Tumor Tumor-infiltrating Lymphocytes Research Articles

Association between stromal tumor-infiltrating lymphocytes (TILs) and pathologic complete response (pCR) in patients with early breast cancer (BC) treated with neoadjuvant chemotherapy and HER2-directed therapies in NSABP B-41.

3017 Background: In the phase 3 NSABP B-41 trial, there was an association between pCR and survival in 519 women with early HER2-positive BC treated with neoadjuvant chemotherapy in combination with trastuzumab (T), lapatinib (L), or both (TL). There is a need to identify prognostic biomarkers to tailor patients’ treatment, and the quantification of stromal TILs represents a promising, accessible, and reproducible option. In this analysis, we studied the association between TILs and pCR. Methods: Eligible patients had a baseline core biopsy sample, known pCR status, and no consent withdrawal for the use of specimens. Slides were scanned using an Aperio GT 450 automated scanner to create whole slide images. TIL analysis was then completed based on the international TIL working group guidelines (RS). Patients were grouped into three TIL score categories based on tumor TIL %: low (1-5%), intermediate (6-30%), and high (31-95%). The dose-response relationship between TIL percentage and pCR was explored via generalized linear models with splines. Results: 257 patients were included. 89 (35%) received T, 92 (36%) L, and 76 (29%) TL. 149 (58%) had ER-positive BC. TIL categories in this cohort: 49% low, 22% intermediate, and 29% high. TIL expression was similar across BC intrinsic subtypes and by ER. There was no association between the TIL group and pCR, irrespective of the treatment group (p=0.98). Among tumors with low TILs, T or TL had similar pCR compared to L alone (51.3 v 49%; p=0.95). Among tumors with high TILs, T or TL had higher pCR than L (61.2 v 40.4%; p=0.04). Among patients with ER-positive BC, high TILs were associated with a numerical increase in pCR rate (56% vs 39.6%; p=0.11). Among patients with ER-negative tumors, high TILs were associated with a numerical decreased rate of pCR (50% v. 66.7%; p=0.15). The p-value of the Breslow test of homogeneity of the odds ratios across ER status was 0.016, suggesting that the association between TILs (0-30 v. >30) and pCR varies across ER status.No interaction between the TIL percentage and age, nodal status, menopausal status, or histologic grade was noted. Conclusions: This analysis revealed that patients with a high TIL percentage treated with T or TL compared to L were more likely to achieve a pCR. These findings can be partially explained by the mechanism of action of the HER2-directed agents. T leads to antibody-dependent cell-mediated cytotoxicity, and a higher percentage of TILs may enhance this mechanism, whereas L has intracellular tyrosine kinase disruption. The association between TILs and pCR varied across ER status. Our next steps include additional studies assessing the interaction between TILs and long-term outcomes, as well as gene expression and TILs to validate this biomarker for future clinical trials in HER2+ early BC. Clinical trial information: NCT00486668 .

Deep learning based quantitative models to distinguish tumoral and stromal tumor-infiltrating lymphocytes and the prognosis patients with high-grade serous ovarian cancer.

84 Background: The better prognostic value of tumor-infiltrating lymphocytes (TILs) has been reported in high-grade serous ovarian cancer (HGSOC). However, the TILs evaluation of previous reports largely depends on manual quantification based on a visual assessment within limited section of pathological slides or tissue microarrays, which is subject to inter-observer variability. Since tumor immune microenvironment (TIME) of HGSOC is highly heterogeneous, there is a need to quantify TILs in large areas of pathological slides in an objective and time-saving manner. Here we present an AI-powered quantification method to distinguish tumoral TILs and stromal TILs using hematoxylin and eosin (H&E)-stained whole-slide images (WSIs) of HGSOC. Methods: 161 H&E stained WSIs of HGSOC patients treated at our hospital between 1998 and 2021 were prepared, and by using these WSIs we generated two AI-powered models. Initially, a TILs detection model (TIL-model) was created using a publicly available dataset of 86104 pan-cancer TILs images of 100 × 100 pixels at magnification of x200 (Saltz et al., 2018, Cell Reports). Secondary, a tumor and stroma segmentation model (T/S seg-model) was developed using 136962 patches of 224 × 224 pixels at magnification of x50 which were manually annotated with tumoral regions and stromal regions in 102 H&E stained WSIs of HGSOC from The Cancer Genome Atlas (TCGA). Deep convolutional neural networks of NASNet-Large for the TIL-model and TransUNet for the T/S seg-model were used. TILs density score (number of TILs positive tiles per tumor area) of each sample was calculated, and association between TILs density score and the prognosis was investigated. Results: The established models achieved high accuracy: the TIL-model showed an area under the curve (AUC) value of 0.896, and the T/S seg-model showed an intersection over union value of 0.967. By combining the two models, we successfully distinguished tumoral TILs and stromal TILs. Using our dataset of 161 WSIs, we split the patients by TILs density score at top quartile into high tumoral TILs subgroup (n=40) and low tumoral TILs subgroup (n=121). High tumoral TILs subgroup showed significantly longer overall survival (OS) and progression free survival (PFS) (OS: median 70 months vs not reached, p < 0.005. PFS: median 34 months vs 21 months, p = 0.02). Conclusions: We developed deep learning based pathological AI models to detect TILs and tumors using H&E stained WSIs, which achieved to quantify tumoral and stromal TILs. The objective assessment of TILs using these models will lead to the development of clinically applicable tools for the evaluation of the immune status in HGSOC.

