Abundance Of Tumor-infiltrating Lymphocytes Research Articles

Endometrial Tumors with MSI-H and dMMR Share a Similar Tumor Immune Microenvironment.

PurposeMicrosatellite instability (MSI) and mismatch repair deficiency (dMMR) are important biomarkers for predicting responses to immune checkpoint inhibitor (ICI) therapies. Although PCR-based tests for high MSI (MSI-H) and dMMR yield highly concordant results in endometrial cancer (EC), it is unclear whether this is true for MSI-H and MMR detected by next-generation sequencing (NGS) and immunohistochemistry (IHC), respectively. This study investigated whether EC with MSI-H identified by NGS and dMMR identified by IHC have similar tumor immune microenvironments.Patients and MethodsEC tissue and corresponding peripheral blood lymphocyte samples were collected from 99 randomly selected patients. MSI status and tumor mutation burden (TMB) were examined by NGS. MMR protein and programmed death ligand (PD-L)1 expression and tumor-infiltrating lymphocyte (TIL) abundance were evaluated by IHC.ResultsOf the 99 EC samples, 29 (29%) had dMMR by IHC, while 18 (18%) had MSI-H by NGS. MSI and MMR status identified by the two methods were discordant in the 99 EC patients, and 2/18 NGS-identified MSI-H patients (11%) retained MMR protein expression. MSI-H and dMMR endometrial tumors had similar numbers of cluster of differentiation (CD)3+ TILs (T cells) and CD8+ TILs (cytotoxic T cells) in the tumor center and periphery, which differed from those in microsatellite stable (MSS) and mismatch repair-proficient (pMMR) EC; they also showed similar TMB, PD-L1 expression, and TIL counts with higher TMB and PD-L1 expression than MSS and pMMR ECs. The abundance of CD3+ and CD8+ TILs was increased in PD-L1-positive EC.ConclusionNGS-identified MSI status and IHC-identified MMR status were inconsistent in EC, and 11% of NGS-identified MSI-H tumors retained MMR protein expression. Conversely, MSI and MMR status determined by the two methods provided similar data on TMB, PD-L1 expression, and TIL abundance, which can guide treatment decisions with ICIs.

Tumor-infiltrating lymphocytes predict survival outcomes in patients with cervical cancer treated with concurrent chemoradiotherapy

<h3>Objectives:</h3> The association between the prognosis of advanced cervical cancer treated with cisplatin-based concurrent chemoradiotherapy (CCRT) and the number of tumor-infiltrating lymphocytes (TILs) in biopsy samples collected before treatment was examined, to (i) identify correlations between selected immunogenic factors and clinicopathological characteristics, (ii) determine whether intra-tumoral abundance of various specific TILs is a prognostic indicator in women with advanced cervical cancer who undergo treatment with CCRT, and (iii) investigate subtypes of FOXP3+ T cells in cervical cancer samples. <h3>Methods:</h3> In this retrospective study, intratumoral lesions in colpo-scopic biopsies from 55 women with advanced cervical cancer who subsequently underwent CCRT were analyzed. The informed consent was obtained from all patients, and approval to conduct this study was granted from the institutional review board. The International Federation of Gynecology and Obstetrics clinical stages of these patients were as follows: Stage IIA1 (n=8), Stage IIA2 (n=6), Stage IIb (n=32), Stage IIIa (n=1), and Stage IIIb (n=8). Biopsy samples were subjected to automatic immunological staining using the following six antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD206, and anti-FOXP3. Associations between the findings on automatic scoring of the number of each type of TIL in each specimen and clinicopathological characteristics or survial status were analyzed. Subtypes of FOXP3+ TILs in 15 additional fresh tumor samples were stained with CD45RA antibody and investigated by flow cytometry. <h3>Results:</h3> Correlations between density of TILs and clinicopathological characteristics were examined. Only weak infiltration of CD8+ lymphocytes was found to be associated with PLN metastasis. No other factors were found to be associated with immune cell infiltration. Better survival was found to be associated with high densities of CD3+, CD4+, CD8+, CD206+, and FOXP3+ T cells, while there were no statistically significant associations with CD20+ B cell density. The subtypes of FOXP3+ TILs were analyzed in 15 fresh tumor samples, and non-Tregs (FOXP3lowCD45RA–) which have anti-tumor activity, were detected in all cases. <h3>Conclusions:</h3> The abundance of various specific intratumoral TILs may be prognostic indicators in patients with advanced cervical cancer undergoing CCRT.

