Presence Of Tumor-infiltrating Lymphocytes Research Articles

Tissue-resident memory T cells in breast cancer control and immunotherapy responses.

The presence of tumour-infiltrating lymphocytes (TILs) is associated with favourable outcomes in patients with breast cancer as well as in those with other solid tumours. T cells make up a considerable proportion of TILs and current evidence suggests that CD8+ T cells are a crucial determinant of favourable clinical outcomes. Studies involving tumour material from numerous solid tumour types, including breast cancer, demonstrate that the CD8+ TILs include a subpopulation of tissue-resident memory T (TRM) cells. This subpopulation has features consistent with those of TRM cells, which have been described as having a role in peripheral immune surveillance and viral immunity in both humans and mice. Patients with early-stage triple-negative breast cancers harbouring greater numbers of TRM cells have a substantially improved prognosis and longer overall survival. Furthermore, patients with advanced-stage breast cancers with higher levels of TRM cells have increased response rates to anti-PD-1 antibodies. These findings have motivated efforts to explore whether CD8+ TRM cells include tumour-specific T cells, their functional responses to cognate antigens and their role in responses to immune checkpoint inhibition. In this Review, we focus on the clinical significance of CD8+ TRM cells and the potential ways that these cells can be targeted to improve the success of immunotherapeutic approaches in patients with breast cancer, as well as in those with other solid tumour types.

The Prognostic Implications of Tumor Infiltrating Lymphocytes in Colorectal Cancer: A Systematic Review and Meta-Analysis

Tumor-infiltrating lymphocytes (TILs) are an important histopathologic feature of colorectal cancer that confer prognostic information. Previous clinical and epidemiologic studies have found that the presence and quantification of tumor-infiltrating lymphocytes are significantly associated with disease-specific and overall survival in colorectal cancer. We performed a systematic review and meta-analysis, establishing pooled estimates for survival outcomes based on the presence of TILs in colon cancer. PubMed (Medline), Embase, Cochrane Library, Web of Science, and ClinicalTrials.gov were searched from inception to April 2017. Studies were included, in which the prognostic significance of intratumoral tumor infiltrating lymphocytes, as well as subsets of CD3, CD8, FOXP3, CD45R0 lymphocytes, were determined within the solid tumor center, the invasive margin, and tumor stroma. Random-effects models were calculated to estimated summary effects using hazard ratios. Forty-three relevant studies describing 21,015 patients were included in our meta-analysis. The results demonstrate that high levels of generalized TILS as compared to low levels had an improved overall survival (OS) with a HR of 0.65 (p = <0.01). In addition, histologically localized CD3+ T-cells at the tumor center were significantly associated with better disease-free survival (HR = 0.46, 95% CI 0.36–0.61, p = 0.05), and CD3 + cells at the invasive margin were associated with improved disease-free survival (HR = 0.57, 95% CI 0.38–0.86, p = 0.05). CD8+ T-cells at the tumor center had statistically significant prognostic value on cancer-specific survival and overall survival with HRs of 0.65 (p = 0.02) and 0.71 (p < 0.01), respectively. Lastly, FOXP3+ T-cells at the tumor center were associated with improved prognosis for cancer-specific survival (HR = 0.65, p < 0.01) and overall survival (HR = 0.70, p < 0.01). These findings suggest that TILs and specific TIL subsets serve as prognostic biomarkers for colorectal cancer.

Programmed Death-Ligand 1 Expression in Non-Small Cell Lung Carcinoma Biopsies and Its Association with Tumor Infiltrating Lymphocytes and the Degree of Desmoplasia

