Tumor Infiltrating Lymphocytes Subpopulations Research Articles

Abstract PO4-16-04: Decoding the immune landscape of breast cancer (BC) during pregnancy (PrBC): Impact of hormone receptors (HR) and tumor-infiltrating lymphocytes (TILs) phenotype on gene expression signatures

Abstract Introduction: PrBC is an uncommon malignancy with aggressive behavior. Its pathogenesis involves distinct immune mechanisms associated with maternal-fetal tolerance and tumor-host immunoediting. PrBC displays specific patterns of TILs with increased CD8+ cells. Gaining a comprehensive understanding of the molecular processes underlying this immune synergy is crucial for enhancing PrBC patients’ clinical management. Here, we sought to identify dysregulated immune-related genes in PrBC and explore their association with HR status and TILs. Methods: A total of n=75 PrBC (age range 26-43 years) and n=67 age-matched early-onset breast cancer (EOBC) in non-pregnant women (controls; age range 28-43 years) were selected from our Institutional registry. For all cases TILs were quantified according to the International TILs Working Group recommendations and profiled by IHC for CD4 and CD8. RNA was extracted from representative FFPE tissue blocks to perform the expression analysis of 395 genes involved in tumor-immune interactions using a targeted NGS panel (Oncomine™ Immune Response Research Assay, Thermofisher). Samples with >1,000,000 mapped reads and >800,000 valid reads were considered adequate. R package DESeq2 software 1.38.3 was used for sequencing depth differences normalization and differential gene expression analysis. Differentially expressed genes (DEGs) were identified based on a significant p-value (p< 0.05). Results: The comparison between PrBC and EOBC revealed a total of n=7 DEGs. All of these genes were upregulated and belonged to distinct superfamilies, including Cancer/Testis (CT) Antigen (MAGEA1/3, XAGE1B), Interferons/Cytokines (IFNA17, IFNB1), Chemokines (CXCL13), and Immunoglobulin (PECAM1/CD31). Notably, the upregulation of Chemokines in respect to EOBC was observed exclusively in HR+ PrBC, whereas triple-negative (TN) PrBC did not exhibit this pattern compared to the control group. Hence, the upregulation of ALOX15B, an enzyme involved in fatty acid peroxidation, was specific to TN PrBC. The immune signatures showed significant variations between PrBC and EOBC also based on TILs density and subpopulations. Indeed, the upregulation of CT genes (MAGEA1, XAGE1B) was exclusively observed in PrBC cases characterized by low TILs levels and prevalence of CD8+ or CD4+ cells. Finally, CD4+ TILs were absent or low in PrBC with upregulated Interferons, Cytokines, Chemokines, and Immunoglobulin genes, but present in cases with increased expression of CT and KLRF1 (NK cells). Conclusion: These findings highlight the heterogeneity and distinct molecular characteristics of PrBC and EOBC. The upregulation of specific immune-related gene families, such as CT and Chemokines, in different PrBC subtypes may suggest their potential role as actionable biomarkers (e.g. MAGEA, a well-known oncogene and potential immunotherapy target). The differences in immune signatures and TILs subpopulations further emphasize the importance of the immune microenvironment in PrBC biology and behavior. Future studies could delve deeper into the clinical implications of these gene expression patterns and explore their relevance in personalized treatment strategies for PrBC. Citation Format: Konstantinos Venetis, Elham Sajjadi, Chiara Frascarelli, Mariia Ivanova, Marianna D'Ercole, Concetta Blundo, Massimo Giroda, Eugenia Di Loreto, Giovanna Scarfone, Stefano Ferrero, Paolo Veronesi, Viviana Galimberti, Fedro Alessandro Peccatori, Nicola Fusco, Elena Guerini-Rocco. Decoding the immune landscape of breast cancer (BC) during pregnancy (PrBC): Impact of hormone receptors (HR) and tumor-infiltrating lymphocytes (TILs) phenotype on gene expression signatures [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-16-04.

Tumor-infiltrating lymphocytes: opportunities to use their potential in clinical practice in breast cancer

Introduction. Tumor-infiltrating lymphocytes are currently considered as a prognostic biomarker of cancer patient survival and response to therapy, as well as a target for immunotherapy. However, tumor-infiltrating lymphocytes are only part of the tumor microenvironment, which consists of cellular and cytokine components. The cellular component also includes tumor-associated macrophages, neutrophils, fibroblasts and other cells. The cytokine component is represented by the products of the activity of all cells of the microenvironment, as well as tumor cells. The purpose of this review is to consider various subpopulations of tumor-infiltrating lymphocytes, show their interaction with tumor cells and other cell populations of the tumor microenvironment, and also evaluate the possibilities of clinical use of this cell population as a biomarker using the example of breast cancer. Material and research methods. Materials for evaluating the importance of tumor-infiltrating lymphocytes as one of the components of the tumor microenvironment for clinical practice were publications in domestic and foreign journals over the past 5-7 years. Results. This review examines various subpopulations of tumor-infiltrating lymphocytes, shows their interaction with tumor cells and other cell populations of the tumor microenvironment, and also describes the potential for clinical use of this cell population as a biomarker for predicting clinical outcomes and the possibility of their use to determine the most effective treatment for cbreast cancer. Conclusion. Obtaining new data on the previously poorly studied components of the tumor microenvironment and tumor-infiltrating lymphocytes, as well as the formation of ideas about the mutual influence of tumor microenvironment cells and tumor cells open up the prospect of developing innovative models of therapy. One of the modern directions is the search for molecular mechanisms of maintaining the antitumor activity of tumor-infiltrating lymphocytes, as well as the polarization of cells in the tumor microenvironment. Advances in the field of molecular oncology will make it possible to reduce the mortality rate of cancer patients to minimal levels while maintaining or even improving their standard of living.