Proteogenomic identification of an immunogenic HLA class I neoantigen in mismatch repair–deficient colorectal cancer tissue

Although CD8+ T cells recognize neoantigens that arise from somatic mutations in cancer, only a small fraction of nonsynonymous mutations give rise to clinically relevant neoantigens. In this study, HLA class I ligandomes of a panel of human colorectal cancer (CRC) and matched normal tissues were analyzed using mass spectrometry–based proteogenomic analysis. Neoantigen presentation was rare; however, the analysis detected a single neoantigen in a mismatch repair–deficient CRC (dMMR-CRC) tissue sample carrying 3967 nonsynonymous mutations, where abundant tumor-infiltrating lymphocytes (TILs) and inflamed gene expression status were observed in the tumor microenvironment (TME). Using the HLA class I ligandome data and gene expression profiles, a set of nonmutated tumor-associated antigen (TAA) candidates was concomitantly identified. Interestingly, CD8+ TILs predominantly recognized the detected neoantigen over the array of TAA candidates. Neoantigen-reactive CD8+ TILs showed PD-1 positivity and exhibited functional and specific responses. Moreover, T cell receptor (TCR) profiling identified the sequence of the neoantigen-reactive TCR clonotype and showed its expansion in the TME. Transduction of the sequenced TCR conferred neoantigen specificity and cytotoxicity to peripheral blood lymphocytes. The proteogenomic approach revealed the antigenic and reactive T cell landscape in dMMR-CRC, demonstrating the presence of an immunogenic neoantigen and its potential therapeutic applications.

Abstract 1675: Leveraging RNA sequencing for scalable tumor immune repertoire profiling

Abstract Background: Therapeutic strategies that modulate the interaction between tumors and tumor-infiltrating lymphocytes (TILs), such as checkpoint blockade, have tremendous promise but yield clinical responses in only a subset of patients. Accumulating evidence indicates that better characterizing TILs using repertoire-sequencing (rep-seq) may aid in predicting immunotherapy outcomes. However, repertoire profiling of the immune infiltrate is often implemented as a stand-alone assay or via single-cell sequencing, which can be difficult to incorporate into diagnostic platforms at scale. We developed and optimized a method to integrate immune repertoire profiling into a high-volume RNA-seq pipeline. Our approach yields accurate quantification of abundant TIL receptors without diminishing the clinical value of the remaining transcriptome. Here, we demonstrate the capabilities of our method in 500 TCR/BCR repertoires from a diverse cohort of tumor samples. Methods: Hybrid capture probes tiling T-cell and B-cell receptor (TCR and BCR) genes were used to enrich immune receptor transcripts detected by the Tempus RNA-seq workflow. Receptor sequences were aligned, assembled, and annotated against IMGT reference sequences using the TRUST4 assembly tool. Shannon Entropy was calculated to evaluate repertoire evenness. Richness (productive clonotypes) and immune infiltration predictions were correlated with a one-tailed Pearson correlation coefficient (95% CI). Results: Immune repertoires were extracted from RNA-seq of 501 tumor samples across 38 different cancer types. TCR and BCR repertoires (including small gamma/delta TCR repertoires) were robustly assembled in our cohort, with over 1.2 million total clonotypes identified. The distribution of repertoire evenness demonstrated expected trends, including dominant monoclonal expansion (&amp;gt;70-90% total receptor reads) of putative tumor cell receptors in T-cell/B-cell-driven malignancies. Repertoire richness also followed tissue-specific expectations where TIL-low cancers (e.g., glioblastoma multiforme) typically exhibited low-richness repertoires, while TIL-high cancers (e.g., non-small cell lung cancer) often yielded thousands of productive clonotypes. Repertoire richness was significantly correlated with RNA-based cell-type-specific transcriptional profiles for T-cells (r = 0.43, P&amp;lt;5e-24) and B-cells (r = 0.2, P&amp;lt;3.6e-6). Benchmarking our method on co-extracted DNA with amplicon-based repertoire sequencing confirmed accurate detection of highly abundant TIL receptors using an orthogonal approach. Conclusions: Our repertoire profiling method effectively leveraged routine RNA-seq to profile TIL receptors, ultimately allowing for the seamless integration of repertoire-based biomarker evaluation and improved immune infiltrate estimation through a high-volume RNA-seq pipeline. Citation Format: Taylor S. Harding, Brittany Mineo, Jenna Malinauskas, Jason Perera, Aly A. Khan. Leveraging RNA sequencing for scalable tumor immune repertoire profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1675.