Immunotherapy blocking the PD-1/PD-L1 signalling pathway has become a dominant treatment modality for patients with non-small cell lung carcinoma (NSCLC). Programmed death-ligand 1 (PD-L1) expression on the membrane of tumour cells and/or tumour infiltrating lymphocytes (TIL) evaluated immunohistochemically is still the only clinically validated predictive biomarker for immunotherapy, but it has its limitations. TIL in the tumour microenviroment was identified as having predictive value. We retrospectively evaluated 134 NSCLC resection specimens, and analysed the association between PD-L1 expression, the presence of TIL, and the degree of desmoplasia in tumours. PD-L1 expression on tumour cells and TIL were evaluated immunohistochemically using the anti-PD-L1 antibody (clone 22C3) and the anti-CD3 antibody (polyclone), respectively. PD-L1 was scored using the &#8220;tumour proportion score&#8221; (TPS) system with three categories: TPS &lt; 1%, 1-49%, and 50%. TIL were evaluated semiquantitatively using the &#8220;percentage of stromal TIL&#8221; (PST) system, and categories of PST &lt; 10%, 10-49% and 50% were recorded. The association between PD-L1 expression in tumour cells and TIL was compared with the PST value. Statistical analysis was conducted using the Cochran-Armitage test, and a p-value &lt; 5% was considered significant. PD-L1 expression was significantly higher in PST 10-49% and 50% categories than in the PST &lt; 10% category in grade 1 and grade 2 adenocarcinomas (p = 0.008), grade 3 adenocarcinomas (p = 0.009), and squamous cell carcinomas (p = 0.028). PD-L1 expression in TIL was associated with the PST value in squamous cell carcinomas (p = 0.025) but not in adenocarcinomas. Desmoplastic tumours had particularly low TPS and PST values. PD-L1 expression in NSCLC is associated with the presence of TIL. Desmoplastic areas in tumours represent immunologically inactive tumour microenviroments. Administration of anti-PD-1/PD-L1 immunotherapy, together with agents blocking the TGF-&#946; signalling pathway, represent a promising combinational therapy for patients with desmoplastic NSCLC. The authors declare they have no potential confl cts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 25. 11. 2019 Accepted: 8. 12. 2019.

Abstract PD5-03: Relationship between tumor-infiltrating lymphocytes (TILs) and outcomes in the KEYNOTE-119 study of pembrolizumab vs chemotherapy for previously treated metastatic triple-negative breast cancer (mTNBC)

