistance to bintrafusp alfa. Given the limited number of patients in this cohort, we intend to study effects of bintrafusp alfa in a larger cohort of patients. Clinical trial information: 02517398 .

SUN-LB132 Frequency and Impact of Mutations in Tumor Suppressor Gene CDC73 in General Population and Malignant Tumors

Background: CDC73 gene is a putative tumor suppressor gene, and somatic and germline mutations of this gene have been linked with parathyroid tumors. There are also some reports suggesting that germline CDC73 mutations may increase risk of jaw, kidney and uterine tumors. However, the incidence and significance of CDC73 somatic mutations is largely unknown in tumors other than parathyroid tumors. Methods/Design: 1) Assess CDC73 germline mutation frequency and genotypes in general population using ExAC (Exome Aggregation Consortium) and gnomAD (Genome Aggregation Database); 2) Assess CDC73 somatic mutation frequency in various type of tumors using the Cancer Genome Atlas (TCGA) and non-TCGA datasets; 3) Examine significance of CDC73 mutation in uterine endometrial carcinoma (UCEC). Overall survival, CDC73 mutation signature, gene expression profile (GEP) and tumor immune microenvironment were compared between the UCEC samples with and without CDC73 mutations. Results: In ExAC and gnomAD databases, most of CDC73 germline mutations were mapped to the intron regions of the CDC73 gene and nonsynonymous CDC73 mutations are very rare, with allele frequency ranging from 8.24e-06 to 9.92e-05 and 3.97e-06 to 7.43e-05, respectively. Missense mutations accounted for more than 95% of the nonsynonymous mutations in the CDC73 gene in both databases. 2) In 155 cancer genomics studies including both TCGA and non-TCGA datasets with no overlapping samples, CDC73 was mutated in 0.7% cases and missense mutations comprised 78% of the CDC73 mutations. CDC73 somatic mutations were undetected or rarely detected in endocrine tumors except for parathyroid tumors. 3) UCEC is the tumor type that has the second highest CDC73 somatic mutation rate (8%) after parathyroid tumors. The UCEC with CDC73 mutations had a significantly better overall survival with a logrank p-value of 0.033 compared to the tumors with CDC73 WT. GEP analysis revealed that the CDC73-mutated UCEC tumors had significant upregulation of immunological markers and enriched immunologic signature compared to the CDC73-WT tumors. In silico analysis of tumor immune microenvironment showed higher fractions of cytotoxic CD8 cells, T follicular helper cells and M1 macrophage in the CDC73-mutated UCEC tumors. The majority of the CDC73-mutated tumors were POLE ultra-mutated and microsatellite instability (MSI) subtypes, which likely account for a better survival and an increased immune-cell infiltrate of these tumors. Conclusion: CDC73 mutations is rare in general population and tumors except for parathyroid carcinoma and UCEC. CDC73 mutation is a marker for better prognosis in UCEC and is associated with increased immune cell infiltrate in tumor microenvironment, likely due to majority of the CDC73-mutated tumors were POLE ultra-mutated and microsatellite instability (MSI) subtypes.

P04.19 Analysis of tumor immune microenvironment and immune checkpoint modulators across infantile and pediatric pilocytic astrocytomas to elucidate the role of immunotherapy in these tumors

