Composition Of Tumor-infiltrating Lymphocytes Research Articles

Abstract PO5-16-07: Unveiling the Landscape of PD-L1 Expression and Tumor-Infiltrating Lymphocyte Subtypes in Advanced Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is known for its aggressive behavior with poor survival outcomes. Influenced by different expression levels of Programmed Death-Ligand 1 (PD-L1) and several subtypes of tumor-infiltrating lymphocytes (TILs), tumor microenvironment (TME) plays a crucial role in TNBC response to treatment and progression. Objective: To assess the prevalence and prognostic role of PD-L1 expression in advanced TNBC, defined as unresectable stage III or stage IV disease, in patients treated with a standard cytotoxic chemotherapy regimen. TILs composition was also evaluated as an exploratory objective. Methods: The internal database of the Brazilian National Cancer Institute was queried out for women diagnosed and treated with advanced TNBC from January 2018 to December 2022. Formalin-fixed paraffin-embedded samples of a maximum of four years old were analyzed. PD-L1 expression using 22C3 pharmDX assay was estimated through combined positive score (CPS) dichotomization (CPS < 10 vs. CPS≥10). Tissue microarrays comprising the samples of biopsies were subjected to immunohistochemistry, targeting specific markers including CD3, CD4, CD8, CD56, CD68, CD117, FOXP3, PD-1, the immune cell profiles were carefully examined and their correlation with the PD-L1 CPS was determined. Results: A total of 150 patients were included. The expression of PD-L1 was undetermined in 2 cases. The median age was 51.5 years (IQR 41.8-60.2) and 20.9% of the cases had CPS≥10. Most patients were < 65 years (73.0%), were postmenopausal (56.8%) and belonged to non-white ethnicity (70.3%). Postmenopausal women predominated in the CPS≥10 subgroup (74.2%) (Table 1). No significant differences in demographic characteristics and clinicopathological variables were observed based on PD-L1 subgroups, 117 patients (79.1%) had CPS < 10, with 91 (77.8%) being de novo stage IV metastatic disease. CD3+ (p=0.037), CD4+ (p=0.005) and CD8+ (p=0.001) TILs had higher expression in tumors with PD-L1 CPS≥10 (Table 2). According to the treatment received, almost half of the patients only received first-line chemotherapy (47%), as the following second line was applied to 28.8% of the total group. The third and fourth lines were administered in 12.9% and 9.1% of patients, respectively. Only 2 patients went through five palliative chemotherapy lines, and only one received up to 6 treatment lines. Between the subgroups CPS≥10 versus CPS < 10 no statistically significant differences were observed in the median progression-free survival (PFS) (5.1 vs. 5.0 months, p=0.89) and overall survival (OS) (8.7 vs. 8.8 months, p=0.6). Conclusion: This study offers insights into the expression patterns of PD-L1 and TILs subtypes in advanced TNBC cases in Brazil. PD-L1 CPS did not influence survival outcomes for this cohort of patients treated with cytotoxic chemotherapy only. The composition of TILs subtypes within the TME appears to vary based on PD-L1 CPS. Table 1. Advanced TNBC population characteristic by CPS (Nf148). Table 2. Tumor Infiltrating Lymphocytes characterization (CD3, CD4, CD8, CD56, CD68, CD 117, FOXP3 and PD-1) and its correlation with CPS status. Citation Format: Alexssandra Lima Siqueira dos Santos, Jesse Lopes da Silva, Lucas Zanetti de Albuquerque, Antônio Lucas Araújo Neto, Cecília Ferreira da Silva, Luana Aguiar Mesquita Cerva, Isabele Ávila Small, Cicera Pimenta Marcelino, Paula de Mendonça Batista, Maria Aparecida do Carmo Rego, Maria Amélia Carlos Souto Maior Borba, Andreia Cristina de Melo. Unveiling the Landscape of PD-L1 Expression and Tumor-Infiltrating Lymphocyte Subtypes in Advanced Triple-Negative Breast Cancer [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 PO5-16-07.

Clinicopathological characteristics and immune microenvironment of breast squamous cell carcinoma