Abstract Background: Several studies show that the presence of TILs has prognostic significance in TNBC, with greater lymphocytic infiltration associated with clinical outcomes. In the phase 2 KEYNOTE-086 study, TIL levels were a surrogate marker of preexisting antitumor immunity and were independent predictors of response to pembrolizumab (pembro) monotherapy. Here, we analyzed the relationship between the presence of TILs and outcomes in the phase 3 KEYNOTE-119 (NCT02555657) study of pembro monotherapy vs single-agent chemotherapy (chemo) in patients (pts) with previously treated mTNBC. Methods: From October 2015 to April 2017, 622 pts from 31 countries were enrolled. Key eligibility criteria included centrally confirmed TNBC, 1 or 2 prior systemic treatments for metastatic disease, documented progression on most recent therapy, prior treatment with an anthracycline and/or a taxane, and provision of a tumor sample for central determination of triple-negative status and PD-L1 expression. Pts with active brain metastases were excluded. Pts were stratified by PD-L1 status (positive vs negative; PD-L1 positivity was defined as a CPS ≥1) and history of prior neoadjuvant/adjuvant treatment vs de novo metastatic disease at initial diagnosis. Pts were randomly assigned 1:1 to pembro 200 mg Q3W or single-agent chemo per investigator’s choice of capecitabine, eribulin, gemcitabine, or vinorelbine, with a maximum enrollment cap of 60% for each, administered per local product label, until progression, intolerable toxicity, or investigator/patient decision. Response was assessed every 9 wk for 1 y and every 12 wk thereafter. Primary end points were OS in the PD-L1 CPS ≥10, CPS ≥1, and total populations. Secondary end points were PFS, ORR, duration of response, and DCR in the PD-L1 CPS ≥10, CPS ≥1, and total populations, and safety. The presence of TILs in archival tumor samples was assessed by light microscopy of hematoxylin and eosin-stained sections using a predefined method. The relationship between TILs and OS and PFS was analyzed using Cox regression; the relationship between TILs and ORR was analyzed using logistic regression. The interaction between treatment and TILs was assessed. Results: The study was negative for the primary end point. TILs were evaluable in 536/622 (86.2%) treated pts (273 [pembro]; 263 [chemo]). The median TILs distribution was 5% (IQR, 14%). TIL levels were significantly higher in responders vs nonresponders in the pembro arm, but not the chemo arm. TIL levels as a continuous variable were significantly (P&amp;lt; 0.05) associated with all clinical outcomes tested in the pembro but not the chemo arm (Table). For pts with TILs &amp;lt;5%, assessment of OS yielded a HR of 1.50 (95% CI, 1.14-1.97); for pts with TILs ≥5%, the HR was 0.75 (95% CI, 0.59-0.96). Median OS and 18-month OS rate (pembro vs chemo) for pts with TILs &amp;lt;5% was 5.9 vs 8.8 mo and 15% vs 27%, respectively; for pts with TILs ≥5%, this was 12.5 vs 11.3 mo and 35% vs 27%. The correlation between TILs vs CPS was moderate at 0.45; multivariate modeling of TILs and CPS showed independent predictive value. Conclusions: High TILs were significantly associated with better clinical outcomes with pembro but not chemo. Efficacy estimates at the prespecified median TIL cut point (≥5%) suggest that a subset of later-line advanced pts with TNBC can derive prolonged survival benefit from pembro over chemo. Association of TILs With Clinical OutcomesTreatment ArmContinuousBORDCRPFSOSVariableORRP*DCRP*Event RateP*Event RateP*Pembro (n=273),TILs25 (9.2)0.000732 (11.7)0.0058241 (88.3)0.0002239 (87.5)0.0001n (%)Chemo (n=263),TILs29 (11.0)0.180950 (19.0)0.3026191 (72.6)0.234236 (89.7)0.3321n (%)*P values are 1-sided for pembro and 2-sided for chemo.The stratification variable, prior (neo)adjuvant therapy vs de novo metastatic disease at initial diagnosis, was used as a covariate in the model.TILs was square root transformed.BOR, best overall response; chemo, chemotherapy; DCR, disease control rate; OS, overall survival; pembro, pembrolizumab; PFS, progression-free survival; TILs, tumor-infiltrating lymphocytes. Citation Format: Sherene Loi, Eric Winer, Oleg Lipatov, Seock-Ah Im, Anthony Goncalves, Javier Cortes, Keun S Lee, Peter Schmid, Laura Testa, Isabell Witzel, Shoichiro Ohtani, Nicholas Turner, Stefania Zambelli, Nadia Harbeck, Fabrice Andre, Rebecca Dent, LingKang Huang, Jaime Mejia, Vassiliki Karantza, Roberto Salgado. Relationship between tumor-infiltrating lymphocytes (TILs) and outcomes in the KEYNOTE-119 study of pembrolizumab vs chemotherapy for previously treated metastatic triple-negative breast cancer (mTNBC) [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 PD5-03.