Tumor-infiltrating CD8+ sub-populations in primary and recurrent glioblastoma: An in-silico study

BackgroundGlioblastoma multiforme (GBM) remains an incurable primary brain tumor. CD8+ tumor-infiltrating lymphocytes (TILs) can target malignant cells; however, their anti-tumoral immune responses mostly do not lead to GBM rejection in GBM patients. We profiled the sub-populations of tumor-infiltrating CD8+ T-cells, i.e., naïve, cytotoxic, and exhausted cells, in primary and recurrent GBM tissues and provided a blueprint for future precision-based GBM immunotherapy. MethodWe re-analyzed the raw data of single-cell RNA sequencing on the cells residing in the GBM microenvironment and leveraged tumor bulk RNA analyses to study the significance of CD8+ TILs sub-populations in primary and recurrent GBM. We investigated cell-cell interaction between exhausted CD8+ TILs and other immune cells residing in the primary and recurrent GBM microenvironments and profiled the expression changes following CD8+ TILs' transition from primary GBM to recurrent GBM. ResultsExhausted CD8+ TILs are the majority of CD8+ TILs sub-populations in primary and recurrent GBM, and cytotoxic CD8+ TILs display decreased expression of inhibitory immune checkpoint (IC) molecules in the primary and recurrent GBM. In the primary and recurrent GBM microenvironment, exhausted CD8+ TILs interact most with tumor-infiltrating dendritic cells. ConclusionThis study demonstrates the profiles of CD8+ TILs sub-populations in primary and recurrent GBM and provides a proof-of-concept for future precision-based GBM immunotherapy.

Subpopulation structure of tumor-infiltrating lymphocytes in early and locally advanced triple negative breast cancer and its effect on the efficiency of neoadjuvant chemotherapy

Recent studies have shown that triple-negative breast cancer (TN BC) is characterized by the highest mutational load and immunogenicity compared to other subtypes, as well as the degree of tumor-infiltrating lymphocytes (TILs) infiltration, which play an important role in the development of antitumor immunity and treatment response. A significant disadvantage of the standard immunohistochemical method for determining TILs is the inability to fully assess the subpopulation structure of the immune infiltration, including minor populations.Aim: The evaluation of the subpopulations of breast cancer lymphoid infiltration in patients receiving neoadjuvant chemotherapy (NACT) and its influence on achieving a complete pathomorphological response (pCR = RCB 0).Materials and methods: The study included 90 patients who received NACT in following regimen: AC (doxorubicin 60 mg/m2 + cyclophosphamide 600 mg/m2 ) every 2 weeks, followed by 12 weekly infusions of paclitaxel 80 mg/m2 + carboplatin AUC2. The TILs subpopulations were evaluated in core-biopsy samples prior to the NACT in all patients. The analysis performed by flow cytofluorimetry. Clinical and immunological analysis was performed for the following 9 lymphocyte subpopulations: CD3+CD4+, CD3+CD8+, CD4+CD25highCD127– / low, CD3–CD19+, CD3–CD16+CD56+, CD3+CD16+CD56+, CD4+CD25+, CD8+CD279+, CD4+CD279+.Results: The frequency of pCR was 51,1 %. The total TILs content in groups with pCR and non-pCR (RCB 0 vs RCB I–III) did not differ statistically (p = 0.271). The subpopulations analysis for CD3+CD8+, CD3–CD16+CD56+, CD3+CD16+CD56+, CD3+CD4+, CD3–CD19+, CD4+CD25+, CD4+CD25highCD127– / low and CD4+CD279+ revealed no statistically significant differences between the median values in the groups with pCR and non-pCR. A study of the CD8+CD279+ population showed a higher level of these cells in patients achieved pCR / RCB 0 (median 18,6 % vs 12,3 % with RCB I–III) (p = 0.033). With CD8+CD279+ above the median (high, > Me), the pCR frequency was 61 % vs 35 % in the subgroup with CD8+CD279+ less than or equal to the median (low, ≤Me). Despite the absence of statistically significant differences in the content of CD3+CD16+CD56+(NKT-cells) in groups with pCR and non-pCR (p = 0.091), numerical differences in medians were revealed: 9,9 % and 8,3 %, respectively. At the same time, with CD3+CD16+CD56+(NKT) > Me (high), the pCR frequency was 63 % vs 36 % in the subgroup with CD3+CD16+CD56 + ≤Me (low). When selecting a narrow subgroup (CD8+CD279+ high and CD3+CD16+CD56+ high), the frequency of pCR was 87,5 % vs 27,3 % in the group with both low indicators.Conclusion: The high content of CD8+CD279+ and CD3+CD16+CD56+ in the tumor sample before the treatment start was a predictor of high sensitivity to NACT and is associated with a higher frequency of pCR.