Objective: To investigate the clinicopathological characteristics of breast squamous cell carcinoma and to analyze the relationship between its immune microenvironment tumor infiltrating lymphocytes (TILs) and prognosis. Methods: Forty-four cases of primary squamous cell carcinoma of the breast diagnosed and treated in the First Affiliated Hospital of Air Force Medical University, Xi'an, China from January 2006 to July 2022 were selected. Their clinicopathological characteristics were analyzed. The cell composition of TILs was evaluated using immunohistochemistry (Mainly markers of B lymphocytes, T lymphocytes and plasma cells). The relationship between TILs and prognosis was also analyzed. Results: The 44 patients of breast squamous cell carcinoma were all female and all were invasive carcinoma. Eight cases (8/44, 18.2%) were squamous cell carcinoma, while 36 cases (36/44, 81.8%) were mixed squamous cell carcinoma. The mixed components included non-specific carcinoma and spindle cell metaplastic carcinoma (17 cases each). One case contained ductal carcinoma in situ of the breast and 1 case contained tubular carcinoma. The proportion of squamous cell carcinoma was 10% to 90%. The cases with pure squamous cell carcinoma often had a large cystic cavity, which was lined by atypical squamous epithelium, while infiltrating squamous cell carcinoma nests were seen in the breast tissue around the cystic cavity. Immunohistochemical staining showed that p63 and CK5/6 were expressed in the squamous cell carcinoma component, but ER, PR and HER2 were not, except for one case of HER2 1+. The positive rates of TRPS1 and PDL-1 were 76% and less than 1%, respectively. Fifteen cases were in the high TILs group (TILs≥30%) and 29 cases were in the low TILs group (TILs<30%). Twenty-three patients were followed up for 5 to 118 months. Among them, 12 died within 3 years and 9 were alive at the end of the follow up. There was no significant difference in TNM stage, TILs and prognosis between simple squamous cell carcinoma and mixed squamous cell carcinoma. Conclusions: Breast squamous cell carcinoma can be divided into simple squamous cell carcinoma and mixed squamous cell carcinoma. There are differences in gross findings and histology between the simple and mixed squamous cell carcinoma of the breast. Sufficient samples should be taken to avoid missing the diagnosis of a minor squamous component. The prognosis of patients with high TILs is significantly better than that of patients with low TILs. The expression rate of TRPS1 in primary squamous cell carcinoma of breast is high and helpful to the differential diagnosis from metastatic squamous cell carcinoma.

Abstract 7176: Optimization of T cell co-stimulatory agonists: A semi-mechanistic PKPD model integrating drug properties and tumor-immune interactions

Abstract Background: Despite the success of some immune checkpoint inhibitors in the treatment of cancer, T-cell co-stimulation has faced limited clinical success due to the intricacies around the optimal engagement of agonistic antibodies to co-stimulatory receptors, and the absence of biomarkers for patient selection. Understanding agonist-binding characteristics, pharmacokinetics, T cell subpopulation activation and differentiation kinetics, T cell life span in the tumor microenvironment, and the impact of T cell/tumor interactions on tumor growth kinetics is crucial for the positive clinical outcomes of T cell agonists. Currently there is not a suitable model to describe these complex interactions. Methods: We investigated the pharmacokinetic (PK), pharmacodynamics (PD), and anti-tumor efficacy of an anti-TNFR2 agonist, HFB3-1, and an anti-OX40 agonist, HFB10-1, in syngeneic tumor models. Flow cytometry was employed to profile post-treatment tumor infiltrating lymphocytes (TILs) in dissociated tumor tissues. In vitro binding affinity was determined using Biolayer Interferometry. A semi-mechanistic model, integrating PK, tumor growth, and immune interaction networks (interactions among Teff, Treg, and tumor cells), was developed in Berkeley Madonna (version 10.5.1). This was subsequently adapted to a human tumor growth model with T cell interaction networks to assess the impact of doses and dosing regimens on tumor growth inhibition. Results: A semi-mechanistic PKPD model was successfully developed incorporating drug properties including binding affinity, agonistic effect, ADCC activity, and pharmacokinetics, and tumor growth characteristics including tumor growth kinetics, TIL composition, interaction, proliferation, and turnover. This model was applied to investigate the effect of FcγR, dose, and dosing frequency on in vivo efficacy. Following incorporation of human PK and human tumor microenvironment properties into the model, tumor growth characteristics were simulated under varying human dosing regimens. Simulation results indicated that lower and less frequent doses optimize T cell co-stimulation for optimal anti-tumor activity. Conclusion: Our semi-mechanistic PKPD model elucidates the intricate interactions between T cell co-stimulatory agonists and the tumor-immune cells in the tumor microenvironment. The model advocates for pulsatile agonism as the optimal anti-tumor activity for T cell co-stimulation agents. This model serves as a valuable tool for guiding dose and dose regimen optimization for T cell co-stimulatory agonists in clinical development. Citation Format: Jinping Gan, William Hedrich, Yun-Yueh Lu, Wenhua Xu, Robert H. Andtbacka, Francisco Adrian, Liang Schweizer. Optimization of T cell co-stimulatory agonists: A semi-mechanistic PKPD model integrating drug properties and tumor-immune interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7176.

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.

Spatial heterogeneity of infiltrating T cells in high-grade serous ovarian cancer revealed by multi-omics analysis

Tumor-infiltrating lymphocytes (TILs), especially CD8+ TILs, represent a favorable prognostic factor in high-grade serous ovarian cancer (HGSOC) and other tumor lineages. Here, we analyze the spatial heterogeneity of different TIL subtypes in HGSOC. We integrated RNA sequencing, whole-genome sequencing, bulk Tcell receptor (TCR) sequencing, as well as single-cell RNA/TCR sequencing to investigate the characteristics and differential composition of TILs across different HGSOC sites. Two immune "cold" patterns in ovarian cancer are identified: (1) ovarian lesions with low infiltration of mainly dysfunctional Tcells and immunosuppressive Treg cells and (2) omental lesions infiltrated with non-tumor-specific bystander cells. Exhausted CD8 Tcells that are preferentially enriched in ovarian tumors exhibit evidence for expansion and cytotoxic activity. Inherent tumor immune microenvironment characteristics appear to be the main contributor to the spatial differences in TIL status. The landscape of spatial heterogeneity of TILs may inform potential strategies for therapeutic manipulation in HGSOC.