The therapeutic potential of multiclonal tumoricidal T cells derived from tumor infiltrating lymphocyte-derived iPS cells

Tumor-infiltrating lymphocytes (TIL), which include tumor-specific T lymphocytes with frequency, are used for adoptive cell transfer therapy (ACT) in clinical practice. The optimization of TIL preparation has been investigated to reduce the senescence and increase the abundance of TIL, as both the quality and quantity of the transferred cells have great influence on the outcome of TIL-based ACT (TIL-ACT). Considering the effects of cell reprogramming on senescence, we expected that the anti-tumor effect could be enhanced by TIL regeneration. To confirm this hypothesis, we established tumor-specific TIL-derived iPS cells (TIL-iPSC) with human colorectal cancer specimens. T cells differentiated from TIL-iPSC (TIL-iPS-T) retained not only intrinsic T cell functions and tumor specificity, but also exhibited improved proliferation capacity and additional killing activity. Moreover, less differentiated profiles and prolonged persistency were seen in TIL-iPS-T compared with primary cells. Our findings imply that iPSC technology has great potential for TIL-ACT.

Dynamic crosstalk within the tumor microenvironment of uterine cervical carcinoma: baseline network, iatrogenic alterations, and translational implications.

Uterine cervical cancer is the fourth most frequent gynecological tumor worldwide. The tumor microenvironment of cervical cancer is the result of persistent high-risk human papillomavirus infection together with stromal activation of estrogen receptor alpha and the pro-angiogenic and pro-inflammatory activity of immune cells, mainly T-helper 17 cells and tumor-associated macrophages. Therapeutic management (e.g., immunotherapy, especially in advanced cases) may be influenced by the translational implications of tumoral stroma crosstalk and an abundance of tumor-infiltrating lymphocytes within the tumor microenvironment. The prognosis of cervical cancer is inversely correlated with microvessel density, making anti-angiogenic strategies with agents such as bevacizumab crucial for improving both progression-free survival and overall survival in patients with advanced and recurrent tumors.

Advances in immunotherapy for pancreatic ductal adenocarcinoma.

Advances in immunotherapy against advanced cancers can be considered stunning and epoch-making. Meanwhile, efficacy of immune-based therapies, especially immune checkpoint inhibitors, remains insufficient in pancreatic ductal adenocarcinoma, differing from other immunogenic cancers. To date, neither immunotherapies targeting immune system acceleration nor release of immunologic brakes have been able to overcome the robust immune barrier in the pancreatic tumor microenvironment, which is characterized by rich fibrotic stroma and accumulation of immunosuppressive myeloid cells. However, by receiving an immune checkpoint blockade, patients with abundant tumor-infiltrating lymphocytes in pancreatic ductal adenocarcinoma clearly have better prognosis, and patients with mismatch repair deficiency have achieved better outcomes, albeit in a small population of pancreatic ductal adenocarcinoma. We overview recent preclinical and clinical studies that have been concerned with immune-based therapies including cancer vaccine and immune checkpoint inhibitors. By providing a deep insight into the immunosuppressive tumor microenvironment, we suggest the possibility of comprehensive immune intensification that could reverse the tumor microenvironment, making it conducive to cytotoxic T lymphocyte activity for overcoming pancreatic ductal adenocarcinoma.

APE1 may influence CD4+ na\xefve T cells on recurrence free survival in early stage NSCLC

BackgroundIt was demonstrated that multifunctional protein APE1 (Apurinic/apyrimidinic endonuclease 1) is closely related to tumor immune microenvironment in a number of investigations, Meanwhile, the abundance of tumor infiltrating lymphocytes (TILs) has been shown as a prognosis indicator in some researches. However, it remains unclear whether APE1 is involved in the process of TILs affecting the prognosis of patients. To this end, we investigated the associations between APE1 and TILs in non-small cell lung cancer (NSCLC) and explored whether APE1 would influence the associations of CD4+ T cells infiltration with the prognosis of patients.MethodsGenome-wide expression datasets were obtained from the Gene Expression Omnibus (GEO) public database under accession number GSE68465, GSE30219, GSE31210 and GSE50081. MCPcounter and CIBERSORT analysis was conducted to evaluate the abundance of TILs in 1006 NSCLC patients of GEO database. Spearman correlation tests were used to evaluate correlations between abundance of various TILs and APE1 expression. RFS (recurrence free survival) was estimated using the Kaplan–Meier method and the Cox proportional-hazards model. The expression level of APE1 and tumor-infiltrating CD4+ T cells was evaluated by immunohistochemistry (IHC).ResultsThe results showed that the abundance of CD4+ naïve T cells was negatively associated with the APE1 expression. CD4+ naïve T cells infiltration was a favorable prognostic factor for RFS, however, there was no effect of CD4+ T cells infiltration on RFS in patients with high APE1 expression. Subsequently, it was further confirmed that CD4+ T cells infiltration was negatively associated with the APE1 expression level in 108 NSCLC tissue samples; high CD4+ T cells infiltration was associated with longer RFS in low APE1 expression group but not in APE1 high expression group.ConclusionThese results suggested that APE1 may affect the relationship between CD4+ T cells infiltration and prognosis in NSCLC. This study provides new insights into predictors of outcome in patients with NSCLC, and suggests that combining immunotherapy and APE1-targeted therapy may be a promising treatment for NSCLC.

