Significance Of Tumor-infiltrating Lymphocytes Research Articles

Significance of tumor-infiltrating lymphocytes in tumor regression in breast cancer: A study in a tertiary care cancer center in South India.

Tumor immunology plays a significant role in predicting tumor biology and how a tumor is going to respond to neoadjuvant chemotherapy (NACT). Tumor-infiltrating lymphocytes (TILs) are the easiest and by far the cheapest method of assessing tumor immunity. Many studies have suggested that TILs play an important role in tumor regression in breast cancer. The aim of the current study was to determine significance of TILs in tumor regression in breast cancer. Patients with newly diagnosed and histologically proven breast cancer who were treated with both NACT and surgery in our institute were included in the study. TILs were assessed both before and after NACT, and were correlated with the relative amount of tumor regression and molecular subtypes based on the immunohistochemistry profile. The study included 43 specimens of carcinoma breast in females. 42 cases were diagnosed with invasive carcinoma, no special type (NST), and one with lobular carcinoma. Pathological complete remission (pCR) was noted in 6 cases, partial remission (PRe) in 12 cases, and no response in 25 cases. TILs were noted before and after NACT in all cases and were correlated with other clinicopathological parameters. The present study highlights that TILs play a vital role in tumor regression and can be included in routine reporting. It can provide an insight into tumor biology.

CD45-positive tumor infiltrating lymphocytes in early-stage hormone-positive, HER2-negative (ER+/HER2-) breast cancer: Correlation with proliferation and prognostic signature scores.

e12555 Background: Tumour infiltrating lymphocytes (TILs), have been associated in higher responses to treatment and are thought to be a prognostic marker in a number of cancer types. The role of TILs in breast cancer varies depending on the molecular subtype of the disease. In hormone positive, HER2-negative breast cancer, the significance of TILs is less clear, partly due to the paucity of data in this area. We aim to assess overall TILs in early-stage, hormone-positive, HER2-negative (ER+/HER2-) breast cancer and its relationship with proliferation index, Ki67, and the 21-gene recurrence score. Methods: Archival patient tissue samples of early-stage, ER+/HER2- breast cancer from the Irish arm of the TAILORx clinical trial were used. Full-face sections of these tumours were stained with anti-CD45 antibody (DAKA clone M0701) via the automated Leica Bond III system. A watershed cell-detection algorithm from the open-source digital pathology software, QuPath, was fine-tuned to identify true cell objects and positive immune markers. Optimised detection parameters were used to calculate the percentage of overall CD45+ cells (CD45%) within the tumour area. Patient TIL categories were dichotomised into High v Low CD45%, with 10% chosen as the threshold. CD45% was further correlated with proliferation index, clinical data, and 21-gene recurrence score. Results: 409 samples were included in the analysis. The mean CD45% was 5.96, with samples ranging from 0.13 to 78.86. The majority of samples were in the range of 0-10% of CD45% positivity with 89% (n = 367) patients in this category. Stratification of the cohort by 21-gene-recurrence score (RS) into Low, Intermediate and High, showed a statistically significantly high CD45% in patients with a High RS compared to Low RS (p = 0.0015). When divided by recurrence score group, high CD45% trended toward a lower disease free survival (DFS) when compared to low CD45%, with statistical significance found in those in the Intermediate RS chemotherapy treatment arm (p = 0.009). Conclusions: The role of TILs in ER+/HER2- breast cancer is still undefined. While a High RS was associated with a higher percentage of CD45-positive lymphocytes, the impact on recurrence and response to treatment remains unclear. Further studies to assess the role of different tumour infiltrating lymphocyte phenotypes, and the spatial distribution of assessed markers, are needed.

Is the presence of tumor-infiltrating lymphocytes predictive of outcomes in patients with melanoma?

The significance of tumor-infiltrating lymphocytes (TILs) in melanoma is debated. This article presents a multicenter, retrospective study assessing the predictive and prognostic value of TILs. The Sentinel Lymph Node Working Group database was queried from 1993 to 2018 for cases with known TIL data. TILs were categorized as absent or present, which included nonbrisk (NB), brisk (B), and present but unspecified TIL levels. Clinicopathologic factors were correlated with TILs, sentinel lymph node (SLN) status, and melanoma-specific survival (MSS). Overall, 3203 patients were included. The median thickness was 1.5 mm, and 469 cases had SLN metastases. TILs were present in 2458 cases (76.7%), with NB, B, and unspecified TILs seen in 1691 (68.8%), 691 (28.1%), and 76 (3.1%), respectively. Multivariable analysis showed that the presence of TILs significantly predicted a negative SLN biopsy (P < .05). The median follow-up was 25.2 months. MSS was significantly better for cases with TILs than cases without TILs (P < .001). According to multivariable analysis, age, gender, thickness, mitotic rate, ulceration, lymphovascular invasion, and SLN status were significantly prognostic of MSS (all P values < .05). Although TILs were not prognostic of MSS, when multiple imputation was used and the SLN status was excluded, the presence of TILs was significantly prognostic of improved MSS (hazard ratio, 0.78; 95% confidence interval, 0.64-0.95; P = .0154). TILs are a favorable marker because their presence significantly predicts a negative SLN, and the absence of TILs may be a prognostic marker of worse survival in patients with a positive SLN but not a negative SLN. TILs may also serve as a prognostic marker of survival when the SLN status is not considered.