NEUROD1 predicts better prognosis in pancreatic cancer revealed by a TILs-based prognostic signature.

It has been well-defined that tumor-infiltrating lymphocytes (TILs) play critical roles in pancreatic cancer (PaCa) progression. This research aimed to comprehensively explore the composition of TILs in PaCa and their potential clinical significance. A total of 178 samples from the TCGA and 63 samples from the GSE57495 dataset were enrolled in our study. ImmuCellAI was applied to calculate the infiltrating abundance of 24 immune cell types in PaCa and further survival analysis revealed the prognostic values of TILs in PaCa. Moreover, the Hallmark enticement analysis of differentially expressed genes (DEGs) between low- and high-risk groups was performed as well. Immunohistochemistry staining was used to evaluate NEUROD1 expression. As result, different kinds of TILs had distinct infiltrating features. In addition, Specific TILs subsets had notable prognostic values in PaCa. We further established a 6-TILs signature to assess the prognosis of PaCa patients. Kaplan-Meier and Cox regression analyses both suggested the significant prognostic value of the signature in PaCa. Based on the prognostic signature, we screened a great deal of potential prognostic biomarkers and successfully validated NEUROD1 as a novel prognostic biomarker in PaCa. Overall, the current study illuminated the immune cells infiltrating the landscape in PaCa and identified a TILs-dependent signature and NEUROD1 for prognostic prediction in PaCa patients.

158P The impact of tumor microenviroment on pathologic complete response after neoadjuvant chemotherapy of early breast cancer

We investigated the impact of baseline tumor infiltrating lymphocytes (TIL) density and TIL composition along with clinicopathological factors on pathologic complete response (pCR) rate in patients treated with standard neoadjuvant chemotherapy ± targeted therapy for early breast cancer.

Proliferative Tumor-Infiltrating Lymphocytes' Abundance within the Microenvironment Impacts Clinical Outcome in Cutaneous B-Cell Lymphomas

Primary cutaneous large B-cell lymphoma, leg-type (PCLBCL-LT) is the most aggressive primary cutaneous B-cell lymphoma (PCBCL). Tumor microenvironment has a crucial role in tumor development, and tumor-infiltrating lymphocytes (TILs) can be targeted by immunotherapies. We characterized TILs in 20 PCBCLs to identify the tumor microenvironment features associated with clinical outcomes. We developed a seven‒multiplex immunofluorescence panel using Opal staining and image analysis using HALO software. In PCLBCL-LT, TILs were sparsely intermingled within tumor infiltrate in contrast to those in indolent PCBCL where TILs were scattered around tumor nodule edges with variable tumor infiltration. In PCLBCL-LT, TILs were composed of CD8 and CD4, whereas CD4 was predominant in indolent PCBCL. Proliferative TILs (CD3+Ki-67+ cells) were more abundant in PCLBCL-LT (P= 0.0036) than in indolent PCBCL. In PCLBCL-LT, proliferative TILs' abundance tended to be associated with better progression-free survival. These data were confirmed in a second independent cohort of 23 cases showing that proliferative TILs were more abundant in PCLBCL-LT (P= 0.0205) and that in PCLBCL-LT, high CD3+Ki-67+ cell density was associated with better progression-free survival (P= 0.002). These distinct TILs composition and distribution among PCBCL suggest that proliferative T lymphocytes represent a good prognostic factor in PCLBCL-LT and that stimulating their functions may represent a therapeutic approach.

A Fraction of CD8+ T Cells from Colorectal Liver Metastases Preferentially Repopulate Autologous Patient-Derived Xenograft Tumors as Tissue-Resident Memory T Cells.

Simple SummaryTreatment options for colorectal cancer (CRC) patients with liver metastases are often limited to liver surgery with or without chemotherapy. However, not all patients present operable colorectal liver metastases (CRLMs). Thus, alternative therapies that exploit the anti-tumor potential of tumor-infiltrating lymphocytes (TILs) are being evaluated. The establishment of markers connecting the phenotype to the function of tumor-reactive CD8+ TILs could aid diagnostic and therapeutic advances. In this regard, tissue-resident memory T cells (TRM cells) could be a potential candidate for therapies targeting TILs. Putative tumor-reactive TRM cells among CD8+ TILs likely co-express CD103 and CD39, since these markers indicate stable tumor residency and repeated response to antigens from the tumor environment, respectively. Our phenotypic and functional analyses of TILs in CRLM, with a specific focus on CD103+CD8+ TRM cells, may guide the improvement of TIL-mediated CRC treatments.The diversity of T cells in the human liver may reflect the composition of TILs in CRLM. Our ex vivo characterization of CRLM vs. adjacent liver tissue detected CD103+CD39+CD8+ TRM cells predominantly in CRLM, which prompted further assessments. These TRM cells responded to cognate antigens in vitro. As functional activities of autologous TILs are central to the implementation of personalized cancer treatments, we applied a patient-derived xenograft (PDX) model to monitor TILs’ capacity to control CRLM-derived tumors in vivo. We established PDX mice with CRLMs from two patients, and in vitro expansion of their respective TILs resulted in opposing CD4+ vs. CD8+ TIL ratios. These CRLMs also displayed mutated KRAS, which enabled trametinib-mediated inhibition of MEK. Regardless of the TIL subset ratio, persistent or transient control of CRLM-derived tumors of limited size by the transferred TILs was observed only after trametinib treatment. Of note, a portion of transferred TILs was observed as CD103+CD8+ TRM cells that strictly accumulated within the autologous CRLM-derived tumor rather than in the spleen or blood. Thus, the predominance of CD103+CD39+CD8+ TRM cells in CRLM relative to the adjacent liver and the propensity of CD103+CD8+ TRM cells to repopulate the autologous tumor may identify these TILs as strategic targets for therapies against advanced CRC.