Tumor-infiltrating lymphocytes and tumor-associated macrophages as potential predictors of lymph node metastases in major salivary gland cancers.

The purpose of this study is to define if tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) could represent potential predictors of lymph node metastases (LNM) in salivary gland cancers (SGC). A selected number of immunohistochemical markers related to TILs (CD3, CD4, CD68, and FOXP3) and TAMs (CD68 and CD163) were investigated on major salivary gland cancers. TIL and TAM densities were measured on digital images using the open-source QuPath both in the tumor interior (TI) and invasive margin (IM). Correlation with pathologic N classification and follow-up clinical data was investigated. A total of 25 consecutive patients (men: 11; median age: 62.0) were included. Densities of CD3+ IM (OR = 7.7, 95% CI 1.2-51.2), CD8+ TI (OR = 7.7, 95% CI 1.2-51.2), CD8+ IM (OR = 7.7, 95% CI 1.2-51.2), FOXP3+ TI (OR = 24.0, 95% CI 2.2-255.9), CD68+ TI (OR = 7.7, 95% CI 1.2-51.2), and CD163+ IM (OR = 7.7, 95% CI 1.2 - 51.2), and the Immunoscore CD8/CD3 (OR = 1.9, 95% CI 1.1-3.4) were significantly associated with LNM (p < 0.05). CD3+ TI density was significantly associated with tumor recurrence and death (HR = 5.8, 95% CI 1.5-22.6; p < 0.05). A high density of specific TIL and TAM subpopulations might be correlated with a higher probability of LNM in SGC.

Abstract 6352: Combination treatment with recombinant murine IL-12 and anti-PD-1 antibody enhanced anti-tumor efficacy through the activation of cytotoxic T cell response

Abstract Introduction: Immune checkpoint inhibitors (ICIs) are widely used in cancer therapy. Among them, anti-PD-1 antibodies are the most used and have been approved by FDA for the treatment of many types of cancer (1). In our previous studies, combination of anti-PD-1 antibody with IL-12 demonstrated significant synergistic effect on tumor growth inhibition. The elevation of IFN-γ mRNA expression suggested the potential roles of CD8 T cells and NK cells in this process. In this study, we used two syngeneic mice tumor models to investigate the mechanisms of synergistic effect by the combination of IL-12 and anti-PD-1 antibody. Methods: We used syngeneic mice tumor models with mice cancer cell lines CT-26 and MC-38. The mice inoculated with tumor cells and were treated until tumors grew to 100 mm3. The mice were divided into three groups to receive the treatment, including rmIL-12 alone, anti-PD1-antibody alone, and the combination of rmIL-12 and anti-PD1-antibody (BIW x 3 weeks). At the end of the study, the mice were sacrificed and tumor resection and splenectomy were performed. The tumor infiltrating lymphocyte subpopulations within the tumor microenvironment were analyzed by flow cytometry and gene expression profile by RNA Seq. Results: The combination of rmIL-12 and anti-PD-1 antibody significantly inhibited tumor growth compared to the treatment with rmIL-12 and anti-PD-1 antibody alone. The combination group demonstrated more CD8+ T cell in the tumor microenvironment via flow cytometry. In addition, RNA Seq showed the combination group increased CD8+ T cells, NK cells, and Th1 populations. The levels of cytotoxicity related genes (IFN-γ, Granzyme B, Perforin) were also higher in combination group. Conclusion: Combination of rmIL-12 and anti-PD-1 antibody increased immune cells infiltration in the tumor microenvironment and enhanced anti-tumor response suggesting the potential of this combination for cancer treatment. Citation:(1). Watson, G.A. et al. (2020) “Novel strategies in immune checkpoint inhibitor drug development: How far are we from the paradigm shift?,” British Journal of Clinical Pharmacology, 86(9), pp. 1753-1768. https://doi.org/10.1111/bcp.14355. Citation Format: Yeng-Jey Yang, Pei-Yi Tsou, Shiuh-Dih Chou, Meng-Na Lee, San-Chi Chen. Combination treatment with recombinant murine IL-12 and anti-PD-1 antibody enhanced anti-tumor efficacy through the activation of cytotoxic T cell response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6352.