The prognostic significance of TILs as a biomarker in triple-negative breast cancer: what is the role of TILs in TME of TNBC?

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancers, and its poor response to treatment has been a major problem in the field of breast cancer. In recent years, the application of immune checkpoint inhibitors has introduced a new era of treatment. The IMpassion 130 trial used PD-L1 as a mature biomarker for immunotherapy in metastatic TNBC, but the population screened, which was only patients with positive PD-L1 expression, was too narrow. Otherwise, it could not be determined whether the PD-L1-positive group benefited from immunotherapy in early TNBC, but this was confirmed in the KEYNOTE-522 and IMpassion 031 studies, in which there was no significant difference in the benefit, whether patients were PD-L1-positive or not. Therefore, how to screen more suitable biomarkers for accurate immunotherapy has become a burning and persistent problem to be solved. In fact, immune infiltration has always been our focus in the process of exploring immunotherapy in TNBC, and tumor infiltrating lymphocytes (TILs) have been well-known for decades as a prognostic factor in early TNBC. Furthermore, TILs are positively correlated with both patient survival and pathological complete response (pCR) after neoadjuvant chemotherapy. Recently, increasingly more foundational experiments and clinical trial verifications suggest that TILs could contain more biomarkers. Thus, in this review, we will assess the composition and heterogeneity of TILs, their evaluation standards, their relationship with TNBC prognosis and the prediction ability of different treatment options in TNBC, and their correlation with other biomarkers in the clinical application. We also summarize new studies that show the future potential of TILs.

Abstract PS6-45: Prognostic significance of tumor-infiltrating lymphocytes and neutrophil-to-lymphocyte ratio in patients with breast cancer receivingneoadjuvant chemotherapy

Abstract Introduction and objectives: Tumor infiltrating lymphocytes (TILs) and neutrophil-to-lymphocyte ratio (NLR) play a prognostic role in early stage breast cancer (BC). There is no evidence about the combined effect of both factors. Our objective was to evaluate the integrated clinical significance of TILs and NLR in patients with early BC treated with neoadjuvant therapy. Materials and methods: Retrospective, single-center analysis of a cohort of patients with early BC treated with neoadjuvant chemotherapy between 2001-2010. Pre-treatment TILs (CD3+-TIL count) was evaluated using a tumor tissue microarray. NLR was calculated within one month of cancer diagnosis. TILs (logarithmic transformed) and NLR were analyzed as continuous variables. Survival analysis was performed using multivariable Cox regression models. Results:A total of 121 patients were included. Median age: 56 years. Cancer stage at diagnosis: 16% IIA, 28% IIB, 33% IIIA, 7% IIIB and 16% IIIC. Molecular subtype: 64% hormone receptor(HR)-positive (12% HER2-positive), 11% HER2-positive HR-negative and 22% triple-negative. Pathological complete response (pCR): 16.5%. Median follow-up: 12 years. Pre-treatment TIL analysis was available in 71 patients (59%) and NLR in 101 (83%). There was no correlation between both variables (Spearman's Rho: 0.03, p = 0.98). In the univariate analysis, the NLR showed a negative prognostic value for overall survival (OS) (HR 1.23, 95%CI 1.11-1.36; p &amp;lt;0.001, C-index: 0.64 95%CI 0.52-0.77, p = 0.69). The effect was opposite for TILs (HR: 0.76 95%CI 0.61-0.95, p = 0.02; C-index: 0.69 95%CI 0.57-0.81, p = 0.69). The linear approximation was adequate, and there was no suspicion of non-proportionality of the hazards. In the multivariate analysis, including or not cancer staging after neoadjuvant therapy, NLR remained as an independent variable (HR 1.18, 95%CI 1.04-1.33; p = 0.01) and a statistic trend for TILs was also observed (HR 0.83, 95%CI 0.65-1.07; p = 0.16). Given the limited sample size, the multivariate analysis did not provide clear evidence of an additive effect. Nevertheless, the combined analysis of both parameters showed a better fit with respect to the two variables separately (Akaike Information Criterion for the combined model, for TILs and for NLR: 106, 133 and 214, respectively). Conclusion:The integrated characterization of TILs and NLR identifies different prognostic subgroups in early BC patients receiving neoadjuvant chemotherapy. Future validation of these findings in large, multicenter cohorts might allow treatment optimization by means of new strategies such as immunotherapy. Citation Format: Esmeralda García-Torralba, Francisco Ayala de la Peña, Beatriz Alvarez-Abril, Pilar de la Morena Barrio, Alejandra Ivars Rubio, Elisa Garcia Garre, Gema Marin Zafra, Alberto Carmona-Bayonas, Elena Garcia Martinez. Prognostic significance of tumor-infiltrating lymphocytes and neutrophil-to-lymphocyte ratio in patients with breast cancer receivingneoadjuvant chemotherapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS6-45.