Microenvironment-related predictive markers of the therapeutic effectiveness of eribulin in patients with locally advanced or metastatic breast cancer: pilot study

Background. Triple negative breast cancer has no specific treatment sites for chemotherapy and is unfavorable in terms of prognosis. One of the drugs widely used in this cohort of patients is eribulin, which in addition to its antimitotic effect has an effect on the tumor microenvironment. The search for biological criteria that will allow predicting the effectiveness of the drug is assumed relevant since it will help to select patients who may receive the most benefit from certain therapy regimens.Objective: identification of immunological predictors of the therapeutic effectiveness of eribulin in patients with locally advanced or metastatic triple-negative breast cancer.Materials and methods. The study included 20 patients with locally advanced and metastatic triple negative breast cancer. 50 % had a short-term response (progression-free survival &lt;3 months) to eribulin therapy, and 50 % had a long-term response (progression-free survival &gt;6 months). Seven-color immunofluorescence was used to determine the subpopulation composition of tumor-infiltrating lymphocytes and their PD1 expression. Image acquisition and analysis were performed using the Vectra® 3.0 system and InForm® software (Akoya Biosciences, USA).Results. It has been shown that the ratio of the number of PD1-negative to PD1-positive CD20+ B-lymphocytes less than 5.5 associated with the long-term effectiveness of eribulin in patients with locally advanced or metastatic triple negative breast cancer.Conclusion. The results showed that the ratio of the number of PD1-negative to PD1-positive CD20+ B-lymphocytes can be considered as a possible marker to predict the effectiveness of eribulin in patients with breast cancer.

Comprehensive analysis of PD-L1 expression, tumor-infiltrating lymphocytes, and tumor microenvironment in LUAD: differences between Asians and Caucasians

BackgroundsThe characteristics of programmed cell death protein-1 (PD-L1) expression, tumor-infiltrating lymphocytes (TILs), and tumor microenvironment (TME) in lung adenocarcinoma (LUAD) patients are closely related to immunotherapy, and there are differences between Asians and Caucasians.MethodsAcquire the transcriptome data of the Cancer Genome Atlas and Chinese LUAD patients. R software was used to analyze the differential expression of genes, prognosis, and gene function. Use CIBERSORT for TIL-related analysis and ESTIMATE for TME-related analysis.ResultsThe expression of PD-L1 in tumor tissues of Caucasian LUAD patients was lower than that in normal tissues, while there was no significant difference in Asians. There was no statistical difference between PD-L1 expression and prognosis. The composition of TILs between Caucasian and Asian LUAD patients was quite different. There was no correlation between TILs and prognosis in Caucasians. However, the higher content of resting mast cells indicated a better prognosis in Asians. The Caucasian patients with higher immune and estimate scores had a better prognosis (p = 0.021, p = 0.025). However, the Asian patients with a higher estimate score had a worse prognosis (p = 0.024). The high expression of COL5A2 (p = 0.046, p = 0.027) and NOX4 (p = 0.020, p = 0.019) were both associated with the poor prognosis in Caucasians and Asians.ConclusionThere are many differences in the characteristics of PD-L1 expression, TILs, and TME between Caucasian and Asian LUAD patients. This provides a certain hint for the selection of specific immunotherapy strategies separately for Caucasian and Asian LUAD patients.

901P Relationship between p16 and tumor-infiltrating lymphocytes in Peruvian patients with head and neck cancer

Human papillomavirus-16 (HPV-16) infection in Head and Neck Squamous Cell Carcinoma (HNSCC) has a different prevalence through worldwide regions, it influences prognosis and treatment response and p16 staining is its surrogate biomarker. The density and composition of tumor-infiltrating lymphocytes (TIL) reflect biology and prognosis in different malignancies. We characterize TIL in Peruvian patients with HNSCC. We evaluate the influence of clinicopathological features including p16 & ki67 staining in tumor cells over TIL level.

Tumor infiltrating lymphocytes associated competitive endogenous RNA networks as predictors of outcome in hepatic carcinoma based on WGCNA analysis.