Abstract P2-11-23: Evaluation of T-cell infiltrating lymphocytes vs. Th2 in triple negative breast cancers (TNBC)

Abstract Purpose. Triple-negative breast cancers (TNBC) are defined as negative for hormonal receptors and human epidermal growth factor receptors 2 and account for 15% of breast cancers. TNBC carry the worst prognosis mainly because of their high proliferation index and the absence of efficient targeted therapies due to their molecular heterogeneity, even though some promising options are emerging. Besides, they do share a very high and distinct tumor-infiltrating lymphocytes (TILs) infiltration. Amongst TILs, CD8+ cytotoxic and CD4+ helper T-cells (TC) proved to be markers of a good outcome. However, pathologists routinely quantify overall TILs density only. Anti-PD-1/PD-L1 immunotherapies recently became available for immunosuppressive TNBC. Unfortunately, neither PD-1/PD-L1 expression nor TILs quantification is able to predict patients’ responses accurately. Thus, we aimed at precisely characterizing TILs sub-populations and evaluating their prognostic significance before their predictive one. Methods. We selected 91 patients at the time of surgery before any adjuvant therapy from January 2013 to December 2018 in Rennes, France. TNBC tumors went through TILs quantification on hematoxylin and eosin slides by a trained pathologist and immune microenvironment characterization using flow cytometry. We then compared the prognostic value of immune microenvironment subpopulations vs total TILs count. Results. TNBCs contained a mean of 22.8±25.9% TILs, including CD4+ TC (14.1%) mainly made of Th2 (11.7%), CD8+ TC (11.1%), and myeloid cells (8.4%) such as antigen presenting cells (APC). TILs groups and percentages were correlated with the abundance of these cellular subpopulations (p≤0.004). TILs percentage was predictive of overall survival (OS), while high APC infiltration was of relapse-free survival (RFS) in univariate analyses (p=0.044 &amp; p≤0.030 respectively). Only Th2 infiltration was predictive of both RFS and OS in univariate (p=0.009 &amp; p=0.008 respectively) and multivariate analyses (p=0.002 &amp; p=0.010 respectively). When considering the different Th populations, only Th2 was a better predictive factor of survival than total leukocytes. Discussion. The development of immune therapies aiming at unlocking the anti-tumor immune response has revealed the desperate need to better characterize the tumor immune infiltrate. TILs quantification by pathologists is the only parameter routinely measured so far but it must be standardized since variability can affect their correlation with pCR. In our study, and contrary to most previous ones, stromal TILs were not significantly associated with RFS and OS. Thus, the characterization of TILs subtypes is critical since they can have either a pro- or an anti-tumorigenic function. Thus, we counteracted the limitations of TILs pathological quantification by finely characterizing the immune infiltrate using flow cytometry. Th2 infiltrate was the most frequent, as previously reported, and counterintuitively, was associated with a favorable prognostic value. This is rarely shown as Th2 infiltration is frequently reported to favor pro-tumorigenic immune tolerance. This proves that a more refined characterization of the intra-tumoral immune landscape is essential to derive interesting therapeutic perspectives, either by recapacitating Th2 response or by targeting their interaction with other immune subsets, like fibroblasts, macrophages, dendritic cells, or eosinophils and IgE. Finally, immunotherapies modulating Th2 response could also be used around the time of surgery that has been proved to induce immunomodulation. Conclusion. The characterization of TILs composition is essential to better understand the potential antitumoral functions of these cells and to substantially improve the associated prognostic and predictive values. Citation Format: Susie Brousse, Florence Godey, Elodie Laffont, Patrick Tas, Boris Campillo-Gimenez, Vincent Lavoué, Matthieu Le Gallo. Evaluation of T-cell infiltrating lymphocytes vs. Th2 in triple negative breast cancers (TNBC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-23.

The Prognostic Impact of Gender, Therapeutic Strategies, Molecular Background, and Tumor-Infiltrating Lymphocytes in Glioblastoma: A Still Unsolved Jigsaw.

Despite the adoption of novel therapeutical approaches, the outcomes for glioblastoma (GBM) patients remain poor. In the present study, we investigated the prognostic impact of several clinico-pathological and molecular features as well as the role of the cellular immune response in a series of 59 GBM. CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) were digitally assessed on tissue microarray cores and their prognostic role was investigated. Moreover, the impact of other clinico-pathological features was evaluated. The number of CD4+ and CD8+ is higher in GBM tissue compared to normal brain tissue (p < 0.0001 and p = 0.0005 respectively). A positive correlation between CD4+ and CD8+ in GBM is present (rs = 0.417-p = 0.001). CD4+ TILs are inversely related to overall survival (OS) (HR = 1.79, 95% CI 1.1-3.1, p = 0.035). The presence of low CD4+ TILs combined with low CD8+ TILs is an independent predictor of longer OS (HR 0.38, 95% CI 0.18-0.79, p = 0.014). Female sex is independently related to longer OS (HR 0.42, 95% CI 0.22-0.77, p = 0.006). Adjuvant treatment, methylguanine methyltransferase (MGMT) promoter methylation, and age remain important prognostic factors but are influenced by other features. Adaptive cell-mediated immunity can affect the outcomes of GBM patients. Further studies are needed to elucidate the commitment of the CD4+ cells and the effects of different TILs subpopulations in GBM.

TIM-3 Is a Potential Immune Checkpoint Target in Cats with Mammary Carcinoma.