Abstract P6-06-13: Racial/ethnic differences in tumor infiltrating lymphocytes

Abstract Background/Rationale: There have been increasing studies regarding the morphologic evaluation of tumor-infiltrating lymphocytes (TILs) and the tumor microenvironment. In breast cancer, TILs are more abundant in stroma (sTILs) compared to intratumoral (iTILs) areas and iTILs have been an unreliable marker due to difficulty in standardizing their evaluation. The International Immuno-Oncology Working Group published recommendations to guide the accurate assessment of TILs. TILs have relevance in the setting of neoadjuvant therapy (NAT), primarily in triple negative and HER2 positive breast cancers. Studies report that sTIL infiltration is predictive of response to NAT in these two breast cancer subtypes, but not in the estrogen positive breast cancer subtype. The presence of TILs is a good prognostic indicator and correlates with axillary lymph node negativity, lower histological grade and improved recurrence-free survival. Therefore, evaluating TILs prior to NAT can provide important predictive and prognostic information for clinicians. There are insufficient data exploring differences in sTILs among different racial/ethnic groups. Previous data showed Asians had more sTILs compared to White and African American patients, consistent with data from Asia that found substantial sTILs in their respective patient populations. Our diverse population has known racial/ethnic disparities, where Pacific Islanders have a higher mortality compared to Asian and White patients. Evaluating sTILs within this population may reveal tumor biology differences that could contribute to health disparities. Research objectives: To determine the stromal tumor infiltrating lymphocytes on core biopsy specimens and correlate them with clinical characteristics: ethnicity, age, BMI and stage To determine differences in pathologic complete response (pCR) with ethnicity, age, BMI, neoadjuvant therapy and stage Table 1. % Stromal tumor infiltrating lymphocytes (sTILs) compared to age, BMI, ethnicity, stage and pCR adjusted for breast cancer subtypeVariableValueNMeanSDPAge30838.615.140927.123.0.30501834.626.6.67601431.121.3.4570+826.822.8.30BMI&amp;lt; 252825.918.825-301638.521.9.07&amp;gt; 301337.228.0.12EthnicityAsian1636.724.2.01Pacific Islander2038.021.3.004White1818.515.9Other348.130.6.02Stage1A818.48.01B933.425.5.192A1631.923.0.172B938.620.6.113A630.823.2.313B937.225.8.10pCRno3233.019.8yes2530.825.6.72 Table 2. Pathologic complete tesponse (pCR) compared to age, BMI, neoadjuvant therapy, ethnicity and stage adjusted for breast cancer subtypeVariableValueNMeanSDPAge3080.5500.5354090.4060.527.5650180.4450.511.6360140.3030.469.2770+80.5860.518.89BMI&amp;lt; 25280.5260.50825-30160.2730.447.11&amp;gt; 30130.4530.519.65Neoadjuvant TherapyACT140.2680.469.88TC110.2370.505TCHP160.7480.479.02THP120.4280.515.41Letrozole40.3850.500.62EthnicityAsian160.4720.512.77Pacific Islander200.3890.503.85White180.4200.511other30.7060.577.36Stage1A80.2410.3541B90.4270.527.412A160.7240.479.022B90.0320.441.403A60.8220.516.023B90.2680.500.90 Results: We evaluated 57 neoadjuvant breast cancer cases for sTILs according to the International Immuno-Oncology Working Group recommendations and reviewed their clinical characteristics. We found significant % sTILs differences in ethnicity, specifically Asian (35%), Pacific Islander (38.5%) and Other (46%) category compared to White (19%) Table 1. There were no differences found in sTILs according to age, BMI or stage. We also found no significant pCR differences according to age, BMI or ethnicity, however for certain stages and neoadjuvant therapy, there is an increased rate of pCR, although these are a small number of cases, no conclusions can be made, Table 2. Future studies to evaluate the significance of sTILs in different ethnicities may be informative to delineate possible mechanisms contributing to known disparities they experience. Citation Format: Jami Fukui, Alana Taniguchi, Madison Meister, Ian Pagano, Jeffrey Killeen. Racial/ethnic differences in tumor infiltrating lymphocytes [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 P6-06-13.

Targeting Histone Deacetylase 6 Reprograms Interleukin-17-Producing Helper T Cell Pathogenicity and Facilitates Immunotherapies for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is often accompanied by resistance to immunotherapies despite the presence of tumor-infiltrating lymphocytes. We report that histone deacetylase 6 (HDAC6) represses interleukin-17 (IL-17)-producing helper T (TH 17) cell pathogenicity and the antitumor immune response, dependent on its deacetylase activity. Adoptive transfer of HDAC6-deficient TH 17 cells impedes HCC growth, dependent on elevated IL-17A, by enhancing the production of antitumor cytokine and cluster of differentiation 8-positive (CD8+) T cell-mediated antitumor responses. Intriguingly, HDAC6-depleted T cells trigger programmed cell death protein 1 (PD-1)-PD-1 ligand 1 expression to achieve a strong synergistic effect to sensitize advanced HCC to an immune checkpoint blocker, while blockade of IL-17A partially suppresses it. Mechanistically, HDAC6 limits TH 17 pathogenicity and the antitumor effect through regulating forkhead box protein O1 (FoxO1). HDAC6 binds and deacetylates cytosolic FoxO1 at K242, which is required for its nuclear translocation and stabilization to repress retinoic acid-related orphan receptor gamma (RoRγt), the transcription factor of TH 17 cell. This regulation of HDAC6 for murine and human TH 17 cell is highly conserved. These results demonstrate that targeting the cytosolic HDAC6-FoxO1 axis reprograms the pathogenicity and antitumor response of TH 17 cells in HCC, with a pathogenicity-driven responsiveness to facilitate immunotherapies.