The important regulatory role of competitive endogenous RNAs (ceRNAs) in hepatocellular carcinoma (HCC) has been confirmed. Tumor infiltrating lymphocytes (TILs) are of great significance to tumor outcome and prognosis. This study will systematically analyze the key factors affecting the prognosis of HCC from the perspective of ceRNA and TILs. The Cancer Genome Atlas (TCGA) database was used for transcriptome data acquisition of HCC. Through the analysis of the Weighted Gene Co-expression Network Analysis (WCGNA), the two modules for co-expression of the disease were determined, and a ceRNA network was constructed. We used Cox regression and LASSO regression analysis to screen prognostic factors and constructed a risk score model. The Gene Expression Omnibus (GEO) was used to validate the model. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used for mRNAs functional analysis. The cell composition of TILs was analyzed by the CIBERSORT algorithm, and Pearson correlation analysis was utilized to explore the correlation between TILs and prognostic factors. We constructed a ceRNA regulatory network composed of 67 nodes through WGCNA, including 44 DElncRNAs, 19 DEGs, and 4 DEmiRNAs. And based on the expression of 4 DEGs in this network (RRM2, LDLR, TXNIP, and KIF23), a prognostic model of HCC with good specificity and sensitivity was developed. CIBERSORT analyzed the composition of TILs in HCC tumor tissues. Correlation analysis showed that RRM2 is significantly correlated with T cells CD4 memory activated, T cells CD4 memory resting, T cells CD8, and T cells follicular helper, and TXNIP is negatively correlated with B cells memory. In this study, a ceRNA with prognostic value in HCC was created, and a prognostic risk model for HCC was constructed based on it. This risk score model is closely related to TILs and is expected to become a potential therapeutic target and a new predictive indicator.

Relationship between stromal regulatory T cells and the response to neoadjuvant chemotherapy for locally advanced rectal cancer.

In addition to the direct power of anticancer drugs, the effectiveness of anticancer therapy depends on the host immune function. The present study investigated whether or not the reduction rate and histological response of preoperative chemotherapy were related to the immune microenvironment surrounding a primary tumor of the rectum. Sixty-five patients received preoperative chemotherapy followed by resection from 2012 to 2014; all of these patients were retrospectively analyzed. CD3, CD8, and FoxP3 were immunohistochemically examined as markers for T lymphocytes, cytotoxic T lymphocytes, and regulatory T lymphocytes (Treg), respectively. The correlation between the tumor-infiltrating lymphocyte composition and the tumor reduction rate and histological response to neoadjuvant chemotherapy was investigated. The average tumor reduction rate was 41.5% ± 18.8%. According to RECIST, 47 patients (72.3%) achieved a partial response (PR), and 1 patient (1.5%) achieved a complete response (CR). Eight patients (12.3%) showed a grade 2 histological response, and 2 (3.1%) showed a grade 3 response. A multivariate analysis demonstrated that a low Treg infiltration in stromal cell areas was significantly associated with the achievement of a PR or CR [odds ratio (OR) 7.69; 95% confidence interval (CI) 1.96-33.33; p < 0.01] and a histological grade 2 or 3 response (OR 11.11; 95% CI 1.37-98.04; p = 0.02). A low Treg infiltration in the stromal cell areas may be a marker of a good response to neoadjuvant chemotherapy in patients with locally advanced rectal cancer.

T cell receptor repertoires of ex vivo-expanded tumor-infiltrating lymphocytes from breast cancer patients.

A higher level of tumor-infiltrating lymphocytes (TILs) is associated with better prognosis in breast cancer patients. Adoptive transfer of lymphocytes coupled with conventional therapies has appealed to many clinicians and investigators as an effective treatment strategy for cancer patients, which necessitates efficient activation and expansion of cytotoxic T lymphocytes precisely targeting cancer cells. To comprehensively understand composition of TILs and to provide a grounding in adoptive T cell therapy, we analyzed the T cell receptor (TCR) repertoires in ex vivo-expanded TILs from nine breast cancer patients via next-generation sequencing. For the three of them, TCR repertoires of TILs gathered after the initial culture during 2weeks were additionally analyzed and compared to those of TILs that underwent ex vivo rapid expansion procedure (REP). Diversity of TCR repertoire was variable among the patients. V/J segment usage in the clonotypes was similar among patients, with variable distribution of read counts for each V/J segment. The top 50% of most frequently observed VJ combinations was present in > 80% of the total clonotypes. Compared with TCGA data, the samples contained a similar amount of recurrent CDR3 sequences, but clonotype expansion was variable among the samples. In terms of clinicopathologic factor, presence of in vitro reactivity among triple-negative breast cancer cases seemed to be related to lower Shannon's index, but p value was not statistically significant. In addition, the proportion of CD45RO+ cells out of CD8+ T cells were negatively correlated with Shannon's diversity index for both TCRα and TCRβ chains (p = 0.010) via Spearman test. In this study, we identified a heterogeneous pattern of expanded T cell clones and stable usage of V/J segments in ex vivo-expanded TILs from breast cancer patients. Further large-scale studies are requisite to elucidate the clinical significance of TCR repertoires.