Recent findings in human breast cancer (HBC) indicate that T-cell immunoglobulin and mucin-domain-containing molecule-3 (TIM-3)-targeted therapies may effectively activate anticancer immune responses. Although feline mammary carcinoma (FMC) is a valuable cancer model, no studies on TIM-3 have been developed in this species. Thus, we evaluated the expression of TIM-3 by immunohistochemistry in total (t), stromal (s), and intra-tumoral (i) tumor-infiltrating lymphocytes (TILs) and in cancer cells, of 48 cats with mammary carcinoma. In parallel, serum TIM-3 levels were quantified using ELISA and the presence of somatic mutations in the TIM-3 gene was evaluated in 19 tumor samples. sTILs-TIM3+ were more frequent than iTILs-TIM-3+, with the TIM-3 ex-pression in sTILs and cancer cells being associated with more aggressive clinicopathological features. In contrast, the TIM-3 expression in iTILs and tTILs was associated with a more benign clinical course. Moreover, the serum TIM-3 levels were lower in animals with FMC when compared to healthy animals (p &lt; 0.001). Only one somatic mutation was found in the TIM-3 gene, at intron 2, in one tumor sample. Altogether, our results suggest that the expression of TIM-3 among TILs subpopulations and cancer cells may influence the clinical outcome of cats with FMC, in line with the previous reports in HBC.

Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer.

BackgroundDespite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear.MethodsTo better...

Spatial architecture of regulatory T-cells correlates with disease progression in patients with nasopharyngeal cancer.

This study aims to investigate the prognostic value of composition and spatial architecture of tumor-infiltrating lymphocytes (TILs) as well as PDL1 expression on TILs subpopulations in nasopharyngeal carcinoma (NPC). A total of 121 patients with NPC were included and divided into two groups: favorable (n = 68) and unfavorable (n = 53). The archived tumor tissues of the included patients were retrieved, and a tissue microarray was constructed. The density and spatial distribution of TILs infiltration were analyzed using the multiplex fluorescent immunohistochemistry staining for CD3, CD4, CD8, Foxp3, cytokeratin (CK), PDL1, and 4',6-diamidino-2-phenylindole (DAPI). The infiltration density of TILs subpopulations and PDL1 expression were compared between the two groups. The Gcross function was calculated to quantify the relative proximity of any two types of cells. The Cox proportional hazards regression model was used to identify factors associated with overall survival (OS) and disease-free survival (DFS). The densities of regulatory T-cells (Tregs), effector T-cells (Teffs), PDL1+ Tregs, and PDL1+ Teffs were significantly higher in patients with unfavorable outcomes. PDL1 expression on tumor cells (TCs) or overall TILs was not associated with survival. Multivariate analysis revealed that higher PDL1+ Tregs infiltration density was independently associated with inferior OS and DFS, whereas Tregs infiltration density was only a prognostic marker for DFS. Spatial analysis revealed that unfavorable group had significantly stronger Tregs and PDL1+ Tregs engagement in the proximity of TCs and cytotoxic T lymphocyte (CTLs). Gcross analysis further revealed that Tregs and PDL1+ Tregs were more likely to colocalize with CTLs. Moreover, increased GTC : Treg (Tregs engagement surrounding TCs) and GCTL : PDL1+ Treg were identified as independent factors correlated with poor outcomes. TILs have a diverse infiltrating pattern and spatial distribution in NPC. Increased infiltration of Tregs, particularly PDL1+ Tregs, as well as their proximity to TCs and CTLs, correlates with unfavorable outcomes, implying the significance of intercellular immune regulation in mediating disease progression.

CD45RO+TILs: cellular biomarkers for larynx squamous cell carcinoma outcome

ObjectiveThe prognostic importance of Tumor-Infiltrating Lymphocytes (TILs) in the tumor microenvironment of various cancers is increasingly recognized. In the present study, we aimed to investigate the prognostic value of CD3+, CD4+, CD8+, and CD45RO + TILs and their relation to histopathological features in larynx squamous cell carcinoma. MethodsFormalin-Fixed and Paraffin-Embedded (FFPE) samples from 63 primary larynx squamous cell carcinoma patients were immunostained for CD3, CD4, CD8, and CD45RO expression. Positive cells in micrographs from Invasive Margin (IM) and Tumor Center (CT) of tissue specimens counted by ImageJ software and their correlation with disease outcome were analyzed. ResultsThe expression level of TILs subpopulations was associated with clinicopathological markers as well as Overall Survival (OS) and Disease-Free Survival (DFS). In multivariate analysis, high frequency of CD45RO + cells in IM were confirmed as an independent prognostic marker for DFS (p = 0.007, HR = 4.968) and OS (p = 0.007, HR = 4.957). Similar findings were observed in the multivariate analysis of the combined frequency of CD45RO+cells in IM and CT. ConclusionTILs are associated with patients clinicopathological features. Also, our findings indicate that CD45RO + TILs are a valuable marker for risk prediction in larynx SCC and could predict patients' outcomes.

The Abundance of Tumor-Infiltrating CD8+ Tissue Resident Memory T Lymphocytes Correlates with Patient Survival in Glioblastoma.