Impact of Tumor-Infiltrating Lymphocytes on Disease Progression in Human Papillomavirus-Related Oropharyngeal Carcinoma.

We aim to explore the prognostic value of tumor-infiltrating lymphocytes (TILs) in the primary tumor and metastatic lymph nodes of patients with HPV(+)OPSCC. We hypothesize that TILS density at both sites is associated with disease-free survival in HPV(+)OPSCC. Matched case-control study among HPV(+)OPSCC patients who underwent intent-to-cure surgery. Cases developed locoregional or distant recurrence. Controls were matched based on age, sex, pathologic T, N, and overall stage, year of surgery, type of adjuvant treatment received, and the Adult Comorbidity Evaluation-27 (ACE-27) score. Single tertiary care center, May 2007 to December 2016. Tumoral TILs (tTILs) density was defined as % TILs; stromal TILs (sTILs) density was defined as absent/sparse or moderate/dense crowding. Associations between TILs and time to disease progression were assessed using Cox regression models. Forty-four case-control pairs (N = 88) were included: 42 (48%) AJCC pStage I, 39 (44%) pStage II, and 7 (8%) pStage III. tTILs density ≥10% (hazard ratio [HR] 0.41, 95% confidence interval [CI] 0.17-0.99, p = .048) and a moderate/dense sTILs density (HR 0.21, 95% CI 0.06-0.75, p = .016) in the primary tumor were significantly associated with decreased risk of progression. TILs density in the lymph node was associated with decreased risk of progressionbut did not reach statistical significance. The tTILs and sTILs density correlated strongly between the primary tumor and lymph node. Concordance between the pathologists' was moderate (60%-70%). In HPV(+)OPSCC, a higher density of tumoral and stromal TILs in the primary tumor and possibly the lymph node may predict a lower risk of disease progression.

Change of tumor-infiltrating lymphocyte of associating liver partition and portal vein ligation for staged hepatectomy for hepatocellular carcinoma.

BACKGROUNDThe role of tumor-infiltrating lymphocytes (TILs) in the growth and progression of hepatocellular carcinoma (HCC) has attracted widespread attention.AIMTo evaluate the feasibility of associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) for massive HCC by exploring the role of TIL in the tumor microenvironment.METHODSFifteen massive HCC patients who underwent ALPPS treatment and 46 who underwent hemi-hepatectomy were selected for this study. Propensity score matching was utilized to match patients in ALPPS and hemi-hepatectomy groups (1:1). Quantitative analysis of TILs in tumor and adjacent tissues between the two groups was performed by immunofluorescence staining and further analyses with oncological characteristics. In the meantime, trends of TILs in peripheral blood were compared between the two groups during the perioperative period.RESULTSContinuous measurement of tumor volume and necrosis volume showed that the proportion of tumor necrosis volume on the seventh day after stage-I ALPPS was significantly higher than the pre-operative value (P = 0.024). In the preoperative period of stage-I ALPPS, the proportion of tumor necrosis volume in the high CD8+ T cell infiltration group was significantly higher than that in the low group (P = 0.048).CONCLUSIONTIL infiltration level maintained a dynamic balance during the preoperative period of ALPPS. Compared with right hemi-hepatectomy, the ALPPS procedure does not cause severe immunosuppression with the decrease in TIL infiltration and pathological changes in immune components of peripheral blood. Our results suggested that ALPPS is safe and feasible for treating massive HCC from the perspective of immunology. In addition, high CD8+ T cell infiltration is associated with increasing tumor necrosis in the perioperative period of ALPPS.

Tumor Infiltrating Lymphocyte Expression of PD-1 Predicts Response to Anti-PD-1/PD-L1 Immunotherapy.

Many studies have focused on the role of programmed death receptor ligand 1 (PD-L1) expression in predicting immunotherapy outcomes. Limited clinical data are available regarding the role of programmed death receptor 1 (PD-1; the PD-L1 receptor) expressing tumor-infiltrating lymphocytes (TILs) in PD-1/PD-L1 antibody responsiveness. However, preclinical studies demonstrate that TILs expressing PD-1 contribute to tumor immune evasion. This study analyzed the association between TIL-PD-1 status and outcome after immune checkpoint blockade (ICB) therapy. We evaluated 123 patients with various solid tumors treated with monoclonal antibodies targeting the PD-1/PD-L1 signaling axis. Additionally, 8706 solid tumor specimens were assessed for TIL-PD-1 and tumor mutational burden (TMB) status. The presence of PD-1-expressing TILs in tumors was associated with increased median progression-free survival (7.0 vs 1.9 months; p = 0.006) and overall survival (18.1 vs 8.0 months; p = 0.04) after treatment with ICB. TIL-PD-1-positive patients had an objective response rate (ORR) of 41% (95% CI, 24-61; N = 12/29) compared with 17% (95% CI, 4-43; N = 3/17) for TIL-PD-1-negative patients (p = 0.18). Analyzed as continuous variables, TIL-PD-1 and TMB showed a weak correlation in 8706 solid tumor samples (Pearson r = 0.074); when analyzed as categorical variables (cutoffs: TIL-PD-1 ≥ 1% and TMB ≥ 10 mutations/Mb), the two variables are correlated (p < 0.0001). TIL-PD-1-positive status is also associated with enrichment of pathologic variants within several genes, most notably TP53 (adjusted p < 0.05). TIL-PD-1 positivity in tumors (≥ 1%) is associated with significantly longer progression-free and overall survival after ICB. ClinicalTrials.gov ID: NCT02478931.