Abstract PD5-07: The immune landscape of residual triple-negative breast cancers after neoadjuvant chemotherapy

Abstract Background: Although neoadjuvant chemotherapy (NAC) induces complete response in 30-40% of triple-negative breast cancers (TNBC), patients with residual disease at surgery have poor prognosis and limited treatment options until recurrence. Tumor-infiltrating lymphocytes (TILs) in the residual disease are a positive prognostic factor, but how specific immune composition of the tumor guides outcome is unclear, resulting in a lack of understanding of how to employ immunotherapies in the adjuvant setting. Methods: We assessed multiple immunologic biomarkers in a series of 99 residual TNBCs after NAC. Immune markers were assessed by multiple methods, including H&amp;E-based TILs analysis, standard immunohistochemistry (IHC; CD8, CD20, CD56, FOXP3, LAG-3, B7-H4, HLA-A) and multiplexed immunofluorescence (mIF; HLA-DR, GZMB, CD4, CD8, PD-L1, pan-CK, PD1, CD3). Association among parameters were assessed (up to n=98) including clinical outcome after surgery (n=94). Results: As previously demonstrated, TILs in residual tumors after NAC predicted both RFS and OS (p=0.0093 and p=0.0376, respectively). H&amp;E-scored TILs were highly correlated to CD3, CD4 and CD8 positive T cells (Spearman r range 0.34-0.4850; all p&amp;lt;0.001), while CD20 and CD56 positive cells only make up a small and non-significant proportion of the TILs composition. Likewise, infiltration by overall T cell populations were associated with significantly improved recurrence free survival (RFS; HR range 0.34-0.54) and overall survival (OS; HR range 0.38-0.43), whereas CD20+ B cells were not. Additionally, infiltration by cells expressing LAG3 (HR 0.49) and CD56 (HR 0.44), were each associated with significantly improved OS. Interestingly, colocalizing GZMB+ and CD8+ T cells by mIF yielded a paradoxical finding that cytotoxic CD8+ T cells (CD8+GZMB+ T cells) were associated with worse RFS and OS (HR 1.8 p=0.0338 and HR=2.8 p=0.0013, respectively). In a multivariate model, only CD8+GZMB+ T cell infiltration remained significant for OS (p=0.0224), suggesting independence. CD8+GZMB+ T cell infiltration inversely correlated with TILs (p=0.0293) and CD8/mm2 counts in the tumor core (p=0.0020) and showed a positive correlation with B7H4 tumor expression (p=0.0183). Additionally, high CD8+GZMB+ cases showed low HLA-A (p=0.0189) expression in tumor cells. In accordance, high CD8+GZMB+ tumors mainly correspond to stroma-restricted, margin-restricted and immune-desert tumor immune microenvironment landscapes. Conclusions: We have found a paradoxical association of GZMB+ CD8+ T cells with a negative prognosis in NAC-treated TNBC. We hypothesize that while these T cells are poised for cytotoxic activity, they remain restricted through sub-localization outside the tumor core, downregulation of HLA-A on tumor cells preventing interaction with the T cell receptor, and upregulation of B7H4 expression, which has been shown to inhibit cytotoxic T cell activity. Patients with high CD8+GZMB+ tumors in the post-NAC setting may benefit from adjuvant immunotherapy, particularly in combination with therapies that enhance MHC-I (e.g. HLA-A) antigen presentation. Citation Format: Paula I Gonzalez-Ericsson, Violeta Sanchez, Roberto Salgado, Jennifer Bordeaux, Ju Y Kim, Christine Vaupel, Henry Gomez, Melinda E Sanders, Justin M Balko. The immune landscape of residual triple-negative breast cancers after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-07.

Abstract 4050: Molecular characterization of residual triple-negative breast cancers after neoadjuvant chemotherapy identifies immune composition and features associated with clinical outcome

Abstract Although neoadjuvant chemotherapy (NAC) induces complete response in 30-40% of triple-negative breast cancers (TNBC), patients with residual disease at surgery have poor prognosis and limited treatment options until recurrence. Tumor-infiltrating lymphocytes (TILs) in the residual disease are a positive prognostic factor, but the composition of TILs has not been explored in granularity, and how specific immune composition of the tumor guides outcome is unclear, resulting in a lack of understanding of how to employ immunotherapies in the adjuvant setting. We assessed multiple immunologic biomarkers in a series of 100 residual TNBCs after NAC. Immune markers were assessed by multiple methods, including H&amp;E TILs analysis, standard immunohistochemistry (IHC; HLA-A, CD4, CD8, LAG-3) and multiplexed immunofluorescence (mIF; HLA-DR, GZMB, CD4, CD8, PD-L1, pan-CK). TILs and IHC were scored by clinical research pathologists and IF was scored by AQUA® analysis. Where multiple markers were multiplexed together, subset analyses was performed. Association among parameters were assessed (n=83) including clinical outcome after surgery (n=79). As previously demonstrated, TILs in residual tumors after NAC predicted both RFS and OS. H&amp;E-scored TILs were correlated to both CD4 and CD8 as scored by IHC or IF (Pearson’s r range 0.41-0.55; all p&amp;lt;0.001). CD4 and CD8 measured by IHC and IF were highly correlated (r=0.68 and 0.71, respectively; p&amp;lt;0.0001). CD4 and CD8 were also highly correlated to one another (r=0.67; p&amp;lt;0.0001). Likewise, infiltration by CD4 or CD8 were each associated with improved recurrence-free (RFS; coxPH p=0.004 and 0.007, respectively) and overall survival (OS; p=0.003 and p=0.005, respectively). Tumor and stromal PD-L1 (E1L3N) staining was associated with marginally poorer RFS (p=0.06) but not OS (p=0.94). Colocalizing GZMB and CD8 cells by mIF yielded a paradoxical finding that cytotoxic CD8 cells were associated with significantly worse RFS and OS (p=0.004 and 0.0004, respectively). In a multivariate model, total PD-L1, total CD4, and GZMB+CD8+ T cell infiltration each remained significant, suggesting independence, and provided a strongly predictive signature for RFS (p=4.6e-06). In the same analysis to predict OS, only GZMB+CD8+ T cells remained significant (p=0.0002). High GZMB+ CD8+ T cells were not associated with PD-L1 expression, but were associated with low LAG-3 expression (p=0.0009). Although the results of this study remain to be validated in larger cohorts, the paradoxical finding that GZMB+ CD8+ T cells are a negative prognostic factor for long term outcome in NAC-treated TNBC is an intriguing result. We hypothesize it may be these patients whose T cells are poised for cytotoxic activity but remain restricted through high PD-1 expression, and thus may benefit from adjuvant immunotherapy. Citation Format: Caroline Nebhan, Paula I. Gonzalez-Ericsson, Roberto Salgado, Jennifer Bordeaux, Ju Young Kim, Christine Vaupel, Henry Gomez, Justin Micah Balko. Molecular characterization of residual triple-negative breast cancers after neoadjuvant chemotherapy identifies immune composition and features associated with clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4050.