Glial tumors alone account for 40% of all CNS tumors and present a low survival rate. The tumor microenvironment is a critical regulator of tumor progression and therapeutic effectiveness in glioma. Growing evidence from numerous studies of human solid tumor-infiltrating CD8+ T cells indicates that tissue-resident memory T cells (TRM) represent a substantial subpopulation of tumor-infiltrating lymphocytes (TILs). Although it is reported that some types of cancer patients with high immune infiltration tend to have better outcomes than patients with low immune infiltration, it seems this does not happen in gliomas. This study aimed to characterize TRMs cells in the glioma tumor microenvironment to identify their potential predictive and prognostic role and the possible therapeutic applications. Fluorescence activated cell sorting (FACS) analysis and immunofluorescence staining highlighted a statistically significant increase in CD8+ TRM cells (CD103+ and CD69+ CD8+ T cells) in gliomas compared to control samples (meningioma). In-silico analysis of a dataset of n = 153 stage IV glioma patients confirmed our data. Moreover, the gene expression analysis showed an increase in the expression of TRM-related genes in tumor tissues compared to normal tissues. This analysis also highlighted the positive correlation between genes associated with CD8+ TRM and TILs, indicating that CD8+ TRMs cells are present among the infiltrating T cells. Finally, high expression of Integrin subunit alpha E (ITGAE), the gene coding for the integrin CD103, and high CD8+ TILs abundance were associated with more prolonged survival, whereas high ITGAE expression but low CD8+ TILs abundance were associated with lower survival.

LAG3 blockade coordinates with microwave ablation to promote CD8+ T cell-mediated anti-tumor immunity

BackgroundThe immune checkpoint inhibitors (ICIs) combined with other therapeutic strategies have shown exciting results in various malignancies, and ICIs have now become the gold standard for current cancer treatment. In several preclinical and clinical investigations, ablation coupled with immunotherapy has proved to be quite effective. Our previous studies have shown that ablation coupled with ICI is a potential anti-cancer regimen for colorectal cancer liver metastases (CRLM). Furthermore, we have reported that following microwave ablation (MWA), the expression of LAG3 is up-regulated in tumor microenvironment (TME), indicating that LAG3 is implicated in the regulation of immunosuppressive immune response, and combination therapy of MWA and LAG3 blockade can serve as a promising therapeutic strategy against cancer.MethodsThe expression of LAG3 was investigated in this study utilizing a preclinical mouse model treated with MWA. Moreover, we monitored the tumor development and survival in mice to assess the anti-cancer effects of MWA alone or in combination with LAG3 blockade. Flow cytometry was also used to phenotype the tumor-infiltrating lymphocytes (TILs) and CD8+ T cell effector molecules. We finally analyzed the single-cell RNA sequencing (scRNA-seq) data of infiltrating CD45+ immune cells in the tumors from the MWA alone and MWA combined with LAG3 blockade groups.ResultsAfter MWA, the expression of LAG3 was up-regulated on sub-populations of TILs, and introducing LAG3 blockade to MWA postponed tumor development and extended survival in the MC38 tumor model. Flow cytometry and scRNA-seq revealed that LAG3 blockade in combination with MWA markedly boosted the proliferation and the function of CD8+ TILs, leading to altered myeloid cells in the TME.ConclusionCombination therapy of LAG3 blockade and MWA was a unique therapeutic regimen for some solid tumors, and such combination therapy might reprogram the TME to an anti-tumor manner.

Prognostic Relevance of Nuclear Receptors in Relation to Peritumoral Inflammation and Tumor Infiltration by Lymphocytes in Breast Cancer.

Simple SummaryThe aim of this study was to investigate the prognostic impact of tumor-infiltrating lymphocytes (TILs) in a panel of 264 sporadic breast cancers by quantifying TIL levels according to Salgado and correlate this with type I and II nuclear receptor expression. Breast cancer cases with a TIL Salgado score of >15% showed a significantly decreased overall survival and peritumoral inflammation (according to Klintrup) determined the prognostic value of ER, PR, and PPARγ in BC. Therefore, the present study demonstrates significant relations between TIL levels, nuclear receptor expression and prognosis in breast cancer.The prognostic impact of tumor-infiltrating lymphocytes (TILs) is intensively investigated in breast cancer (BC). It is already known that triple-negative breast cancer (TNBC), the most aggressive type of BC, has the highest percentage of TILs. In addition, there is an influence of steroid hormone receptor expression (type I nuclear receptors) on TIL subpopulations in breast cancer tissue. The link between type II nuclear receptors and the level of TILs is unclear. Therefore, the aim of this study was to quantify TILs in a panel of 264 sporadic breast cancers and investigate the correlation of TIL levels with type I and II nuclear receptors expression. TIL levels were significantly increased in the subgroup of TNBC. By contrast, they decreased in estrogen (ER)- or progesterone receptor (PR)-positive cases. Moreover, TIL levels were correlated with type II nuclear receptors, including PPARγ, with a significant inverse correlation of the nuclear form (r = −0.727, p < 0.001) and a weak positive correlation of the cytoplasmic form (r = 0.202, p < 0.002). Surprisingly, BC cases with a TIL Salgado score of >15% showed a significantly decreased overall survival. In addition, peritumoral inflammation was also quantified in BC tissue samples. In our cohort, although the level of peritumoral inflammation was not correlated with OS, it determined the prognostic value of ER, PR, and PPARγ in BC. Altogether, the present study provides a differentiated overview of the relations between nuclear receptor expression, TIL levels, peritumoral inflammation, and prognosis in BC.