Systemic immune checkpoint blockade and intraperitoneal chemo-immunotherapy in recurrent ovarian cancer: An interim analysis (317)

Systemic immune checkpoint blockade and intraperitoneal chemo-immunotherapy in recurrent ovarian cancer: An interim analysis (317)

Abstract 6106: The effect of neoadjuvant chemotherapy on tumor immune microenvironment in ovarian cancer

Abstract Background: Ovarian cancer (OC) is a highly lethal gynecologic malignancy, neoadjuvant chemotherapy (NACT) with interval de-bulking surgery (IDS) is an established strategy for woman with advanced epithelial ovarian cancer (EOC). Tumor infiltrating lymphocytes (TILs) play an important role in the pathogenesis of OC. However, the effect of NACT on tumor immune microenvironment (TIME) has not been evaluated. Methods: All patients underwent NACT with IDS. Formalin-fixed paraffin-embedded tissue samples were collected from biopsy specimens and the corresponding post-NACT surgical specimens. The densities and percentage of specific TILs [CD8+, CD3+, CD4+, CD20+, tumor associated macrophages (TAMs), dim CD56+NK, bright CD56+NK, PD-1+ etc.] within the TIME were analyzed by multiplex immunohistochemistry (3DMedcines Inc.). Serum CA 125 was measured 3-weekly per protocol and evaluated the response to NACT using GCIG CA125 response criteria. Results: A total of 28 patients were enrolled in this study with planned IDS after NACT. Of 25 patients evaluated by GCIG CA125 response criteria, 76% (19/25) had a CA125 response. In CA125 response patients, we observed significant recruitments of the densities and percentage of CD8+ in tumor, percentage of PD-1+ cell in tumor after-NACT vs before-NACT (34.85/mm3 vs 110.54/mm3, p=0.0258; 0.50% vs 1.73%, p=0.0132; 0.53% vs 2.66%, p=0.0576, respectively), while no significant change was found in TAMs, NK cells and CD20+ cells. However, in CA125 non-response patients, the TILs in tumor did not change significantly. Conclusion: NACT could release the tumor associated antigen by killing tumor cells for uptake by tumor-infiltrating dendritic cells and promote the infiltration of CD8+ and PD-1+ T lymphocyte in CA125 response OC patients. Given the limited clinical prognostic information and the number of patients in this study, the results of this study will need confirmation in larger studies in the future. Citation Format: Guangming Cao, Dingchao Hua, Lina Cui, Xiaochen Zhao, Shuzhen Wang, Lei Zhu. The effect of neoadjuvant chemotherapy on tumor immune microenvironment in ovarian cancer [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 6106.

Prognostic Significance of Primary Tumor-Infiltrating Lymphocytes in a Contemporary Melanoma Cohort.

The prognostic impact of tumor-infiltrating lymphocytes (TILs) on outcomes and treatment efficacy for patients with melanoma in the contemporary era remains poorly characterized. Consecutive patientswho underwent wide excision and sentinel lymph node biopsy forcutaneous melanoma 1 mm thick or thicker at a single institution were identified (2006-2019). The patients were stratified based on primary tumor TIL status as brisk (bTILs), non-brisk (nbTILs), or absent (aTILs). Associations between patient factors and outcomes were analyzed using multivariable analysis. Of the 1017patients evaluated, 846(83.2 %) had primary TILs [nbTILs (n = 759, 89.7 %) and bTILs (n = 87, 10.3 %)]. In the multivariable analysis, the patients with any type of TILs had higher rates of regression [odds ratio (OR), 1.86; p = 0.016], lower rates of acral lentiginous histology (OR, 0.22; p < 0.001), and lower rates of SLN positivity (OR, 0.64; p = 0.042) than those without TILs. The multivariable analysis found no association between disease-specific survival and bTILs [hazard ratio (HR), 1.04; p = 0.927] or nbTILs (HR, 0.89; p = 0.683). An association was found between bTILs and recurrence-free survival (RFS) advantage [bTILs (HR 0.46; p = 0.047), nbTILs (HR 0.71; p = 0.088)], with 5-year RFS rates of 84 % for bTILs, 71.8 % for nbTILs, and 68.4 % for aTILs (p = 0.044). For the 114 immune checkpoint blockade (ICB)-naïve patients who experienced a recurrence treated with ICB therapy, no association was observed between progression-free survival and bTILs (HR, 0.64; p = 0.482) or nbTILs (HR, 0.58; p = 0.176). The prognostic significance of primary TILs in the contemporary melanoma era appears complex. Further studies characterizing the phenotype of TILs and their association with regional metastasis and responsiveness to ICB therapy are warranted.

Efficient recovery of potent tumour-infiltrating lymphocytes through quantitative immunomagnetic cell sorting.

Adoptive cell therapies require the recovery and expansion of highly potent tumour-infiltrating lymphocytes (TILs). However, TILs in tumours are rare and difficult to isolate efficiently, which hinders the optimization of therapeutic potency and dose. Here we show that a configurable microfluidic device can efficiently recover potent TILs from solid tumours by leveraging specific expression levels of target cell-surface markers. The device, which is sandwiched by permanent magnets, balances magnetic forces and fluidic drag forces to sort cells labelled with magnetic nanoparticles conjugated with antibodies for the target markers. Compared with conventional cell sorting, immunomagnetic cell sorting recovered up to 30-fold higher numbers of TILs, and the higher levels and diversity of the recovered TILs accelerated TIL expansion and enhanced their therapeutic potency. Immunomagnetic cell sorting also allowed us to identify and isolate potent TIL subpopulations, in particular TILs with moderate levels of CD39 (a marker of T-cell reactivity to tumours and T-cell exhaustion), which we found are tumour-specific, self-renewable and essential for the long-term success of adoptive cell therapies.