Abstract P1-10-08: Assessing immune biomarkers of response to anthracyclines in breast cancer

Abstract Background: Pathologists have long recognized that the interaction between immune and tumour cells is critical in the development and progression of breast cancer. Studies have demonstrated the presence of tumour-infiltrating lymphocytes (TILs) correlates with improved clinical outcome in breast cancer especially in the triple negative and HER-2 positive subtypes. TILs predict for improved response to certain therapies including chemotherapy and trastuzumab. The predictive value of TILs in ER positive tumours is less clear. It has been demonstrated the higher presence of the immune microenvironment is associated with a better prognosis and as a result a higher likelihood of benefit from chemotherapy and possibly from immunotherapy, whereas cold immune microenvironment carries greater risk of relapse and lower benefit from chemotherapy and possibly immuno-therapies. In this study, we evaluated whether TILs could be used to predict chemotherapy response and characterize the pre-existing tumour microenvironment (TME) using NanoString’s GeoMx Digital Spatial Profiling (DSP) platform. Methods: We assessed haematoxylin and eosin stained slides from the phase III BR9601 adjuvant breast cancer trial using software used in the international ring study 2 for standardized evaluation of TILs integrated in VMscope slide explorer. Evaluation of stromal TILs was based on international guidelines. NanoString’s DSP platform was used to analysis 256 patient samples from the BR9601 clinical trial. For analysis, region of interest were selected and compared for the TME (CD45+ve) and tumour rich (pan cytokeratin) in tissue microarrays. A panel of 56-antibodies were analysed in each ROI. Results: The mean TIL score in this cohort of patients was 15.58% (ranging from 0 to 66.67%). The presence of higher levels of TILs was significantly associated with ER negativity (p&amp;lt;0.001), high grade (p=0.01) and increased lymph nodal involvement (p=0.002). In multivariate analysis, patients whose tumours had medium/high levels of TILS expression had better DRFS (HR: 0.49, 95%CI 0.24-1.02, p=0.057) when treated with E-CMF than those treated with CMF alone. Highest levels of TILs were found in Basal and HER2-like tumours. A T-cell score was generated using the average expression of CD3, CD4 and CD8. The T-cell score was examined in both the tumour and TME. Using the T cell score it was apparent that the cohort had a range of immune “hot” and immune “cold” tumours. It was demonstrated that immune “hot” TME doesn’t not always correlate with immune “hot” tumour expression. Proteins that were most associated with T-cell exclusion (p&amp;lt;0.01) in the TME were Fibronectin, B7-H3, PTEN, ER-α, TGFB1, FAPα and CD34. This would indicate that these proteins are causal inhibitors of T-cell invasion. Conclusion: In conclusion, this study highlights the significance of assessing the entire tumour since TILs, tumour and stromal cells collectively engage in a complex interplay that contributes to disease development and progression. NanoString’s GeoMx DSP is a promising technology for multiplexed analysis. TILs, whether measured using automated software, or estimation by protein profiling, are predictive of chemotherapy benefit. Citation Format: Melanie Spears, Carsten Denkert, Sonia L Villagas, Nicola Lyttle, Linda Liao, Mary Anne Quintayo, Christopher J Twelves. Assessing immune biomarkers of response to anthracyclines in breast cancer [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 P1-10-08.

Immune Response Assessed by Flow-Cytometry and Immunhistochemistry is Linked to Pathological Response after Preoperative Chemotherapy in Patients Undergoing Resection for Colorectal Liver Metastases

Background: Preoperative chemotherapy followed by curative-intent resection represents the mainstay in treatment of advanced colorectal cancer liver metastases (CRCLM). The host immune response classified by the presence of tumour-infiltrating lymphocytes (TILS) has been verified as a major contributor to oncological outcome in several malignant diseases including colorectal cancer. This study characterizes the immune cell distribution in different tumour compartments by flow cytometry and immunohistochemistry to establish a link between immune response and pathological tumour regression grading (TRG) in patients undergoing liver resection for CRCLM.