Abstract PS17-21: Characterization of the immune microenvironment in ductal carcinoma in situ of the breast

Abstract Background Ductal carcinoma in situ (DCIS) is considered a low-risk disease of the breast. Current increases in its incidence have resulted in many women either being under- or over-treated due to our limited findings on independent prognostic and predictive biomarkers. The Van Nuys Prognostic Index, based on tumor size, margin status, grade and age, is one tool used in treatment decisions. Patients with a low score show no significant benefit from radiotherapy, in contrast to those with an intermediate score, while patients with high scores should be considered for mastectomy. In contrast, for invasive ductal carcinoma (IDC) of the breast there is a strong consensus for the prognostic and predictive value of tumor infiltrating lymphocytes (TIL). As very little is known about TIL in DCIS, the goal of this study is to fully characterize the immune infiltrate and compare it to IDC, examine differences in the balance between effector and regulatory subpopulations and potentially discover new biomarkers for risk stratification. Material and Methods Fourteen patients were prospectively enrolled at the St. Luc hospital in Brussels, including 4 pure DCIS, 5 mixt DCIS and IDC, and 5 normal breast tissues. Formalin-fixed paraffin-embedded sections were stained with three fluorescent multiplex immunohistochemistry (mIHC) panels that combined antibodies to CD45, CD4, CD8, CD20, FOXP3, CD68, GZMB, PD-1, Ki67 and cytokeratin. InForm® Tissue Finder™ software and PhenoptrReports (Akoya Biosciences®) were employed for TIL quantification and spatial distribution. Freshly resected DCIS tissues were used to isolate tumor-infiltrating CD4 and CD8 T cells for single cell RNAseq analysis to determine the T cell clonotypes present (A. Devaux’s poster) Results Our analyses reveal the DCIS stroma has a significant immune infiltrate dominated by CD4+ helper T cells and B cells (140 and 115 cells/mm2, respectively) followed by CD8+ cytotoxic T cells (72 cells/mm2), regulatory T cells (Treg) (27 cells/mm2) and to a lesser extent macrophages (23 cells/mm2). The immune pattern in DCIS is similar to IDC except there are fewer macrophages in the tumor areas and Treg increase in the stroma. Tumor areas are generally less infiltrated than the stroma but some DCIS cells are in direct contact with T cells and macrophages. Spatial distribution analysis within a radius of 30 μm confirms that Treg are in close proximity to the DCIS cells and in the proximity of CD4+ helper and CD8+ cytotoxic T cells. Moreover, proliferating GZMB+ cells, mainly CD8+ cytotoxic T cells, were observed in direct contact with DCIS cells. Only one patient out of 4 had PD1+ TIL in the stroma. A comparison of pure and mixt DCIS reveals lower stromal infiltration by T and B cells in the former, which is also associated with an increase in macrophages. Finally, the abundance of stromal TIL was frequently organized in tertiary lymphoid structures (TLS), composed by a B cell follicle surrounded by a T cell zone containing both CD4+ helper and CD8+ cytotoxic T cells. TLS were characterized by the presence of proliferating B cells and PD1high T follicular helper cells. FOXP3+ and GZMB+ cells were also observed in the T cell zone. Conclusions Examination of the immune infiltrate in DCIS shows an abundance of helper T cells, B cells and active cytotoxic T cells in association with stromal TLS. These observations reveal an active tumor immune microenvironment in DCIS and suggest that the immune response plays an active role in DCIS pathogenesis. Citation Format: Soizic Garaud, Alix Devaux, Anais Boisson, Céline Naveaux, Pierre Coulie, Ahmad Awada, Karen Willard-Gallo. Characterization of the immune microenvironment in ductal carcinoma in situ of the breast [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 PS17-21.