Tumor Infiltrating Lymphocytes (TILs) and T Cell Fraction in Human Papillomavirus (HPV) Associated Oropharyngeal Squamous Cell Carcinoma

Purpose/Objective(s)The percentage of tumor infiltrating lymphocytes (TILs) is indicative of immune response and prognostic in many malignancies, although TILs data within HPV associated oropharyngeal cancer are limited. Our prior analyses of HPV associated malignancy revealed that both a higher fraction of T cells (TCF) to total cells present in the primary tumor specimen and a higher percentage of TILs were associated with a decreased chance of cancer recurrence. Herein, we investigated the relationship between these two immune related prognostic factors, TILs and TCF.Materials/MethodsPatients had HPV(+) oropharyngeal cancer and underwent primary surgery between 2007 and 2016. One representative H&E slide of the primary tumor was independently reviewed by two pathologists blinded to outcome for TILs count and presence/absence of desmoplastic stroma. The Brandwein-Gensler pattern of invasion (POI) score was used to grade the primary tumor. For TCF, immunosequencing of the CDR3 regions of human TCRβ chains was performed using an immunosequencing assay. TCF was determined as the proportion of T cells compared to the total number of nucleated cells in a sample. The TCF for patients by primary tumor TILs density, desmoplastic stroma, and POI were evaluated using Wilcoxon rank sum tests.ResultsTwenty-four patients were identified who had both TCF and TILs count available for comparison. Median age was 55 years (IQR 47-61), 96% were male, 42% had a > 10 pack year history of smoking, and 83% underwent adjuvant therapy. For patients with < 10% TILS, median TCF was 0.29 (IQR 0.17-0.36) as compared to TCF of 0.40 (IQR 0.34-0.57) for patients with ≥10% TILs (P = 0.09). There was no statistically significant difference (P = 0.5) in TCF for patients with desmoplastic stroma absent (median TCF 0.41; IQR 0.36-0.48) versus desmoplastic stroma present (median TCF 0.34; IQR 0.23-0.50). Patients with POI I or II had similar TCF to those with type III or IV, median TCF 0.37 (IQR 0.30-0.48) versus 0.35 (IQR 0.23-0.50), respectively (P = 0.8).ConclusionIn this series of HPV+ oropharyngeal cancer patients, TILs ≥10% was associated with a higher fraction of T cells to total cells (TCF) (n = 24, P = 0.09). Additional investigation is warranted including the relationship of TCF as characterized by sequencing and pathologist quantification of TILs, and whether the combination of these factors improves prognostication over each alone. The percentage of tumor infiltrating lymphocytes (TILs) is indicative of immune response and prognostic in many malignancies, although TILs data within HPV associated oropharyngeal cancer are limited. Our prior analyses of HPV associated malignancy revealed that both a higher fraction of T cells (TCF) to total cells present in the primary tumor specimen and a higher percentage of TILs were associated with a decreased chance of cancer recurrence. Herein, we investigated the relationship between these two immune related prognostic factors, TILs and TCF. Patients had HPV(+) oropharyngeal cancer and underwent primary surgery between 2007 and 2016. One representative H&E slide of the primary tumor was independently reviewed by two pathologists blinded to outcome for TILs count and presence/absence of desmoplastic stroma. The Brandwein-Gensler pattern of invasion (POI) score was used to grade the primary tumor. For TCF, immunosequencing of the CDR3 regions of human TCRβ chains was performed using an immunosequencing assay. TCF was determined as the proportion of T cells compared to the total number of nucleated cells in a sample. The TCF for patients by primary tumor TILs density, desmoplastic stroma, and POI were evaluated using Wilcoxon rank sum tests. Twenty-four patients were identified who had both TCF and TILs count available for comparison. Median age was 55 years (IQR 47-61), 96% were male, 42% had a > 10 pack year history of smoking, and 83% underwent adjuvant therapy. For patients with < 10% TILS, median TCF was 0.29 (IQR 0.17-0.36) as compared to TCF of 0.40 (IQR 0.34-0.57) for patients with ≥10% TILs (P = 0.09). There was no statistically significant difference (P = 0.5) in TCF for patients with desmoplastic stroma absent (median TCF 0.41; IQR 0.36-0.48) versus desmoplastic stroma present (median TCF 0.34; IQR 0.23-0.50). Patients with POI I or II had similar TCF to those with type III or IV, median TCF 0.37 (IQR 0.30-0.48) versus 0.35 (IQR 0.23-0.50), respectively (P = 0.8). In this series of HPV+ oropharyngeal cancer patients, TILs ≥10% was associated with a higher fraction of T cells to total cells (TCF) (n = 24, P = 0.09). Additional investigation is warranted including the relationship of TCF as characterized by sequencing and pathologist quantification of TILs, and whether the combination of these factors improves prognostication over each alone.