Relationship between tils and neoadjuvant chemotherapy response /in breast cancer

Introduction: Breast cancer corresponds to 25% of new cancer cases each year. In Brazil, the rate is 29.5% and 59,700 new cases were estimated in 2019. Tumor infiltrating lymphocytes (TIL) are observed in some solid tumors, including breast cancer, the amount of TIL is an indirect marker of the pre-existing antitumor activated T cell response, reflecting the host’s antitumor immunity. Studies show that its presence is an independent predictor of complete pathological response (PRC) to neoadjuvant chemotherapy (QTneo) and its presence provides a favorable prognosis in breast cancer. Objectives: To evaluate the relationship between the presence and value of TIL with the pathological response after QTneo in patients with breast cancer. Materials and methods: An analytical, descriptive, retrospective, case-control study was carried out with 40 patients diagnosed with breast cancer who underwent QTneo between 01/01/2016 to 01/01/2019 at the ISPON complex in the city of Ponta Grossa, Paraná. Data collection was carried out in electronic medical records. The association between variables was tested with the χ2 test and the intensity of the association with the Odds Ratio estimate with a 95% confidence interval. TIL were considered absent when 0, low when 1% to 10%, moderate when 11% to 59%, and high if 60% or more; this division was based on other articles that quantify TIL. Results: The mean age of the patients in the study was 50 years old, 13 of them (32.5%) had PRC, 15 (37.5%) had a partial response, and 13 (30%) had no response to QTneo. Of the 13 that obtained PRC, 7 (53.8%) were triple negative (TN), 5 (38.4%) HER 2, and 1 (7.6%) Luminal. PRC was more frequent in triple negative invasive ductal carcinoma (53.8%), the frequency of PRC in the luminal subtype was significantly lower than that of TN (p=0.03), with the worst response to QTneo. The mean age of patients who had PRC was 48 years. TIL were present in 27 (67.5%) patients, whose value was low in 57.5%, moderate in 3 patients, and high in one case. The presence of TIL, regardless of their value, increased the probability of PRC by 7.7 times when compared to their absence (OR 7.7 CI 1.15‒51.17 p=0.03). When considering its value, it was found that higher values conferred a significant association with PRC (p=0.01). There was a statistically significant association between the clinical stage (CS) and the presence of response to QTneo, patients with locally advanced neoplasia were 6.44 times more likely to respond than patients in an early stage (p=0.015). Conclusion: TIL can be used as a predictor of response to QTneo, being an aid in choosing this treatment and an indicator of better prognosis. The evaluation of TIL should be performed routinely in the preparation of anatomopathological reports, as it is an indication of the investigation of PD-L1 and its blockade, if present.

Prediction of Response to Neoadjuvant Chemotherapy in Egyptian Patients with Locally Advanced Breast Cancer: The Evolving Role of Tumour Infiltrating Lymphocytes (TILs)

Background & objectives: Presence of TILs in breast cancer indicates better therapeutic responses to neoadjuvant chemotherapy (NAC), increased pCR and improved outcome. We aim at evaluation of TILs in breast cancer biopsies in correlation with pathological response after NAC in locally advanced breast cancer Methods. This study was conducted at Ain Shams university hospital and Maadi Military hospital in Egypt. 45 Female patients with locally advanced breast cancer were treated with NAC; pathological response was assessed. Tumours were categorized into: luminal A, luminal B, Her2 enriched and TNBC. Assessment of TILs (CD4 and CD8 lymphocytes) was based on Immuno-Oncology Biomarker Working Group guidelines. Results: Tumours were classified into high and low TIL groups using the interquartile range cutoff (29%). High CD4 group showed increased pCR (p = 0.003) and smaller residual tumours (p = 0.04). High CD8 group showed a significant association with smaller residual tumours (p = 0.003). At follow-up of 24-months, CD4 and CD8 high groups showed significantly higher 2-years DFS. The difference between CD4 high and low groups was significant in regards to estrogen receptor status, showing higher levels in hormone-negative tumors (p = 0.029). Patients with Her2 subtype showed higher CD4 (p = 0.007) and CD8 expression (p = 0.018). In CD4 & CD8 low groups, more patients developed local recurrence and distant metastasis (p = 0.025). Conclusion: We concluded that TILs may predict response to NAC and overall prognosis of breast cancer. The evaluation of TILs in correlation with morphological or genomics-based parameters helps to stratify patients of breast cancer.