Tumour-infiltrating lymphocytes and response to neoadjuvant letrozole in patients with early oestrogen receptor-positive breast cancer: analysis from a nationwide phase II DBCG trial

BackgroundThe presence of tumour-infiltrating lymphocytes (TILs) is associated with response to neoadjuvant chemotherapy among patients with triple-negative and HER2-positive breast cancer. However, the significance of TILs is less clear in luminal breast cancer. Here, we in postmenopausal patients with primary oestrogen receptor-positive (ER+), HER2 normal, operable breast cancer assessed the importance of inducing TILs during 4 months of letrozole on response in a neoadjuvant phase II study.MethodsParticipants were postmenopausal women with ER+, HER2 normal operable breast cancer assigned to 4 months of neoadjuvant letrozole. Pretreatment core biopsies and surgical specimens were assessed centrally for the percentage of TILs on haematoxylin and eosin-stained slides according to the International Immuno-Oncology Biomarker Working Group on Breast Cancer guidelines. Pathological response was assessed by the Residual Cancer Burden (RCB) index and a modified Miller-Payne grading system and was analysed according to change in TILs.ResultsTumour specimens were available from 106 of the 112 patients treated per protocol. TIL concentration increased with mean 6.8 percentage point (p < 0.0001) during treatment (range − 39 to 60). An increase in TILs was significantly associated with pathological response with OR = 0.71 (95% CI 0.53–0.96; p = 0.02) per 10% absolute increase for pathological response and correspondingly OR = 0.56 (95% CI 0.40–0.78; p = 0.0007) for lower RCB index per 10% increase.ConclusionIncreasing TILs during letrozole was significantly associated with a poor treatment response. An increase in TILs during endocrine therapy might imply immunogenicity, and these patients could be targetable by immunotherapy.Trial registrationClinicalTrials.govNCT00908531, registered 27 May 2009.

Abstract A41: Antitumor reactivity of human breast tumor-infiltrating lymphocytes (TILs)

Abstract Background: Clinical significance of tumor-infiltrating lymphocytes (TILs) was demonstrated in many tumor types. In the clinical trials, immune checkpoint inhibitors showed significant clinical efficacy in some tumor types with high mutation burden such as melanoma, bladder cancer, and smoking-induced lung cancer, while there were little effects in cancers with lower mutation rates such as breast cancer. Recently, selection and adoptive transfer of autologous mutation specific TILs showed successful regression of metastatic cancer in an estrogen receptor-positive breast cancer patient. In this study, we analyzed in vitro reactivity of cultured TILs against autologous breast cancer cells. Methods: We cultured TILs and cancer cells derived from 31 breast tumor tissues obtained from the surgery (13 of hormone receptor-positive, one of HER2-positive, and 17 of triple-negative breast cancer [TNBC]). Reactivity of TILs against cancer cells was determined by IFN-gamma ELISA analysis after co-culture of TILs and cancer cells for 24 hours. Epithelial cell markers (cytokeratin and EpCAM) for cancer cells and T cell markers [memory types (CD45RO, CCR7, and CD62L), T-cell activation and exhaustion markers (PD-1, 4-1BB, OX40, CD39, CD107a, TIM3, TIGIT, KLRG1, and LAG3), and regulatory T cells markers (CD25, CD127, and FOXP3)] for expanded TILs were analyzed by flow cytometry. Results: Seven cases showed in vitro reactivity, which was defined by increased secretion (more than twice) of IFN-gamma from TILs upon cancer cell interaction. Interestingly, most of reactive TILs were from TNBC (6/7) and all of TNBCs showing in vitro reactivity were derived from resection specimens after neoadjuvant chemotherapy (NAC, 6/11). The remaining one case with in vitro reactivity was derived from HER2-positive breast cancer. TNBC cancer cells of cases with in vitro reactivity had more EpCAM-positive cells (p = 0.02) than those of nonreactive cases. Most T cells in TIL cultures showed effector memory phenotype (CD45RO+/CCR7-; CD4 T cells, median 97.4 % (73.7-99.9); CD8 T cells, median 98.8% (93.8-99.8)). TILs of TNBC cases with in vitro reactivity expressed higher levels of PD-1 (p value &amp;lt; 0.01) on CD4 T cells. PD-1 expression levels on CD8 T cells in reactive group tended to be higher than nonreactive group (p = 0.05). Expression of other markers was not different between reactive and nonreactive groups. Conclusion: Among 17 TNBC cases, 35% showed in vitro TIL reactivity against autologous tumor cells. All reactive TNBCs were NAC-resistant tumors, which have high risk of tumor recurrence and high mortality rates. Adoptive TIL therapy might be a promising therapeutic modality for TNBC patients. Citation Format: Heejae Lee, Young-Ae Kim, Hye Seon Park, Won Seon Bang, In Ah Park, Miseon Lee, Gyungyub Gong, Hee Jin Lee. Antitumor reactivity of human breast tumor-infiltrating lymphocytes (TILs) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A41.