Abstract 2709: Modulation of the tumor-infiltrating lymphocyte population by PARP inhibitor talazoparib in combination with anti-PD1 treatment significantly enhances overall survival in a murine BRCA1-/- breast cancer model

Abstract Targeted therapy of BRCA-deficient cancers has been achieved using poly(ADP-ribose) polymerase (PARP) inhibitors, which block BRCA-independent DNA repair. With first approval in 2014 of Olaparib the concept of tumor-specific synthetic lethality was added to the treatment portfolio of cancer patients. Although the effects of PARPi have shown promising results in multiple cancer types, how and whether patients might benefit from combination with compounds modulating the immune landscape of a tumor is largely unknown. In the current study, we investigate the cross-talk between PARPi and immune checkpoint inhibition, in particular, anti PD-1 and anti CTLA-4, as the most advanced targets in the field. PARP inhibitors Niraparib, Rucaparib, Talazoparib and Olaparib were investigated in vivo using the murine EMT6/BRCA1-/- model in monotherapy as well as in combination with anti-CTLA-4 or anti-PD1 treatment. The four PARPi showed distinct activity profiles in the two breast cancer models. Talazoparib was the most active compound in the BRCA1-/- model (optimal T/C (test/control) of 60%), followed by Niraparib and Rucaparib (65% and 67%, respectively). Olaparib was considered inactive with a T/C value of 80% in monotherapy. The EMT6/BRCA1-/- turned out to be sensitive towards anti CTLA-4 treatment (optimal T/C of 35% - 38% in three independent experiments). Anti PD-1 treatment in monotherapy induced no significant reduction in tumor growth (optimal T/C of 80% - 82%, in two independent experiments). The combination of PARPi and anti CTLA-4 induced a transient but significant reduction of tumor load early in the treatment phase (p&amp;lt; 0.002, one-way ANOVA, day 8). However, anti PD-1 treatment significantly prolonged overall survival in combination with Talazoparib (p&amp;lt; 0.011 log-rank test). The analysis of tumor infiltrating lymphocytes (TILs) by flow cytometry revealed that Talazoparib as monotherapy and in combination with checkpoint (CP) inhibitors enhanced the number of gMDSC as well as the number of CD3/CD11b double positive T cells. Weekly cytokine analysis in the serum of tumor bearing mice will elucidate possible interactions between PARP and CP inhibitors and give guidance to optimal combination and schedules. First results indicate that PARPi as well as CPi induce unique cytokine profiles correlating well with the modified TIL composition of the respective treatment groups. Further mechanistic studies as well as comparative studies with the EMT6/BRCAwt model will elucidate the tumor biology behind these observations and might lead to beneficial combination strategies in patients suffering from triple negative and BRCA1-/- breast cancer. Citation Format: Anya Avrutskaya, Cordula Tschuch, Astrid Jensen, William Durham, Maycee Robinson, Charles Krause, Emily O’Koren, Gerhard Kelter, Anne-Lise Peille, Armin Maier, Anne-Marie Zuurmond, Julia Schüler. Modulation of the tumor-infiltrating lymphocyte population by PARP inhibitor talazoparib in combination with anti-PD1 treatment significantly enhances overall survival in a murine BRCA1-/- breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2709.

Relationship between tumor-associated immune infiltrate and p16 staining over clinicopathological features in acral lentiginous melanoma.