TIGIT blockade enhances functionality of peritoneal NK cells with altered expression of DNAM-1/TIGIT/CD96 checkpoint molecules in ovarian cancer

ABSTRACT Advanced ovarian cancer (OC) patients have a poor 5-year survival of only 28%, emphasizing the medical need for improved therapies. Adjuvant immunotherapy could be an attractive approach since OC is an immunogenic disease and the presence of tumor-infiltrating lymphocytes has shown to positively correlate with patient survival. Among these infiltrating lymphocytes are natural killer (NK) cells, key players involved in tumor targeting, initiated by signaling via activating and inhibitory receptors. Here, we investigated the role of the DNAM-1/TIGIT/CD96 axis in the anti-tumor response of NK cells toward OC. Ascites-derived NK cells from advanced OC patients showed lower expression of activating receptor DNAM-1 compared to healthy donor peripheral blood NK cells, while inhibitory receptor TIGIT and CD96 expression was equal or higher, respectively. This shift to a more inhibitory phenotype could also be induced in vitro by co-culturing healthy donor NK cells with OC tumor spheroids, and in vivo on intraperitoneally infused NK cells in SKOV-3 OC bearing NOD/SCID-IL2Rγnull (NSG) mice. Interestingly, TIGIT blockade enhanced degranulation and interferon gamma (IFNγ) production of healthy donor CD56dim NK cells in response to OC tumor cells, especially when DNAM-1/CD155 interactions were in place. Importantly, TIGIT blockade boosted functional responsiveness of CD56dim NK cells of OC patients with a baseline reactivity against SKOV-3 cells. Overall, our data show for the first time that checkpoint molecules TIGIT/DNAM-1/CD96 play an important role in NK cell responsiveness against OC, and provides rationale for incorporating TIGIT interference in NK cell-based immunotherapy in OC patients.

Topographic distribution of lymphocyte infiltrate in triple negative tumors and its impact on the survival of the patient with breast cancer

Introduction: Tumor infiltrating lymphocytes (TIL) are generally measured using subjective methods unable to differentiate subpopulations and to locate immune cells in the tumor microenvironment. The identification and location of these cells is of great importance as they present different responses to immune stimuli. Objective: to analyze the presence of TIL in early-stage breast cancer of the triple negative molecular subtype, and to evaluate the association of TIL with the overall survival time (OS). Methods: a cross-sectional study was carried out at the Hospital de Câncer de Pernambuco (HCP), where patients diagnosed with triple negative breast cancer were selected between 2009 and 2013. TIL evaluation was performed by two methods: on slides stained with hematoxylin and eosin (H&amp;E), by two pathologists blindly and independently, and on slides submitted to immunohistochemistry, with CD3 and CD8 lymphocyte marking. In this case, the sample was then subjected to computerized histophotometry and morphometric analysis. Results: 87 patients were included, of which 22 patients had an event in the follow-up period. The evaluation of TIL, by two pathologists, showed regular agreement between the evaluators, with an intraclass correlation coefficient of 0.574 and p=0.001. CD3+ and CD8+ lymphocytes were in greater quantity in the intra tumor area compared to those outside the tumor margin (extra tumor). When the association between the presence of lymphocytes and the patient’s OS was analyzed, a directly proportional relationship with this survival was observed, that is, the greater the amount of lymphocytes, the lower the risk of death. Conclusion: results suggest that there is a correlation between the tumor lymphocyte infiltrate of triple negative breast cancer and OS. As this tumor subtype has a poor prognosis and does not have specific target therapy, TIL analysis can be explored as a prognostic marker for the treatment of breast cancer.