Prognostic Significance of Tumor-Infiltrating Lymphocytes on Survival Outcomes of Patients with Resected Pancreatic Ductal Adenocarcinoma: A Systematic Review and Meta-Analysis

Prognostic Significance of Tumor-Infiltrating Lymphocytes on Survival Outcomes of Patients with Resected Pancreatic Ductal Adenocarcinoma: A Systematic Review and Meta-Analysis

Prognostic Impact of Tumor-Infiltrating Lymphocytes in Primary and Metastatic Colorectal Cancer: A Systematic Review and Meta-analysis.

There is increasing literature emerging on the significance of tumor-infiltrating lymphocytes in colorectal cancer. However, there have been inconsistent findings, secondary to small patient numbers and varied methods for identifying these lymphocytes. The aim of this study was to determine the prognostic and predictive power of tumor-infiltrating lymphocytes in colon, rectal (in neoadjuvant setting), and metastatic colorectal cancer. A comprehensive search of PubMed and Embase was undertaken from January 2006 to December 2016. The inclusion criteria included a description of the tumor-infiltrating lymphocyte subset(s) assessed with reporting of associated short- and long-term outcomes. The main outcome measures, were disease-free and overall survival. A total of 25 studies were included, 15 for primary colorectal cancer (4719 patients), 7 for locally advanced rectal cancer (727 patients), and 3 studies for metastatic colorectal cancer (418 patients). High CD3, CD8, FoxP3, and CD45RO densities were associated with improved overall survival for primary colorectal cancer, with pooled estimated HRs of 0.88, 0.81, 0.70, and 0.63 (all p < 0.001) respectively. Furthermore, in locally advanced rectal cancer, the levels of CD8 cells were a significant predictor of good tumor regression grade after chemoradiotherapy. The retrospective nature of included studies and the significant interstudy heterogeneity were limitations. There is increasing evidence that tumor-infiltrating lymphocytes play an important role in predicting prognosis in colorectal cancer and tumor regression after neoadjuvant chemoradiotherapy in locally advanced rectal cancer. Clinical researchers are now in a unique position to build on this work to identify robust predictive markers to stratify patients not only to currently available therapies but also to immunotherapy, which has demonstrated success in improving patient outcomes.

Abstract A118: Clinical impact of PD-L1 expression and epithelial-mesenchymal transition in the tumor microenvironment of extrahepatic cholangiocarcinoma

Abstract Background: Extrahepatic cholangiocarcinoma (eCCA) has a poor prognosis. Although the possibility of immunotherapy has been studied, immune checkpoint molecules such as programmed death ligand 1 (PD-L1) in eCCA are not well understood. Epithelial-mesenchymal transition (EMT) has recently been shown to regulate PD-L1 expression. Our aims were to assess the clinicopathological significance of tumor-infiltrating lymphocytes (TILs) and tumor PD-L1 expression in eCCA and to compare these immune responses with EMT marker expression. Material and Methods: An immunohistochemical evaluation using a TMA method at a single large center was performed. Patients (n = 117) underwent surgical resection in the Department of Gastroenterological Surgery II at Hokkaido University Hospital between January 1995 and November 2006 and eCCA tumors were confirmed histopathologically. We investigated the level of infiltration of immune cells positive for CD4, CD8, Foxp3, PD-L1 in addition to E-cadherin, N-cadherin, Vimentin, ZEB1, ZEB2, SNAIL, TWIST expression, statistically comparing correlations among these factors. Results: High numbers of CD4+ and CD8+ TILs correlated with node-negative (P = 0.009 and P = 0.046, respectively) and low SNAIL expression (P = 0.016 and P = 0.022, respectively). High PD-L1 expression was associated with poor histopathologic classification (P = 0.034), and low E-cadherin (P = 0.001), high N-cadherin (P = 0.044), high vimentin (P &amp;lt; 0.001) and high ZEB1 (P = 0.036) expression. Multivariate analysis showed that CD4+ TILs, PD-L1 expression and N-cadherin expression were independent prognostic factors (hazard ratio (HR) = 0.61; 95% confidence interval (CI) = 0.38-1.00; HR=4.27; 95% CI = 1.82-9.39; HR = 2.20; 95% CI = 1.18-3.92, respectively). Summary: Present data suggested that CD4+ T lymphocyte tumor infiltration and PD-L1 expression on tumor cells were independent prognostic factors in eCCA. We also provided the first evidence that high tumor PD-L1 expression correlates with a mesenchymal phenotype. Citation Format: Takahiro Tsuchikawa, Takashi Ueno, Osamu Sato, Toru Nakamura, Yoshitsugu Nakanishi, Toshimichi Asano, Takehiro Noji, Keisuke Okamura, Toshiaki Shichinohe, Satoshi Hirano. Clinical impact of PD-L1 expression and epithelial-mesenchymal transition in the tumor microenvironment of extrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A118.