DIMEimmune: Robust estimation of infiltrating lymphocytes in CNS tumors from DNA methylation profiles

ABSTRACT The interaction of CNS tumors with infiltrating lymphocytes plays an important role in their initiation and progression and might be related to therapeutic responses. Gene expression-based methods have been successfully used to characterize the tumor microenvironment. However, methylation data are now increasingly used for molecular diagnostics and there are currently only few methods to infer information about the microenvironment from this data type. Using an approach based on differential methylation and principal component analysis, we developed DIMEimmune (Differential Methylation Analysis for Immune Cell Estimation) to estimate CD4+ and CD8+ T cell abundance as well as tumor-infiltrating lymphocytes (TILs) scores from bulk methylation data. Well-established approaches based on gene expression data and immunohistochemistry-based lymphocyte counts were used as benchmarks. The comparison of DIMEimmune to the previously published MethylCIBERSORT and MeTIL algorithms showed an improved correlation with both gene expression-based and immunohistological results across different brain tumor types. Further, we applied our method to large datasets of glioma, medulloblastoma, atypical teratoid/rhabdoid tumors (ATRTs) and ependymoma. High-grade gliomas showed higher scores of tumor-infiltrating lymphocytes than lower-grade gliomas. There were overall only few tumor-infiltrating lymphocytes in medulloblastoma subgroups. ATRTs were highly infiltrated by lymphocytes, most prominently in the MYC subgroup. DIMEimmune-based estimates of TILs were a significant prognostic factor in the overall cohort of gliomas and medulloblastomas, but not within methylation-based diagnostic subgroups. To conclude, DIMEimmune allows for robust estimates of TIL abundance and might contribute to establishing them as a prognostic or predictive factor in future studies of CNS tumors.

CD147 regulates antitumor CD8+ T-cell responses to facilitate tumor-immune escape.

Negative regulation of antitumor T-cell-immune responses facilitates tumor-immune escape. Here, we show that deletion of CD147, a type I transmembrane molecule, in T cells, strongly limits in vivo tumor growth of mouse melanoma and lung cancer in a CD8+ T-cell-dependent manner. In mouse tumor models, CD147 expression was upregulated on CD8+ tumor-infiltrating lymphocytes (TILs), and CD147 was coexpressed with two immune-checkpoint molecules, Tim-3 and PD-1. Mining publicly available gene-profiling data for CD8+ TILs in tumor biopsies from metastatic melanoma patients showed a higher level of CD147 expression in exhausted CD8+ TILs than in other subsets of CD8+ TILs, along with expression of PD-1 and TIM-3. Additionally, CD147 deletion increased the abundance of TILs, cytotoxic effector function of CD8+ T cells, and frequency of PD-1+ CD8+ TILs, and partly reversed the dysfunctional status of PD-1+Tim-3+CD8+ TILs. The cytotoxic transcription factors Runx3 and T-bet mediation enhanced antitumor responses by CD147-/- CD8+ T cells. Moreover, CD147 deletion in T cells increased the frequency of TRM-like cells and the expression of the T-cell chemokines CXCL9 and CXCL10 in the tumor microenvironment. Analysis of tumor tissue samples from patients with non-small-cell lung cancer showed negative correlations between CD147 expression on CD8+ TILs and the abundance of CD8+ TILs, histological grade of the tumor tissue samples, and survival of patients with advanced tumors. Altogether, we found a novel function of CD147 as a negative regulator of antitumor responses mediated by CD8+ TILs and identified CD147 as a potential target for cancer immunotherapy.

Tumor-infiltrating lymphocyte abundance and programmed death-ligand 1 expression in metaplastic breast carcinoma: implications for distinct immune microenvironments in different metaplastic components.