Prognostic significance of immunoscore related markers in bladder cancer

BackgroundThe significance of total and specific subpopulations of tumor-infiltrating lymphocytes (TILs) in cancer is now well-documented. In the present study, we investigated the relevance of CD3+, CD8 +, CD45RO +, and FOXP3 + TILs to the prognosis and survival of patients with bladder cancer and the disease's clinical-pathological parameters.MethodsInfiltration of each subset was immunohistochemically evaluated in both stromal and intratumoral regions of tumor tissues from 85 patients with urothelial cell carcinoma of the bladder, with known survival.ResultsOur results indicated that intratumoral CD45RO+ lymphocytes were significantly higher in high-grade tumors than in low-grade ones (P = 0.028). The frequencies of intratumoral CD3+ (P = 0.002), CD8 + (P = 0.008), intratumoral (P = 0.002), and stromal (P = 0.017) CD45RO+ lymphocytes were also higher in patients with muscular invasion than those without invasion. The frequencies of intratumoral CD3+ (P = 0.043), CD8+ (P = 0.003), CD45RO+ (P = 0.023), and total CD45RO+ (P = 0.015), showed variation in patients with different T-stage, as well; mostly increased in T2 versus Ta and T1. Comparing patients in different stages revealed an increase in the frequencies of total CD3+ (P = 0.011), intratumoral CD3+ (P = 0.006), total CD8+ (P = 0.012), intratumoral CD8+ (P = 0.009) and stromal CD8+ (P = 0.034), as well as total and stromal CD45RO+ lymphocytes (P = 0.01 and P = 0.034, respectively) in stage II comparing to stage I, while the frequencies of stromal CD3+ (P = 0.077) and CD8+ (P = 0.053) cells tended to be decreased in stage III compared to stage II.ConclusionsWe collectively observed that the frequency of immune cells, especially CD45RO+, CD3+, and CD8+ lymphocytes, were significantly higher in early-progressed tumors. This observation could be explained by continuous and prolonged stimulation of immune cells with tumor antigens during tumor progression or an increase in the recruiting factors, especially in the early stages, to eliminate tumor cells. However, with tumor progression to the late stages, the inhibitory microenvironment provided by tumor cells suppresses or changes the functionality of the effector and memory immune cells to help tumor growth. However, more functional studies with larger sample sizes are needed to reveal the real status of the immune system in patients with bladder cancer.

Immune Lymphocyte Infiltrate and its Prognostic Value in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) occurs more frequently in young (<50 years) non-Hispanic black and Hispanic/Latina women. It is considered the most aggressive subtype of breast cancer, although, recently, immune infiltrate has been associated with long-term survival, lower risk of death and recurrence, and response to neoadjuvant chemotherapy. The aim of this review was to evaluate the clinical impact of the immune infiltrate in TNBC by discussing whether its prognostic value varies across different populations. A comprehensive systematic search in databases such as PubMed and Web of Science was conducted to include papers focused on tumor-infiltrating lymphocytes (TILs) in TNBC in different population groups and that were published before January 2021. TNBC patients with higher levels of TILs had longer overall survival and disease-free survival times compared with TNBC patients with low TIL levels. Similar results were observed for CD4+, CD8+ TIL populations. On the other hand, patients with high TIL levels showed a higher rate of pathological complete response regardless of the population group (Asian, European, and American). These results altogether suggest that TIL subpopulations might have a prognostic role in TNBC, but the underlying mechanism needs to be elucidated. Although the prognosis value of TILs was not found different between the population groups analyzed in the revised literature, further studies including underrepresented populations with different genetic ancestries are still necessary to conclude in this regard.

Abstract 1722: Role of tumor infiltrating lymphocytes and spatial immune heterogeneity in sensitivity to PD-1 axis blockers in non-small cell lung cancer (NSCLC)