This study aims to evaluate the association between composition of tumor-infiltrating lymphocytes (TIL) and expression of p16 in acral lentiginous melanoma (ALM), and their impact on prognosis. A cohort of 148 surgical pathology specimens of ALM was studied. TIL were evaluated by immunohistochemical detection of CD3 and CD8, along with CD20, CD4, CD68, and CD163 in a subset of 43 cases. p16 protein expression was also investigated in all the cases. The median age was 66years, median Breslow thickness was 6.0mm, grade III TIL was found in 28.4% and lymph nodes were involved in 54.2%. Breslow thickness (p < 0.001), stage I-II (p < 0.001), negative lymph nodes (p < 0.001) and <10% p16 (p = 0.01) were associated with longer survival. Grade III of TIL was associated with thinner Breslow thickness (p = 0.008) and lower mitosis (p = 0.047). A higher density of CD3 TIL was associated with male gender (p = 0.008), thinner Breslow thickness (p = 0.047), negative lymph node (p = 0.031), early stage (p = 0.046), and p16 nuclear expression of >10% (p = 0.045). Higher CD8 TIL was associated with >p16 (p = 0.03). Survival analysis found that longer survival had a trend to be associated with high TIL (p = 0.090). Levels of CD3+ and CD8+ cells were correlated with those of CD4+, CD20+, CD68+ and CD163+ immune cells. Higher levels of TIL tend to be associated with better overall survival in ALM. Loss of expression of p16 is associated with lower levels of CD3+ and CD8+ TIL, indicating a probable relationship between p16 and TIL immune response in ALM .

Abstract P5-12-09: The ≥5% cut-off reveals tumor PD-L1 positivity as potential selection biomarker for patient enrollment into the trials evaluating anti-PD-1 or anti-PD-L1 agents in neoadjuvant treatment of triple negative breast cancer

Abstract Background: Durable responses of triple negative breast cancer (TNBC) to pembrolizumab (anti-PD-1) or atezolizumab (anti-PD-L1) have been reported in the metastatic setting. Moreover, it is currently being hypothesized that immune checkpoint inhibitors might be more effective in the neoadjuvant setting, due to better preserved anti-tumor immunity in early TN disease. However, biomarkers predictive of response to anti-PD-1 or anti-PD-L1 agents, as well as biomarker-based strategies for testing those drugs in the neoadjuvant setting are still lacking. We evaluated PD-L1 protein expression and the composition of tumor-infiltrating lymphocytes (TILs) in untreated TNBC, in order to get a better insight into the TNBC sub-population(s) which would be suitable for neoadjuvant anti-PD-1 or anti-PD-L1 therapy evaluation. Methods: TNBC patients consecutively treated at the Jean Perrin Comprehensive Cancer Centre (Clermont-Ferrand, France), from 01/01/2010 to 12/31/2014, were included. FFPE tumor tissues were assessed for PD-L1 expression by immunohistochemical (IHC) laboratory-developed test (clone 28-8, Abcam), in tumor cells (tPD-L1) and in TILs. Positivity cut-offs evaluated were ≥1%, ≥5% and ≥10%. The amount CD8+, CD4+, FoxP3+ or PD-1+ TILs was determined by counting those cells, detected by IHC methods, within 5 consecutive HPFs (x400), from tumor invasive front toward tumor center. Clinical disease stage was determined using the TNM system. Results: One hundred and two TNBCs were assessed. There were 28.4%, 23.5% or 16.7% tPD-L1-positive cases (cs), for cut-offs ≥1%, ≥5% or ≥10%, respectively. Similarly, 32.4%, 15.7% or 5.9% of cs were positive for PD-L1 in TILs, using the same cut-offs. With ≥5% as cut-off, positivity for tPD-L1 significantly correlated with the amount of CD8+ (p=0.023), FoxP3+ (p=0.0036) or PD-1+ TILs (p=0.043). The same cut-off, applied to TILs, revealed significant correlations between PD-L1 positivity and the amount of each CD8+, CD4+ or PD-1+ TILs (p=0.025, 0.039 and 0.0042, respectively). Interestingly, when the ≥5% cut-off was used, tumors of T2 size were most frequently tPD-L1+ (11/41 cs, 26.8%), compared with the T1 (3/35 cs, 8.6%) and T3+T4 (3/18 cases, 16.7%) (p=0.04). With regards to TILs, with the ≥5% cut-off, the PD-L1+ cases belonged exclusively to the T1+T2 group (15/76), whereas the T3+T4 group was PD-L1-negative (0/18 cs). Other two cut-offs revealed only occasional correlations. Conclusion: This single-center, real-world TNBC cohort contained a high number of smaller tumors (T1-T2). The IHC-based PD-L1 assessment, with ≥5% as the positivity cut-off, revealed that approximately 1/4 of TNBC could be candidates for neoadjuvant anti-PD-1/anti-PD-L1 approaches. Combined with the amount of CD8+ and PD-1+ TILs, tumor PD-L1 positivity might make an easy-to-use composite biomarker for the 1st-level patient selection for PD-1 or PD-L1 inhibitors in neoadjuvant TNBC treatment. The 2nd level could exploit, for example, the assessment of mutation burden in tumors with low tPD-L1 or amount of CD8+ or PD-1+ TILs. Such tumors might be more frequent among larger TNBC (T3-T4). Citation Format: Finck W, Passildas J, Poirier C, Kwiatkowski F, Abrial C, Durando X, Penault-Llorca F, Radosevic-Robin N. The ≥5% cut-off reveals tumor PD-L1 positivity as potential selection biomarker for patient enrollment into the trials evaluating anti-PD-1 or anti-PD-L1 agents in neoadjuvant treatment of triple negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-09.