Both stromal tumor-infiltrating lymphocytes (sTILs) and programmed death-ligand 1 (PD-L1) affect responses to immunotherapy; however, the extent of sTIL and PD-L1 expression within various metaplastic components in metaplastic breast carcinoma (MBC), which are critical for the characterization of immune microenvironments, remains unreported. We profiled sTIL infiltration and PD-L1 expression in different metaplastic components of specimens from 82 MBC patients. The overall positivity for high or intermediate (H/I) sTIL, immune cell-PD-L1 (IcPD-L1), and tumor cell-PD-L1 (TcPD-L1) was 34.1%, 47.6%, and 17.1%, respectively, but differences specific to MBC subtypes and each metaplastic component existed. Squamous cell carcinoma exhibited the highest positivity rates of sTIL(H/I) (50.0%) and IcPD-L1 (66.7%), while matrix-producing carcinoma had the lowest respective rates (14.3% and 28.6%). The positivity rates of sTIL(H/I) and IcPD-L1 were the highest in squamous component (Sq) and the lowest in chondroid component (Ch). All cases that had discordant sTIL categories between carcinoma of no special type (NST) and metaplastic components showed sTIL(H/I) positivity higher in Sq, but lower in spindled component (Sp) and Ch. While there was no pattern of higher IcPD-L1-positivity in Sp, six of the seven cases that were TcPD-L1-discordant between NST and Sp were TcPD-L1-positive in Sp, suggesting a trend for higher TcPD-L1 in Sp. The diagnostic predictability of total tumor IcPD-L1 positivity based on IcPD-L1 positivity in Sq and Ch was 95.2% and 33.3%, respectively. Multivariate analysis showed that sTIL(H/I) positivity, but not PD-L1 positivity, correlated with better survival. Our data implicate distinct immune microenvironments in different metaplastic components in MBC, which may have immunopathologic, diagnostic, and therapeutic significance.

Predicting Relapse in Patients With Triple Negative Breast Cancer (TNBC) Using a Deep-Learning Approach

The abundance and/or location of tumor infiltrating lymphocytes (TILs), especially CD8+ T cells, in solid tumors can serve as a prognostic indicator in various types of cancer. However, it is often difficult to select an appropriate threshold value in order to stratify patients into well-defined risk groups. It is also important to select appropriate tumor regions to quantify the abundance of TILs. On the other hand, machine-learning approaches can stratify patients in an unbiased and automatic fashion. Based on immunofluorescence (IF) images of CD8+ T lymphocytes and cancer cells, we develop a machine-learning approach which can predict the risk of relapse for patients with Triple Negative Breast Cancer (TNBC). Tumor-section images from 9 patients with poor outcome and 15 patients with good outcome were used as a training set. Tumor-section images of 29 patients in an independent cohort were used to test the predictive power of our algorithm. In the test cohort, 6 (out of 29) patients who belong to the poor-outcome group were all correctly identified by our algorithm; for the 23 (out of 29) patients who belong to the good-outcome group, 17 were correctly predicted with some evidence that improvement is possible if other measures, such as the grade of tumors, are factored in. Our approach does not involve arbitrarily defined metrics and can be applied to other types of cancer in which the abundance/location of CD8+ T lymphocytes/other types of cells is an indicator of prognosis.

Chloride intracellular channel protein 2: prognostic marker and correlation with PD-1/PD-L1 in breast cancer.

Immune checkpoint inhibition has emerged as an effective treatment for multiple solid tumors, including advanced-stage breast cancer (BC). During the past decade, the US Food and Drug Administration has approved a number of agents for immune checkpoint blockade (ICB). However, the limited data on monotherapy anti-tumor activity in BC underscores the need for robust predictive biomarker development. Here, we used weighted gene coexpression network analysis of genes differentially expressed between BC and normal tissue to identify genes coexpressed with programmed death-1 (PD-1) and its ligand (PD-L1). Tumor Immune Estimation Resource and Gene Expression Profiling Interaction Analysis were used to assess the relationship between gene expression and the abundance of tumor-infiltrating lymphocytes (TILs). We found that chloride intracellular channel protein 2 (CLIC2) was not only coexpressed with PD-1 and PD-L1, but its increased expression was associated with a favorable prognosis and enrichment of multiple TIL types, particularly CD8+ T cells. These results suggest that CLIC2 is a potentially useful biomarker for identifying BC patients who could benefit from ICB.

Tumor-infiltrating lymphocytes predict survival outcomes in patients with cervical cancer treated with concurrent chemoradiotherapy

ObjectivesTo (i) identify correlations between selected immunogenic factors and clinicopathological characteristics, (ii) determine whether intratumoral abundance of various specific tumor-infiltrating lymphocytes (TILs) is a prognostic indicator in women with Stage II and III cervical cancer who undergo treatment with cisplatin-based concurrent chemoradiotherapy (CCRT), and (iii) investigate subtypes of FOXP3+ T cells in 15 fresh samples of cervical cancer. MethodsIn this retrospective study, intratumoral lesions in colposcopic biopsies from 55 women with advanced cervical cancer who subsequently underwent CCRT at our institution were subjected to automatic immunological staining using the following six mouse monoclonal antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD206, and anti-FOXP3. Associations between the findings on automatic scoring of the number of each type of TIL in each specimen and various clinicopathological characteristics were analyzed, as were associations between the abundance of various specific types of TIL and survival. Subtypes of FOXP3+ TILs in 15 additional fresh tumor samples were also investigated using flow cytometry. ResultsInfiltration with CD8+ TILs was associated with pelvic lymph node metastasis. Abundant infiltration by CD3+, CD4+, CD8+, CD206+, and FOXP3+ TILs were statistically significant indicators of better progression-free and overall survival. Regarding subtypes of FOXP3+ TILs, non-Tregs (Fr-III) were found in all samples tested for this. ConclusionsThe abundance of various specific intratumoral TILs may be prognostic indicators in patients with advanced cervical cancer undergoing CCRT.