Abstract The rapid clinical expansion of immune checkpoint blockers (ICB) has transformed the therapeutic arsenal for non-small cell lung cancer (NSCLC). However, only a relatively small fraction of patients benefits from these treatments and those who respond can develop acquired resistance. This underlines the need to identify factors or biomarkers associated with treatment sensitivity and resistance to guide clinical decisions. It has been proposed that elevated levels of tumor infiltrating lymphocytes (TIL) in the tumor microenvironment could be associated with favorable clinical outcomes after treatment with ICB. However, few studies have explored this relationship and they used semi-quantitative scoring methods and/or analyzed small tumor areas. Here, we used multiplexed quantitative immunofluorescence (QIF) panels to study major TIL subsets (DAPI, CK, CD4, CD8 and CD20) and T-cell exhaustion markers (DAPI, CD3, LAG-3, PD-1, TIM-3) in full-face baseline whole-tumor slides from a large multi-institutional patient cohort (n=179). The analysis of numerous fields of view per case allowed us to examine the spatial distribution of immune cells and spatial immune heterogeneity. Our results demonstrate that CD8+ effector T-cells are the largest TIL subpopulation and that they are preferentially located in the stromal compartment. We also show that tertiary lymphoid structures have limited association with survival and their detection depends on the size of the tissue area analyzed. Additionally, higher levels of baseline CD8+ T-cells in the stroma were significantly associated with better survival in PD-L1 positive patients but not in PD-L1 negative. To validate these results, we used whole-exome sequencing to analyze the TCR-burden in an independent cohort of ICI-treated NSCLC patients. Increased expression of the exhaustion markers LAG-3 and TIM-3 in CD3 positive T-cells was associated with reduced survival. Finally, we used a novel multiparametric heterogeneity metric termed Rao’s quadratic entropy that enabled us to measure the spatial immune heterogeneity and was associated with worse survival. In summary, we quantitatively measured the density, functional properties, and spatial distribution of major TIL subsets in a large cohort of NSCLC patients treated with ICB. Our results highlight the prominent role of TILs in NSCLC and their potential role as a biomarker. These results could be easily translated into the clinic and used to guide optimal treatment options. Citation Format: Miguel Lopez de Rodas Gregorio, Venkata Nagineni, Arvind Ravi, Ila J. Datar, Mari Mino-Kenudson, German Corredor, Cristian Barrera, Lindsey Behlman, David L. Rimm, Roy S. Herbst, Anant Madabhushi, Jonathan W. Riess, Vamsidhar Velcheti, Matthew Hellmann, Justin F. Gainor, Kurt A. Schalper. Role of tumor infiltrating lymphocytes and spatial immune heterogeneity in sensitivity to PD-1 axis blockers in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1722.

Tumor-infiltrating lymphocytes for adoptive cell therapy: recent advances, challenges, and future directions

Introduction Adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) is a highly personalized type of cancer immunotherapy. TIL-based ACT exploits naturally occurring TILs, derived from the patients’ tumor. This treatment has shown consistent clinical responses in melanoma, and recent results point toward a potential use in multiple cancer diagnoses. However, several limitations have restricted the clinical development and adaptation of TIL-based ACT. Areas covered In this review, we present the principles of TIL-based ACT and discuss the most significant limitations for therapeutic efficacy and its widespread application. The topics of therapeutic resistance (both innate and acquired), treatment-related toxicity, and the novel research topic of metabolic barriers in the tumor microenvironment (TME) are covered. Expert opinion There are many ongoing areas of research focusing on improving clinical efficacy and optimizing TIL-based ACT. Many strategies have shown great potential, particularly strategies advancing TIL efficacy (such as increasing and harnessing ex vivo the sub-population of tumor-reactive TILs) and manufacturing processes. Novel approaches can help overcome current limitations and potentially result in TIL-based ACT entering the mainstream of cancer therapy across tumor types.

The ratio of CD8\u2009+\u2009lymphocytes to tumor-infiltrating suppressive FOXP3\u2009+\u2009effector regulatory T cells is associated with treatment response in invasive breast cancer

PurposeFOXP3 + and CD8 + are recognized markers of tumor-infiltrating lymphocytes (TILs) for breast cancer. FOXP3 + TILs are composed of effector Tregs (eTregs) and other subpopulations that are classified by their differences in suppressive function. In this prospective study, we evaluated Treg subpopulations and CD8 + TILs in breast cancer.Methods84 patients with breast cancer were enrolled. Fresh TILs were extracted andTregs were classified into eTregs (CD4+FOXP3highCD45RA−), other FOXP3+ Treg subsets (naïve and non-Tregs), and total CD8+CD4− TILs using flow cytometry. The suppression strength of each Treg subpopulation was analyzed. The association between TIL subpopulations, clinicopathological characteristics, and response to chemotherapy was evaluated.ResultsThe mean CD8/eTreg ratio value was 7.86 (interquartile range: 4.08–12.80). The proliferation function of eTregs was significantly suppressed compared with that of the other subpopulations (proliferation rates: control: 89.3%, + naiiveTreg: 64.2%, + non-Treg: 78.2% vs eTreg 1.93%; all P < 0.05). The patients with high with a high CD8 + /eTreg ratio achieved excellent pathological complete response (pCR) rate of neoadjuvant chemotherapy (90.2%) and the CD8/eTreg ratio were independent predictive factors for pCR (odds ratio:18.7(confidence interval 1.25–279) P < 0.05). A detailed assessment of the CD8/eTreg ratio for each patient who underwent NAC revealed that high CD8/eTreg ratio showed a significantly higher pCR rate compared to patients with a low CD8/FOXP3 ratio (39.6% vs 13.3, P < 0.05) in triple negative subtype patients with stromal TILs < 50%.ConclusionsA high CD8/eTreg ratio enhances pCR rate in patients with invasive breast cancer.