Immune markers of response to pembrolizumab and guadecitabine in platinum resistant ovarian cancer utilizing multiplex immunohistochemistry (mIHC)

<h3>Objectives:</h3> Treatment options for platinum-resistant ovarian cancer (PROC) are limited by poor response (poor response) rates and short progression-free survival. Immune checkpoint inhibitors induce modest response rates (8-11%) in phase II trials for PROC, although responders can have prolonged disease control. An immunoreactive subtype of ovarian cancer (OC) was proposed from TCGA genomic analysis and susceptibility to immune checkpoint blockade seems to correlate with specific populations of tumor infiltrating lymphocytes (TILs). This study sought to identify potential markers of response to immunotherapy by using multiplex immunohistochemical staining (mIHC) to phenotype TILs in ovarian tumors obtained before and after treatment with guadecitabine and pembrolizumab in a phase II clinical trial (NCT02901899). <h3>Methods:</h3> OC core needle biopsies were obtained at baseline and after 2 cycles of treatment from patients with PROC enrolled in a phase 2 trial testing the combination of guadecitabine and pembrolizumab. The Opal Multiplex IHC kit (Akoya Biosciences) was used to identify the TIL markers CD3, CD8, CD20, CD68 and FoxP3, and the epithelial marker pan-cytokeratin. A total of 12 paired (pre- and post-treatment) specimens from responders (PR or stable disease > 4 months, n=6, R) vs. non-responders (progression <4 months, n=6, NR) were stained and scanned by using the Vectra digital laser microscope and analyzed with the inForm software. The following lymphocytes were phenotyped and analyzed for spatial relationships to each other and to epithelial tumor nests: T cells, cytotoxic T cells, B cells, macrophages, and T regulatory cells (Tregs) in relationship to response to treatment. Additionally, archival tissue from 36 patients enrolled in this trial was stained with the monoclonal antibody 22C3 to assess PD-L1 expression. <h3>Results:</h3> In this study, 48 pts were enrolled, 43 were treated, and 33 were evaluable for response. Overall, there were 3 PRs (RR=9.9%) and 16 pts had stable disease (SD) [48%]. The clinical benefit rate (PR + SD > 3 months) was 27%. Analysis of TILs in the tumor microenvironment identified an increased total and stromal numbers of CD8 cells in baseline biopsies in R vs. NR (p=0.05). Likewise the total and stroma-infiltrating numbers of CD8 cells post-treatment were higher in R vs. NR (p=0.05). The total, tumor, and stromal numbers of B cells at baseline and after treatment were increased in R vs. NR (p<0.05). The numbers of total, stromal, and tumor infiltrating macrophages post-treatment samples were increased in R vs. NR (p<0.05). There were no differences in numbers of Tregs between R and NR at baseline of after treatment. Of the 36 archival specimens, 16 (45.7%) showed PD-L1 tumor modified H score/modified percent score staining greater than 0; 20 specimens (59%) had present staining at the tumor/stroma interface. <h3>Conclusions:</h3> Immunophenoyping using mIHC identified treatment induced changes in immune cell subpopulations predictive of response to immunotherapy in PROC.

The impact of tumor infiltrating lymphocytes (TILs) on disease progression in human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma.

6049 Background: In the head and neck, human papillomavirus-related oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) has a better prognosis and more tumor infiltrating lymphocytes (TILs) compared to its HPV(-) counterpart. Within HPV(+)OPSCC, the prognostic value of TILs in the primary tumor and in metastatic lymph nodes is not well understood. Methods: This is a matched case-control study at a tertiary care center of HPV(+)OPSCC patients who underwent primary surgery between 05/2007–12/2016. Cases developed locoregional recurrence or distant metastases during follow-up, while controls did not during a similar duration of follow-up. Pairs were matched on age, American Joint Committee on Cancer (AJCC) 8th edition pathologic stage, sex, year of surgery, degree of adjuvant treatment, comorbidities, and smoking status. One representative H&amp;E slide of the primary tumor and lymph node (when nodal disease was present) from each patient was independently reviewed by two pathologists (JG, MR) blinded to outcome, for tumor TILs (tTILs) density (defined as % TILs), presence/absence of desmoplastic stroma, and when stroma was present, for stromal TILs (sTILs) density (defined as relative crowding of TILs). The Brandwein-Gensler pattern of invasion (POI) score was used to grade the primary tumor. Interrater agreement was assessed using Cohen’s kappa. Associations between TILs and time to disease progression were assessed using Cox proportional hazards regression models. Results: 41 case-control pairs (N=82) were included in the study: 38 (46%) were AJCC pStage I, 37 (45%) were pStage II, and 7 (9%) were pStage III; 22 (27%) underwent surgery alone, 15 (18%) underwent surgery with adjuvant radiotherapy, and 45 (55%) underwent surgery with adjuvant chemoradiation. Interrater agreement was fair for tTILs density in the primary tumor ( k=0.24) and lymph node ( k=0.23), moderate for desmoplastic stroma in the primary tumor ( k=0.58) and lymph node ( k=0.64), moderate for sTILs density in the primary tumor ( k=0.58) and lymph node ( k=0.48), and fair for the POI score ( k=0.17). tTILs density ≥10% (HR 0.35, 95% CI 0.14-0.90, p=0.028) and a moderate/dense sTILs density (HR 0.15, 95% CI 0.04-0.68, p=0.014) in the primary tumor were significantly associated with decreased risk of disease progression. An aggressive POI score of III or IV was significantly associated with increased risk of disease progression (HR 4.00, 95% CI 1.34-11.96, p=0.013). None of the study measures in the lymph node were significantly associated with disease progression. Conclusions: In HPV(+)OPSCC, a higher density of tumor and stromal TILs and nonaggressive POI in the primary tumor specimen may indicate a lower risk of disease progression. TILs may serve as a powerful prognostic marker for the adaptive immune response to this disease.