Abstract BACKGROUND Pilocytic astrocytomas are the most common central nervous system tumors in pediatric age-group. Although grade I, some of the cases show recurrence and progression, and few might not be amenable to surgery due to location or size, and hence have a less favorable prognosis. Drugs blocking immune check-point interactions such as those including programmed cell death ligand-1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) are now in clinical use for certain tumors. We performed this study to understand the potential candidature of pilocytic astrocytomas in infants and children for immunotherapy by analyzing the expression of immune checkpoint proteins and immune infiltrate, and correlating with clinical details, wherever possible. MATERIALS AND METHODS Cases with adequate tissue (2010–2017) diagnosed in pediatric age-group (<18 years) were retreived from the archives of Department of Pathology, AIIMS, New Delhi. Immunohistochemistry for PD-L1 (SP263, Ventana), CTLA-4, CD3, CD8, CD4 and CD68 was performed. Quantification of cytotoxic lymphocytes was done using digital imaging in the core of the tumor. RESULTS A total of 50 pilocytic astrocytomas were included, 14 of them were <3 years (infants), while 36 were of pediatric age-group (3–18 years). Overall, male preponderance was noted. Cerebellum was the most common location, followed by 4th venrticle, optic pathway, hypothalamus, cerebrum and thalamus. Almost all CD3 lymphocytes were cytotoxic T-lymphocyes (CD8 positive, CTLs). Helper T-lymphocyte infiltration was not seen. Median CTL density/mm3 was 13/mm3(Range:1–85/mm3). CTLA-4 was positive in 4 cases, positivity ranged from 1–4 cells/lpf. PD-L1 was found to be positive in 7 cases, and the positivity ranged from 1+ to 2+ in 1 to 5% of tumor cells. A median TAM (tumor associated macrophages) density of 44/hpf (range: 1–98/hpf) was noted. There was no correlation of CTL density with PD-L1 or CTLA-4 expression, and neither with TAM density. On correlation with clinical parameters, a higher density of CTLs and TAMs was noted in infants, and a higher proportion of cases revealed PD-L1 positivity, though not statistically significant. There was no correlation of TILs or TAMs with the tumor location. CONCLUSION Immune check point blockade using PD-(L)1 or CTLA4 inhibitors may not be a potential therapeutic option for unresectable or recurrent pilocytic astrocytomas, as low positivity rate as well as extremely low percentage of tumor/ immune cells found to be positive. However, alternate forms of immunotherapy might be helpful as most of the cases showed immune infiltrates and a high density of tumor-associated macrophages (TAMs). Large scale studies with larger numbers and longer follow-up periods including in-vitro and clinical studies are warranted for decoding the tumor immunogram.

Anti-PD1 treatment to induce M1 polarization of tumor infiltrating macrophages in a 3D ex vivo system of lung cancer patients.

e23090 Background: Nivolumab and pembrolizumab treatment targeting the PD1/PD-L1 axis has demonstrated increased survival benefit in subpopulations of patients with advanced non-small cell lung cancer (NSCLC). The impact of these therapeutics on tumor immune microenvironment is not fully understood. Classical M1 macrophages are critical components involved in the inflammatory response and antitumor immunity. In this study, we evaluated the effect of PD1/PD-L1 blockade on M1 polarization of tumor-associated macrophages (TAMs) and activation of cytotoxic T-cells in a 3D ex vivo system of NSCLC. Methods: Fresh tumor tissues obtained from consented patients with NSCLC at the time of surgical resection were utilized in a 3D ex vivo tumor miscrosphere assay. 3D tumor microspheres were treated with nivolumab or pembrolizumab at 10 mg/ml for 36 hours within an intact tumor microenvironment. Flow cytometry analysis was performed to evaluate treatment-mediated TAM polarization, activation of T-cells and changes in CD4 and CD8 subpopulations. A multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines. Additionally, a NanoString platform containing probes to quantitate 770 immune function genes was used to determine potential positive or negative associations between expression of immune function genes and TIL activation by ex vivo treatment. Results: Both nivolumab and pembrolizumab treatment increased population of M1 macrophages (CD68+, CD80+, CD163-) and simultaneous release of MIP1b, IFN-ɣ, TNF-a, and GM-CSF cytokines as well as expression of genes related to the M1 phenotype that was accompanied by activation of CD8 cells assessed by Ki67 and CD107a expression and increased expression of genes involved in the IFNg pathway. Conclusions: Our studies showed that anti PD1/PD-L1 treatment leads to M1 macrophage polarization and T-cell activation in subgroups of NSCLC patients emphasizing the importance of comprehensive analysis of tumor immune microenvironment for a better understanding of the mechanism of action of immuno-oncology drugs that may help developing rationale combination treatments in NSCLC.