Abstract D121: Predicting African American and European American patient response to kidney cancer immunotherapy by stage using the tumor microenvironment

Abstract Background Kidney cancer ranks as a top ten leading site of new cancer cases (~64,000) in the US each year. Renal Cell Carcinoma (RCC) is the predominant type of kidney cancer (~90% cases). The most common RCC subtypes include clear cell RCC (ccRCC, 75%), papillary RCC types I and II (pRCC I and II, 5% and 10%), and chromophobe RCC (chRCC, 5%). RCC subtypes have distinct responses to immunotherapy. Combination checkpoint inhibitors are the first-line treatment for advanced stage ccRCC. Unfortunately, response varies in advanced stage patients, suggesting stage-specific differences in tumor biology. Tumor infiltrating lymphocytes (TILs) in the tumor microenvironment (TME) have been shown to directly influence checkpoint inhibitor response. African Americans (AAs) have higher RCC incidence rates than European Americans (EAs) for reasons that are unclear. Biological determinants of this cancer health disparity have been largely understudied. To our knowledge, no study to date has profiled the TME by stage in AA and EA RCC patients and explored its usefulness as a predictor of checkpoint inhibitor response. Hypothesis TIL abundances and composition varies by stage between AAs and EAs with RCC. Methods Downloaded mRNA-sequencing and clinical data for 531 ccRCC patients, 52 Type I pRCC patients, 66 Type II pRCC patients, and 66 chRCC patients in the TCGA discovery cohort. Differential expression analyses for checkpoint inhibitor drug targets (PD-1, PD-L1, and CTLA-4) were carried out. CIBERSORT, a computation tool that quantifies abundances for 22 TILs based on gene expression, was used to characterize the TME. 1-way ANOVAs between Stages I-IV were performed with GraphPad Prism 8. Significant racial differences for relevant TILs were determined using two-tailed unpaired Welch t tests. Kidney cancer immunotherapy drug responses were inferred based on published clinical studies. Results PD-1 and CTLA-4 significantly increased in advanced stage ccRCC patients. 6/22 TILs showed stage-specific increases and decreases in ccRCC patients. Interestingly, three of these (follicular helper T cells, regulatory T cells, and CD8+ T cells) showed the same trend as the checkpoint inhibitor targets. In pRCC I and II, some TILs also demonstrated a linear increase by stage (Both: regulatory T cells, M1 macrophages; Type II only: naive B cells). M2 macrophage abundance increased significantly by stage in chRCC patients. When comparing AA (n=55) and EA (n=461) ccRCC patients, follicular helper T cells and regulatory T cells increased by stage in EA only. CD8+ T cells increased in abundance in both racial groups. Regulatory T cells and M2 macrophages have been reported to mediate clinical response to checkpoint inhibitor immunotherapy. Conclusions/Future Directions Our data suggests AA and EA RCC patients with advanced stage disease will respond differently to kidney cancer immunotherapy due to TME differences, namely TIL abundance and composition. Future studies include validating these findings in the expO cohort. Citation Format: Jacquelyn Cobb, Heinric Williams, Khadijah A. Mitchell. Predicting African American and European American patient response to kidney cancer immunotherapy by stage using the tumor microenvironment [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D121.

Surfactant Expression Defines an Inflamed Subtype of Lung Adenocarcinoma Brain Metastases that Correlates with Prolonged Survival.

To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease. Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data (n = 230) and single-cell RNA-sequencing (scRNA-seq) data (n = 52,698). TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment. The expression of surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches.