CD45RO⁺ Memory T Lymphocytes As A Candidate Marker for Non-small Cell Lung Cancer

背景与目的肺癌是世界范围内最常见的恶性肿瘤之一。肿瘤微环境中多种多样的免疫浸润细胞，是肿瘤免疫的重要组成，对患者预后具有临床意义。CD45RO+肿瘤浸润淋巴细胞（tumor infiltrating lymphocytes, TILs），即记忆T细胞，其表达与多种肿瘤预后相关。本研究旨在探讨非小细胞肺癌（non-small cell lung cancer, NSCLC）中评估肿瘤和基质区CD45RO+ TILs密度与患者临床特征和预后的关系，及其联合程序性死亡受体配体1（programmed cell death-ligand 1, PD-L1）作为预后标志物的临床价值。方法对167例NSCLC患者的组织微阵列进行多重荧光免疫组织化学染色，标记CD45RO、细胞角蛋白（cytokeratin, CK）和PD-L1。利用人工智能图像识别技术和肿瘤细胞特异性CK染色，划分组织中的肿瘤区和基质区，评估肿瘤区和基质区CD45RO+ TILs的密度以及肿瘤细胞的PD-L1表达水平。采用非参检验分析CD45RO+ TILs与患者临床特征的关系，使用Kaplan-Meier方法和Cox风险比例模型分析CD45RO+ TILs独立或与PD-L1联合与肿瘤预后的关系。结果CD45RO+ TILs的密度与患者年龄、吸烟、肿瘤分期和病理类型显著相关。在NSCLC和肺腺癌（lung adenocarcinoma, LUAD）患者中，基质区高密度CD45RO+ TILs具有更长的总生存期（overall survival, OS）（NSCLC: P=0.007; LUAD: P < 0.001），并且是OS的独立预后因素（NSCLC: HR=0.559, 95%CI: 0.377-0.829, P=0.004; LUAD: HR=0.352, 95%CI: 0.193-0.641, P=0.001）。联合肿瘤细胞的PD-L1评分以及所有区域CD45RO+ TILs的浸润评分将患者分为四组：其中PD-L1+/CD45RO+患者无病生存期（disease-free survival, DFS）最长，PD-L1+/CD45RO-的患者DFS时间最短，并可作为DFS预后的独立因素（HR=2.221, 95%CI: 1.258-3.919, P=0.006）。结论肿瘤组织中CD45RO+ TILs密度以及CD45RO+ TILs联合肿瘤区PD-L1，与NSCLC的临床病理特征及预后显著相关，可作为新的生存预后标志物。

Surprising impact of stromal TIL’s on immunotherapy efficacy in a real-world lung cancer study

IntroductionImmune checkpoint inhibitors (ICI), such as anti-PD-1 agents, have become part of the standard of care treatment of advanced non-small cell lung cancer (NSCLC). Predictive biomarkers are needed to identify patients that benefit from anti-PD-1 treatments. Tumor infiltrating lymphocytes (TILs) and PD-L1 are major players in the ICI mechanism of action. In this study, we assess the impact of real-world clinicopathological variables, including TILs and PD-L1, on anti-PD-1 efficacy. MethodsWe performed a monocenter retrospective study in advanced NSCLC treated with nivolumab or pembrolizumab between January 2015 and February 2019. The impact of baseline clinical and pathological variables was assessed by univariate and multivariate models. TILs, defined as CD8+T-cells, and PD-L1 were scored in tumor and stroma, and correlated with progression free survival (PFS) and overall survival (OS). ResultsWe included 366 patients of whom 141 were assessed for tumor and stromal TILs. The median follow-up time was 487 days. In the whole cohort, PFS was associated with high tumor PD-L1, high albumin and good performance. OS was associated with low LDH, high albumin, good performance and ‘first-line treatment’. In the TILs subcohort, stromal TILs had the strongest impact on PFS and OS. Stromal TILs were a stronger marker for PFS and OS than tumoral TILs, tumoral PD-L1 or stromal PD-L1. Remaining factors for PFS and OS were albumin and albumin with LDH, respectively. ConclusionsThis real-world study on clinicopathological features shows that stromal CD8 + TILs were the strongest predictor for PFS and OS in patients with advanced NSCLC on anti-PD-1 therapy. Other predictors for PFS and OS included albumin and albumin together with LDH, respectively. This study highlights the pivotal role of the stromal compartment in the mechanisms of action of ICI, and the need for further studies aiming to overcome this stromal firewall.

Predictive value of tumor-infiltrating lymphocytes for pathological response to neoadjuvant chemotherapy in breast cancer patients with axillary lymph node metastasis

We investigated the role of tumor-infiltrating lymphocytes (TILs) in pretreatment primary breast cancer to predict pathological response to neoadjuvant chemotherapy (NAC) in patients with clinical node-positive disease (cN +). The subjects of this study were 60 patients with cN + , who received NAC followed by breast surgery with axillary lymph node dissection (ALND). We conducted a semi-quantitative assessment of TILs in pretreatment primary tumors and their association with clinicopathological factors and axillary lymph node metastasis. We observed a higher number of TILs in tumors with negative hormone receptors, positive human epidermal growth factor receptor 2, or high Ki67. TILs were associated with a favorable response to NAC in primary tumors. The rate of axillary pathologic complete response (Ax-pCR) was significantly higher in patients with a high number of TILs than in patients with a low number of TILs (72.0% versus 17.1%, p < 0.001). In multivariable analysis, a high number of TILs was a significant predictor of Ax-pCR as well as of pCR of the primary tumor after NAC. Importantly, all patients with HER2-positive tumors in the high TILs group showed Ax-pCR on ALND. TILs in pretreatment primary breast cancer had the potential to predict therapeutic efficacy of NAC in patients with clinical node-positive disease.