Tumor Infiltrating Lymphocytes May Predict for Distant Metastasis in Soft Tissue Sarcomas

The presence of tumor infiltrating lymphocytes (TILs) has been shown to be prognostic in malignancies such as breast, ovarian and colorectal cancer. Moreover, hypofractionated radiation therapy (RT) has been shown to improve tumor control and lead to an increased number of TILs in tumors. However, the significance of TILs in soft tissue sarcoma (STS) remains to be determined. In this study, we aim to determine if TILs are prognostic in patients undergoing pre-operative RT for STS of the extremity and chest-wall, and to evaluate if hypofractionated RT is associated with increased TILs when compared to standard fractionation. H&E slides from resected specimens of extremity and trunk STS patients who underwent pre-operative standard fractionation at 2 Gy/fx or hypofractionation at 7 Gy/fx were reviewed by a blinded pathologist to quantify TILs. All hypofractionated patients were enrolled on an institutional clinical trial. TILs were defined as lymphocytes infiltrating in areas of viable tumor cells. TILs were quantified utilizing 40x and 10x objectives lens (Olympus BX40 microscope) at the tumor hot spots (areas of tumors with highest concentration of TILs) and expressed as a percentage; TILs%/ 40x and TILs%/ 10x. Receiver-operating curve (ROC) analysis was performed to assess the best cut-off that predicted for distant metastasis (DM) in patients undergoing pre-operative RT. Distant metastasis-free survival (DMFS) was calculated using Kaplan-Meier survival analysis. A t-test was performed to assess the difference in means between the 2 groups. Seventy-nine patients with stage I-III STS of the extremity and chest-wall were analyzed, 61 who underwent standard and 18 who underwent hypofractionated RT. Median overall age was 56. Median overall tumor size was 9.8 cm. Median dose of the standard and hypofractionated groups were 50 Gy and 35 Gy, respectively. 30% of patients developed DM in the standard fractionated group vs 11% in the hypofractionated group (p=0.07). The median overall % TILs at 10x and 40x was 20% and 25%, respectively. The mean % TILs at 10x and 40x for the standard and hypofractionated RT was 19.68% vs 31% (p=0.06) and 26.2% vs 42.8% (p=0.03), respectively. ROC analysis revealed that % TILs (10x and 40x) ≤10 was associated with increased risk of distant metastasis (p<0.0001). Median DMFS for patients with % TILs ≤10 was 44.3 months vs >54 months for % TILs > 10% (p=0.0009). In localized STS of the extremity and chest-wall, increasing % TILs was associated improved DMFS. Hypofractionated RT appears to be protective with these patients demonstrating higher % TILs and a decreased rate of developing DM. A cut-off ≤10 % TILs of was shown to be associated with poorer DMFS in this study for all patients treated with pre-operative RT. Further validation of the clinical utility of TILs in this context is warranted.

Prognostic significance of tumor-infiltrating lymphocytes in patients with operable tongue cancer

BackgroundOur aim was to investigate the prognostic significance of tumor-infiltrating lymphocytes (TILs) in operable tongue cancer patients.MethodsThe presence of CD3+, CD4+, CD8+, and forkhead box protein P3-positive (FOXP3+) TILs in tumor tissues obtained from 93 patients during surgery was examined using immunohistochemistry.ResultsThe 3-year overall survival (OS) of patients with a low CD8/FOXP3 ratio was significantly lower than that of patients with a high CD8/FOXP3 ratio (63.8% vs. 87.3%, p = 0.001). Patients with high FOXP3 had a significantly lower 3-year regional recurrence-free survival (RRFS) than did patients with low FOXP3 (49.3% vs. 87.3%, univariate log rank p = 0.000). A low CD4/FOXP3 ratio (68.4% vs. 93.7%, univariate log rank p = 0.002) was significantly unfavorable prognostic factors for 3-year distant metastasis-free survival (DMFS).ConclusionsIn addition to clinicopathological characteristics, TIL markers represent prognosticators for clinical outcomes.

Prediction of Treatment Response to Neoadjuvant Chemotherapy in Breast Cancer by Subtype Using Tumor-infiltrating Lymphocytes