Molecular profile reveals immune-associated markers of lymphatic invasion in human colon adenocarcinoma

Lymphatic invasion (LI) is an early event of metastasis and closely associated with overall survival in colon adenocarcinoma (COAD). Our aim was to gain deeper insight into the mechanism of lymphatic invasion in COAD. Subtype-specific somatic mutations and differentially expressed genes (DEGs) screening were based on The Cancer Genome Atlas (TCGA). Gene Ontology (GO) enrichment analysis was utilized to explore the biological function. The condition of tumor-infiltrating lymphocytes was performed by TIMER online database. Survival analysis was based on Kaplan-Meier curve method. Lymphatic invasion was associated with poor prognosis of patients with COAD. Nine mutations were enriched in lymphatic invasion-negative group. A total of 50 were differentially expressed between LI-positive tissues and LI-negative tissues. The DEGs were enriched in lipoprotein-related functions. MUC4 in-frame deletion at A4166-S4181 was associated with favorable prognosis of COAD patients. BMPR2 frameshift mutation g.chr2:202555407delA played cis and trans functions in downregulation of itself and CTLA4 upregulation. And it was associated with higher mutational burden. LAMP5, CUBN and TCHH were DEGs associated with prognosis and abundance of tumor-infiltrating lymphocytes. In conclusion, our study provides LI-associated genetic and transcriptional alterations, which helps to better understand the potential mechanisms and microenvironment in COAD.

Clinicopathological significance of deficient DNA mismatch repair and MLH1 promoter methylation in endometrioid endometrial carcinoma.

The pathogenesis of DNA mismatch repair (MMR)-deficient endometrial carcinoma (EC) is driven by inactivating methylation or less frequently mutation of an MMR gene (MLH1, PMS2, MSH2, or MSH6). This study evaluated the prognostic and clinicopathologic differences between methylation-linked and nonmethylated MMR-deficient endometrioid ECs. We performed MMR immunohistochemistry and methylation-specific multiplex ligation-dependent probe amplification, and classified 682 unselected endometrioid ECs as MMR proficient (MMRp, n = 438) and MMR deficient (MMRd, n = 244), with the latter subcategorized as methylated (MMRd Met) and nonmethylated tumors. Loss of MMR protein expression was detected in 35.8% of the tumors as follows: MLH1 + PMS2 in 29.8%, PMS2 in 0.9%, MSH2 + MSH6 in 1.3%, MSH6 in 2.8%, and multiple abnormalities in 0.9%. Of the 244 MMRd cases, 76% were methylation-linked. MMR deficiency was associated with older age, high grade of differentiation (G3), advanced stage (II-IV), larger tumor size, abundant tumor-infiltrating lymphocytes, PD-L1 positivity in immune cells and combined positive score, wild-type p53, negative L1CAM, ARID1A loss, and type of adjuvant therapy. MMRd-Met phenotype correlated with older age and larger tumor size, and predicted diminished disease-specific survival in the whole cohort. In the MMRd subgroup, univariate analysis demonstrated an association between disease-specific survival and disease stage II-IV, high grade (G3), deep myometrial invasion, lymphovascular invasion, ER negativity, and L1CAM positivity. In conclusion, MMR methylation profile correlates with clinicopathologic characteristics of endometrioid EC, and MMRd-Met phenotype predicts lower disease-specific survival. MMR deficiency, but not MLH1 methylation status, correlates with T-cell inflammation and PD-L1 expression.

Clonality, Antigen Recognition, and Suppression of CD8+ T Cells Differentially Affect Prognosis of Breast Cancer Subtypes.

In breast cancer, response rates to immune therapies are generally low and differ significantly across molecular subtypes, urging a better understanding of immunogenicity and immune evasion. We interrogated large gene-expression data sets including 867 node-negative, treatment-naïve breast cancer patients (microarray data) and 347 breast cancer patients (whole-genome sequencing and transcriptome data) according to parameters of T cells as well as immune microenvironment in relation to patient survival. We developed a 109-immune gene signature that captures abundance of CD8 tumor-infiltrating lymphocytes (TIL) and is prognostic in basal-like, her2, and luminal B breast cancer, but not in luminal A or normal-like breast cancer. Basal-like and her2 are characterized by highest CD8 TIL abundance, highest T-cell clonality, highest frequencies of memory T cells, and highest antigenicity, yet only the former shows highest expression level of immune and metabolic checkpoints and highest frequency of myeloid suppressor cells. Also, luminal B shows a high antigenicity and T-cell clonality, yet a low abundance of CD8 TILs. In contrast, luminal A and normal-like both show a low antigenicity, and notably, a low and high abundance of CD8 TILs, respectively, which associates with T-cell influx parameters, such as expression of adhesion molecules. Collectively, our data argue that not only CD8 T-cell presence itself, but rather T-cell clonality, T-cell subset distribution, coinhibition, and antigen presentation reflect occurrence of a CD8 T-cell response in breast cancer subtypes, which have been aborted by distinct T-cell-suppressive mechanisms, providing a rationale for subtype-specific combination immune therapies.