Abstract 3853: The anti-tumor immune responses by active and quiescent tertiary lymphoid structures to breast cancer

Abstract There is a growing interest in active immune responses generated by tertiary lymphoid structures (TLS) arising in solid tumors; however, their clinical impact in breast cancer (BC) remains unclear. Several studies show that transcription factors contribute to TLS formation via their regulation of cytokine and chemokine production. The Forkhead box (FOX) protein 1 (FOXP1) has been shown to play critical roles in regulating immune cells, including our recent work revealing its effects on TIL migration. These data lead us to further investigate FOXP1 expression in tumor infiltrating lymphocytes (TIL) and TLS. We identify two types of TLS based on FOXP1 expression: 1) those that contain a germinal center (GC+) and those that do not (GC-). Comparative analysis of FOXP1 expression in secondary lymphoid organs, including more immune active tonsils (many GC) and less immune active spleens (primarily without GC) confirm differences in FOXP1 expression associated with GC. In BC, TLS containing tumors were more frequently GC- than GC+ (n=49), with triple-negative tumors having higher numbers of GC+ TLS compared to luminal or HER2+ tumors. Immunofluorescence and multiplex immunohistochemistry was used to closely examine the GC+ and GC- TLS, finding an immune active profile in the former, characterized by T follicular helper cells (PD1+CD4+ T), mature dendritic cells (CD21+ and CD23+), actively proliferating (Ki67+) B cells undergoing immunoglobulin (Ig) class switch recombination (AID+) and a plasma cell presence (CD138+). Analysis of Ig's in the primary tumor supernatants revealed that BC with ≥1 GC+ TLS (n=20) were characterized by increases in total Ig, IgG1, IgG2 and IgA, reflecting active humoral immunity, compared to BC containing only GC- TLS (n=29). Gene expression analysis of individual micro-dissected TLS demonstrated upregulation of Th1, Th2 and Tfh immune genes in the GC+ compared to the GC- TLS, suggesting the former also sustain cell-mediated immune responses. Immune infiltrates in tumors with ≥1 GC+ TLS are specifically characterized by high global TIL, CD3+, CD4+ or CD8+ T cell TIL and CD20+ TIL-B (n=29). Analysis of BC TIL spatial distribution identified increased stromal TIL (all subpopulations) while intratumoral TIL increases were predominantly CD3+ and CD8+ T cell TIL in tumors with GC+ TLS. Overall, our data indicate that GC+ TLS house active immune responses in BC while GC- TLS are quiescent. Citation Format: Pushpamali De Silva, Soizic Garaud, Cinzia Solinas, Grégory Noël, Mireille Langouo Fontsa, Anaïs Boisson, Alexandre de Wind, Vinu Jose, Gert Van den Eynden, Noemie Thomas, Hugues Duvillier, Céline Naveaux, Ligia Craciun, Dominique Bron, Martine Piccart-Gebhart, Denis Larsimont, Karen Willard-Gallo. The anti-tumor immune responses by active and quiescent tertiary lymphoid structures to breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3853.

Correlation Between Mammographic Radiomics Features and the Level of Tumor-Infiltrating Lymphocytes in Patients With Triple-Negative Breast Cancer.

Objectives: Tumor-infiltrating lymphocytes (TILs) have been identified as a significant prognostic indicator of response to neoadjuvant therapy and immunotherapy for triple-negative breast cancer (TNBC) patients. Herein, we aim to assess the association between TIL levels and mammographic features in TNBC patients.Methods: Forty-three patients with surgically proven TNBC who underwent preoperative mammography from January 2018 to December 2018 were recruited. Pyradiomics software was used to extract 204 quantitative radiomics features, including morphologic, grayscale, and textural features, from the segmented lesion areas. The correlation between radiological characteristics and TIL levels was evaluated by screening the most statistically significant radiological features using Mann–Whitney U-test and Pearson correlation coefficient. The patients were divided into two groups based on tumor TIL levels: patients with TIL levels <50% and those with TIL levels ≥50%. The correlation between TIL levels and clinicopathological characteristics was assessed using the chi-square test or Fisher's exact test. Mann–Whitney U-test and Pearson correlation coefficient were used to analyze the statistical significance and Pearson correlation coefficient of clinical pathological features, age, and radiological features.Results: Of 43 patients, 32 (74.4%) had low TIL levels and 11 (25.6%) had high TIL levels. The histological grade of the low TIL group was higher than that of the high TIL group (p = 0.043). The high TIL group had a more negative threshold Ki-67 level (<14%) than the low TIL group (p = 0.017). The six most important radiomics features [uniformity, variance, grayscale symbiosis matrix (GLCM) correlation, GLCM autocorrelation, gray level difference matrix (GLDM) low gray level emphasis, and neighborhood gray-tone difference matrix (NGTDM) contrast], representing qualitative mammographic image characteristics, were statistically different (p < 0.05) among the low and high TIL groups. Tumors in the high TIL group had a more non-uniform density and a smoother gradient of the tumor pattern than the low TIL group. The changes in Ki-67, age, epidermal growth factor receptor, radiomic characteristics, and Pearson correlation coefficient were statistically significant (p < 0.05).Conclusion: Mammography features not only distinguish high and low TIL levels in TNBC patients but also can act as imaging biomarkers to enhance diagnosis and the response of patients to neoadjuvant therapies and immunotherapies.