Recent interest has focused on the significance of tumor-infiltrating lymphocytes (TILs) on the efficacies and outcomes of the treatment in breast cancer (BC). Based on the recent international recommendation to standardize the evaluation method, the clinical validity and utility of TILs in patients who underwent neoadjuvant chemotherapy (NAC) were investigated in the present study. TILs were evaluated in 177 patients with BC treated with NAC and subsequent curative surgery. The correlation between TILs evaluated according to the standard method and prognosis, including the efficacy of NAC, was investigated retrospectively. In the high-TIL group (n=96) compared to the low-TIL group (n=81), triple-negative breast cancer (TNBC) (p<0.001) and human epidermal growth factor receptor 2-enriched breast cancer (HER2BC) (p=0.040) were significantly more frequent, and the pathological complete response (pCR) rate was significantly higher (p=0.003). Among patients with TNBC and those with HER2BC, the pCR rate was significantly higher in the high-TIL group than in the low-TIL group (p=0.013 and p=0.014, respectively). Multivariable analysis also showed that high-TIL status was an independent factor predicting favorable prognosis (hazard ratio(HR)=0.24, p=0.023 and HR=0.13, p=0.036). Biopsy specimens from local recurrence after successful NAC frequently showed TILs decreased. TILs may be a biomarker for predicting treatment response to NAC in patients with TNBC and HER2BC. A decrease in TILs may also be associated with tumor recurrence.

Significance of tumor-infiltrating lymphocytes before and after neoadjuvant therapy for rectal cancer.

Neoadjuvant therapy for locally advanced rectal cancer is becoming increasingly common. However, biomarkers predicting the response to neoadjuvant therapy have not been established. Tumor‐infiltrating lymphocytes (TILs) have a crucial effect on tumor progression and survival outcome as the primary host immune response, and an antitumor immune effect has been reported to contribute to the response to radiotherapy and chemotherapy. We investigated the significance of TILs before and after neoadjuvant treatment and the change in the density of those TILs. Sixty‐four patients who underwent radical resection after neoadjuvant treatment for locally advanced rectal cancer were enrolled. The number of TIL subsets was examined using immunohistochemical staining of pretreatment biopsy samples and post‐treatment resected specimens. In both the neoadjuvant chemotherapy cohort and the neoadjuvant chemoradiotherapy cohort, a low density of CD8+ TILs in pretreatment biopsy samples was associated with a poor response, and a low density of CD8+ TILs in post‐treatment resected specimens was similarly associated with a poor response. In the neoadjuvant chemoradiotherapy cohort, the density of CD8+ TILs in post‐treatment resected specimens was significantly increased compared with that in pretreatment biopsy samples. We concluded that T lymphocyte‐mediated immune reactions play an important role in tumor response to neoadjuvant treatment for rectal cancer, and the evaluation of TILs in pretreatment biopsy samples might be a predictor of the clinical effectiveness of neoadjuvant treatment. Furthermore, neoadjuvant therapy, especially chemoradiotherapy, could induce the activation of the local immune status.

The prognostic significance of tumour-infiltrating lymphocytes and programmed death ligandexpression in patients with squamous cell carcinoma of bladder

The prognostic significance of tumour-infiltrating lymphocytes and programmed death ligandexpression in patients with squamous cell carcinoma of bladder

Immunologic profile and prognostic significance of tumor-infiltrating lymphocytes in renal cell carcinoma.

673 Background: Renal cell carcinoma (RCC) has been shown to be a genetically and morphologically heterogeneous cancer. As studies show the significance of tumor-infiltrating lymphocytes (TIL) in cancer, it is critical to examine how the phenotype of TILs manifests in RCC. Such analysis may help explain the role of the immune landscape in developing resistance and its contribution to the high treatment failure observed in immunotherapy for RCC. This study compares the immune phenotype of primary tumors, venous tumor thrombi (VTT), and metastases, as well as multiple sites within primary tumors, and investigates whether inter- and intra-tumoral immune heterogeneity is present in RCC. The association of TIL levels and clinical prognosis will also be assessed and compared with current RCC prognostic scores. Methods: Our cohort included 15 VTT and 6 metastases (3 adrenal, 2 bone, 1 liver) with 20 matched primary tumors, as well as 25 non-metastatic primary tumors. The RCC tissue was collected and digested into single cell suspensions. Suspensions were stained for TIL surface markers and processed using flow cytometry. The data was analyzed to capture %CD8+ and %CD4+ in total tumor. The %CD28+ and %PD1+ of CD8+ cells were measured to describe the co-stimulatory and co-inhibitory receptor expression, respectively. Clinical data was obtained to calculate the Mayo Stage, Size, Grade, and Necrosis (SSIGN) and UCLA Integrated Staging System (UISS) scores for each patient and to evaluate recurrence. Results: Immunologic concordance was observed in all measured parameters (%CD8+, %CD4+, %CD28+, and %PD1+) for primary tumors and VTT, for primary tumors and their metastases, and for different sites within 10 primary tumors. In our overall cohort, %CD8+ was found to be inversely associated with recurrence (p = 0.02). Furthermore, %CD8+ was not significantly associated with patient Mayo SSIGN and UISS scores. Conclusions: Despite the genetic and morphologic heterogeneity seen in RCC, immunologic concordance was observed for both inter- and intra-tumoral analysis. The association between %CD8+ and recurrence presents a novel and objective immune-based prognostic factor for RCC, independent of Mayo SSIGN and UISS scores.

Clinical impact of tumour-infiltrating B-cells in periampullary adenocarcinoma in relation to adjuvant chemotherapy.

330 Background: The prognostic significance of tumour-infiltrating lymphocytes of the B lineage in solid cancers has only recently begun to receive attention. Herein, we explored the relationship between B cell and plasma cell density and survival in patients with periampullary adenocarcinoma, including pancreatic cancer, with particular reference to morphological type. Methods: Immunohistochemistry was applied to quantify the abundance of B cells (CD20+), and plasma cells (CD138+) in tissue microarrays with tumours from a retrospective consecutive cohort of 175 patients who had undergone pancreaticoduodenectomy for periampullary adenocarcinoma. Cox regression was applied to calculate univariable and multivariable hazard ratios (HR) for recurrence and death within five years up until March 2017. Multivariable HRs were adjusted for established clinical parameters. Results: High CD20+ cell density was an independent prognostic factor for a prolonged overall survival in both intestinal-type (HR 0.32; 95% CI 0.12-0.86) and pancreatobiliary-type (HR 0.38; 95% CI 0.23-0.66) tumours. CD138+ was an independent prognostic factor in pancreatobiliary-type tumours (HR 0.51; 95% CI 0.32-0.82) only. A significant positive interaction was seen between adjuvant chemotherapy and CD20+ cells in relation to OS for PB-type tumours (pinteraction= 0.027). Conclusions: These results demonstrate that dense infiltration into the tumour by cells of the B lymphocyte lineage confers an improved prognosis in periampullary adenocarcinoma including pancreatic cancer. The impact of B cells in the local anti-tumour response is gradually being unravelled, and their potential influence on adjuvant chemotherapy response in the more aggressive pancreatobiliary-type tumours may be clinically relevant and merits further study.

Significance of Tumor-Infiltrating Lymphocytes and the Expression of Topoisomerase IIα in the Prediction of the Clinical Outcome of Patients with Triple-Negative Breast Cancer after Taxane-Anthracycline-Based Neoadjuvant Chemotherapy

Purpose: The aim of this study was to determine factors able to predict chemotherapeutic responses and clinical outcomes in patients with triple-negative breast cancer (TNBC) after neoadjuvant chemotherapy (NAC). Methods: Fifty-two TNBC patients on taxane-anthracycline-based NAC were included. The expression of Ki67, topoisomerase IIα (TOPOIIα), and p53, as well as the presence of CD4+ tumor-infiltrating lymphocytes (TILs) and CD8+ TILs were evaluated in biopsy specimens by immunohistochemistry. The expression of Ki67, TOPOIIα, and p53, as well as CD4 and CD8 in TILs was calculated according to the pathological response to NAC, disease-free survival (DFS), and overall survival (OS). Results: Fourteen (26.9%) TNBC patients demonstrated a pathological complete response (pCR). According to univariate analyses, significant factors associated with pCR were high infiltration of CD4+ TILs (p = 0.004), high infiltration of CD8+ TILs (p = 0.010), and high expression of topoisomerase IIα (TOPOIIα) (p = 0.006). CD4+ TILs and TOPOIIα were significantly positively correlated with CD8+ TILs. Multivariate analyses indicated that TOPOIIα was an independent predictor of pCR. Although TNBC patients with high infiltration of CD4+ TILs, CD8+ TILs, or with high expression of TOPOIIα exhibited a significantly good 5-year DFS, only TNBC patients with a high infiltration of CD8+ TILs exhibited significantly positive 5-year OS probabilities. Conclusion: Our study demonstrated that CD4+ TILs and TOPOIIα in pretreated cancer tissues were significantly correlated with CD8+ TILs. CD4+ TILs, CD8+ TILs, and TOPOIIα expression were predictors of pCR and 5-year DFS of TNBC patients who were treated with NAC, and TOPOIIα was an independent predictor of pCR. CD8+ TILs were a key factor in the prediction of good 5-year OS rates of TNBC patients after taxane-anthracycline-based NAC.

Abstract LB-082: Endoplasmic reticulum stress induces secretion of high mobility group (HMG) proteins and is associated with tumor-infiltrating lymphocytes in triple-negative breast cancer