Clinicopathologic Features and Prognostic Role of CD3, CD8, and PD-1 positive Tumor-Infiltrating Lymphocytes and the Association with COX-2 Overexpression in Endometrial Carcinoma

Background: Determining prognostic factors of endometrial cancer is important for the management of this disease. Objectives: The aim of this study was to identify the association between tumor- infiltrating lymphocytes (TILs) and programmed cell death protein-1 (PD-1) expression in intra-tumoral inflammatory cells and clinicopathological features of endometrial carcinoma. Methods: This observational study was conducted on 56 patients who underwent hysterectomy with diagnosis of endometrial carcinoma and available 5-year follow-up data from 2006 to 2013 in Imam Khomeini Hospital, Tehran, Iran. The TILs scoring was calculated based on the mean percentage of stroma occupied by mononuclear inflammatory cells at the invasive border of the tumor in H & E stained slides in 10 representative high-power fields. The mean number of CD3, CD8, and PD-1 positive lymphocytes in 10 high-power fields, as well as cyclo-oxygenase-2 (COX-2) expression, were assessed. Results: TILs were significantly higher in endometrioid carcinoma compared to non-endometrioid carcinomas (P = 0.001). There was no significant association between TILs percentage, myometrial invasion, lymphovascular invasion, and tumor grade (P > 0.05). High TILs tumors showed better prognosis (P = 0.046). On the immunohistochemical (IHC) study, TILs were higher in endometrioid tumors compared to non-endometrioid tumors (P < 0.05). The expression of PD-1 was higher in endometrioid tumors compared to non-endometrioid carcinomas (P = 0.001). The COX-2 expression was not associated with prognosis and the other clinicopathologic features. Conclusions: High TILs and PD-1 expression were associated with better prognosis in endometrial carcinoma. Endometrioid carcinomas had a higher TILs, CD3, CD8, and PD-1 lymphocytes. This study failed to identify a clinically significant cut off for these inflammatory biomarkers.

Multi-panel immunofluorescence analysis of tumor infiltrating lymphocytes in triple negative breast cancer: Evolution of tumor immune profiles and patient prognosis.

The evolutionary changes in immune profiles of triple negative breast cancer (TNBC) are not well understood, although it is known that immune checkpoint inhibitors have diminished activity in heavily pre-treated TNBC patients. This study was designed to characterize immune profile changes of longitudinal tumor specimens by studying immune subsets of tumor infiltrating lymphocytes (TILs) in paired primary and metastatic TNBC in a cohort of “poor outcome” (relapsed within 5 years) patients. Immune profiles of TNBCs in a cohort of “good outcome” (no relapse within 5 years) patients were also analyzed. Immune subsets were characterized for CD4, CD8, FOXP3, CD20, CD33, and PD1 using immuno-fluorescence staining in stroma, tumor, and combined stroma and tumor tissue. TIL subsets in “good outcome” versus “poor outcome” patients were also analyzed. Compared with primary, metastatic TNBCs had significantly lower TILs by hematoxylin and eosin (H&E) staining. Stromal TILs (sTILs), but not tumoral TILs (tTILs) had significantly reduced cytotoxic CD8+ T cells (CTLs), PD1+ CTLs, and total PD1+ TILs in metastatic compared with matched primary TNBCs. Higher PD1+ CTLs, PD1+CD4+ helper T cells (PD1+TCONV) and all PD1+ T cells in sTILs, tTILs and total stromal and tumor TILS (s+tTIL) were all associated with better prognosis. In summary, TIL subsets decrease significantly in metastatic TNBCs compared with matched primary. Higher PD1+ TILs are associated with better prognosis in early stage TNBCs. This finding supports the application of immune checkpoint inhibitors early in the treatment of TNBCs.

CTLA-4 blockade boosts the expansion of tumor-reactive CD8+ tumor-infiltrating lymphocytes in ovarian cancer

Adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) can induce durable complete tumor regression in patients with advanced melanoma. Efforts are currently underway to expand this treatment modality to other cancer types. In the microenvironment of ovarian cancer, the engagement of co-inhibitory immune checkpoint molecules such as CTLA-4 can lead to the inactivation of TILs. Thus, approaches that directly manipulate co-inhibitory pathways within the tumor microenvironment might improve the expansion of tumor-reactive TILs. The initial expansion of TILs for ACT from tumor fragments provides a window of opportunity to manipulate an intact tumor microenvironment and improve CD8+ T-cell output and TIL tumor reactivity. To exploit this, we used a CTLA-4-blocking antibody, added during the initial TIL culture, and found that the blockade of CTLA-4 favored the propagation of CD8+ TILs from ovarian tumor fragments. Interestingly, adding the CTLA-4 blocking antibody in the initial phase of the TIL culture resulted in more potent anti-tumor TILs in comparison to standard TIL cultures. This phenotype was preserved during the rapid expansion phase. Thus, targeting CTLA-4 within the intact tumor microenvironment of tumor fragments enriches tumor-reactive TILs and may improve clinical outcome of TIL-based ACT in ovarian cancer.