Abstract Purpose: Even though prognostic and predictive significance of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have been demonstrated widely, the cause of TIL influx is not clear. High mobility group (HMG) N1, one of the three subtypes of HMGs which are nucleosome-binding proteins is recently known as a potent alarmin that binds to TLR4 and induces Th1 immune responses. The mechanisms which involve transportation of HMGN1 are not identified. We, in this study, suggest that extracellular secretion of HMGN1 might be associated with influx of TILs and subsequent formation of TLSs, as well as dendritic cell maturation and activation in human TNBC. Experimental Design: We reviewed the hematoxylin and eosin (H&amp;E)-stained slides of 767 patients with TNBC and evaluated the degree of TILs. We also measured the degree of expression of HMGs (HMGB1 and HMGN1) and several endoplasmic reticulum stress (ERS)-associated molecules (PERK, p-eIF2a, and XBP1) by immunohistochemical (IHC) staining in tissue microarrays of the patients. The hypothesis that HMGN1 migrates from nucleus to cytoplasm and extracellular space as the cell gets variable ER stresses was confirmed by Western blot analyses using human TNBC cell lines and pharmacological ER stress inducers. Results: On IHC staining, high cytoplasmic expression of HMGB1, HMGN1, PERK, p-eIF2a, and XBP1 were identified in 43.1%, 25.7%, 45.9%, 46.9%, and 41.8% of TNBCs, respectively. Cytoplasmic expression of HMGB1 was significantly correlated with cytoplasmic expression of HMGN1, amount of TILs, expression of PERK and XBP1 (p&amp;lt;0.001∼0.039). Cytoplasmic expression of HMGN1 was also significantly correlated with amount of TILs, expression of p-eIF2a and XBP1 (p&amp;lt;0.001∼0.004). Higher nuclear expression of both HMGB1 (p&amp;lt;0.001) and HMGN1 (p&amp;lt;0.001) showed significant association with ER stress. On Western blot analyses using TBNC cell lines, treatment of a ERS inducer resulted in the secretion of HMG proteins to the extracellular space. Conclusions: To the best of our knowledge, it is the first study that elucidated the associations among ERS, extracellular secretion of HMGs, and degree of TILs in TNBCs. Understanding the mechanisms of TIL influx will help to develop effective immunotherapeutic agents for treatment of TNBC. Citation Format: Sun-Hee Heo, In Ah Park, Hee Jin Lee, Young-Ae Kim, Gyungyub Gong. Endoplasmic reticulum stress induces secretion of high mobility group (HMG) proteins and is associated with tumor-infiltrating lymphocytes in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-082.

Abstract P5-08-15: Prognostic value of aldehyde dehydrogenase 1 (ALDH1) and tumor infiltrating lymphocytes (TIL) to predict the late recurrence in ER positive, HER2 negative breast cancer

Abstract Introduction: Aldehyde dehydrogenase 1(ALDH1) is known to be cancer stem cell marker. Also, tumor infiltrating lymphocytes (TILs) are known to be prognostic factor for triple negative breast cancer. It is reported that these factors have the correlation with chemosensitivity. Meanwhile, the late recurrence (LRec; 5 years after primary surgery) of ER positive breast cancer is the major problem. Significance of expressions of ALDH1 and TILs in primary tumor as predictive factors for late recurrence in ER positive, HER2 negative breast cancer is still unknown. Methods: ER-positive, and HER2-negative breast cancer patients who underwent surgery or received neoadjuvant chemotherapy between January 2000 and December 2004 were registered from nine institutes belonging the Collaborative Study Group of Scientific Research of the Japanese Breast Cancer Society. For each LRec patient, approximately two matched control patients without relapse for more than ten years were selected. Expression of ALDH1 was assessed by immunohistochemistry. Positive ALDH1 was defined as tumor including more than 1% cancer cells with ALDH1 expression. TIL was assessed by single whole section according to Denkert's definition. A tumor showing high ki67 and/or low PgR expressions was categorized into Luminal B-like group. Results: 639 patients (184 with early recurrence (ERec), 134 with LRec and 321 with no recurrence (NoRec)) were analyzed. The rates of positive ALDH1 in ERec, LRec and NoRec groups were 18%, 13% and 8%, respectively. ALDH1 positivity was significantly higher in ERec compared with NoRec group (p&amp;lt;0.01). There was no significant difference between LRec and NoRec group (p=0.12). Positive ALDH1 showed significantly shorter DFS and OS in multivariate analyses (DFS: p=0.03, OS: p&amp;lt;0.01). Especially, that was the significantly prognostic factor in the Luminal B like tumor with adjuvant or neoadjuvant chemotherapy (p=0.01), but not in those without any chemotherapy (p=0.53). High TILs in ERec, LRec and NoRec was 1.1%, 1.5% and 3.7%, respectively. There was no significant difference among three recurrent groups (p=0.13). High TILs was not significantly associated with DFS (p=0.09) and OS (p=0.72). However, there was significant correlation between High TILs and DFS in Luminal B like group (p=0.04) and ALDH1-negative group (p=0.02). Conclusion: In ER-positive, and HER2-negative breast cancer, ALDH1 was an independent prognostic factor (a predictor of ERec, but not LRec). ALDH1 might be a predictor of benefit from chemotherapy in Luminal B like subtype. TILs was neither a predictor of ERec nor LRec. However, significance of TILs as prognostic factor might differ depending on subtypes and cancer stemness. Citation Format: Miyoshi Y, Shien T, Ogiya A, Ishida N, Yamazaki K, Horii R, Horimoto Y, Masuda N, Yasojima H, Inao T, Osako T, Takahashi M, Tomioka N, Hagio K, Endo Y, Hosoda M, Yamashita H. Prognostic value of aldehyde dehydrogenase 1 (ALDH1) and tumor infiltrating lymphocytes (TIL) to predict the late recurrence in ER positive, HER2 negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-08-15.