Clinicopathologic determinants of pathologic treatment response in neoadjuvant treated rectal adenocarcinoma

Neoadjuvant treatment (NAT) followed by total mesorectal excision is currently considered the standard of treatment for rectal adenocarcinoma. The degree of pathologic treatment response (pTR) correlates significantly with the recurrence free survival and overall survival (OS). However, it remains unclear which clinical and pathologic factors are associated with a more robust response to NAT, including showing pathologic complete response (pCR). Chemokine receptor 4 (CXCR4) overexpression has been associated with unfavorable OS in some studies. In this study, we sought to evaluate the clinicopathologic determinants of pTR in neoadjuvant treated rectal adenocarcinoma (NAT-RA). We retrospectively identified 91 patients who underwent pre-treatment diagnostic biopsy, NAT, and surgical resection at our institution. The archival slides were reviewed for pathologic features in the pre-treatment biopsies and for assessment of pTR in the resection specimens according to the current College of American Pathologist (CAP)'s guidelines. pCR was obtained in 16.5% of the cases, whereas 20.9% had near pCR, 30.8% had partial response, and 31.9% had a poor/no response. CXCR4 immunohistochemical analysis was also performed on the pre-treatment biopsies. Lower pre-treatment cT-stage (p = 0.019) and pre-treatment AJCC cTNM stage groups (p = 0.004), longer time interval between completion of NAT and resection (p = 0.022), and presence of tumor-infiltrating lymphocytes in the pre-treatment biopsies (p = 0.019) were significantly associated with a better pTR. CXCR4 nuclear expression was associated with a lower percentage of residual tumor (p = 0.036). Pre-treatment CEA levels, tumor differentiation, CAP treatment response groups and lower percentage of residual tumor were associated with a better OS.

Abstract A43: Spatial organization of pancreatic ductal adenocarcinoma (PDAC)–associated immune cells from the Adjuvant Pancreatic Adenocarcinoma Clinical Trial (APACT) study

Abstract Introduction: It is strikingly difficult to develop successful treatments for PDAC; even with curative resection, most patients die from early occult metastases. Prior studies identified the presence of tumor-infiltrating lymphocytes (TILs) in primary PDAC tumors as having prognostic significance in the PDAC adjuvant setting, sharpening the questions of what fraction of patients have immune-infiltrated tumors and what therapeutic strategies should be pursued in these patients vs. the non-infiltrated group. The phase 3 APACT trial evaluated the use of adjuvant nab-paclitaxel plus gemcitabine vs. gemcitabine in 866 patients with PDAC who had undergone primary tumor resection, with the primary endpoint of disease-free survival evaluated by independent review. We extended studies of the tumor microenvironment of PDAC to a large set of resected APACT primary tumors in an effort to further refine features of tumor or immune infiltrate that influence disease progression and to determine if chemotherapy regimen–specific predictive signatures are identifiable. Tissue analyses for a large subset of APACT samples included RNA-seq, DNA-seq, multiplexed immunohistochemistry (IHC), and proteomics. Methods: We imaged and quantified markers for tumor cells, 7 different immune cells, and 2 immune checkpoint markers using bright-field chromogenic multiplexed IHC from pretreatment samples for more than 500 APACT primary tumor samples. We computationally defined the tumor, tumor margin, and distal stromal (&amp;gt; 150 μm from tumor boundary) regions, and quantified densities and distributions of immune cells in these regions. As part of an initial analysis of more than 400 samples, we applied both unsupervised clustering and supervised classification to these IHC measurements to identify patient subgroups with similar spatial arrangements of immune cells relative to tumor regions. Results: The preliminary analysis of normalized cell densities across all 3 tissue regions revealed 3 patient subgroups: one in which immune cells are mixed within the tumor regions; a second where immune cells approach the tumor boundary but are depleted within the tumor; and a third in which immune cells are depleted in both tumor and its margin, remaining at high densities only in the distal stromal regions. Within these latter subgroups, CD20+, CD4+, and CD8+ cells were more prevalently depleted from tumor and/or margin, whereas CD163+ and CD163+CMAF+ cells showed less of this arrangement. Nearly 85% of patients fell in the second or third patient group. Conclusions: We are pursuing analyses of these data in conjunction with upcoming molecular and genetic profiling data to further elucidate the association of the immune cell populations and these subgroups with clinical outcomes. These data will provide an unprecedented opportunity for exploratory analysis and discovery of immune, molecular, and genetic biomarkers for PDAC patient stratification. Citation Format: David J. Reiss, Thomas Lila, Suzana Couto, Sitharthan Kamalakaran, Yan Ren, Doug Bowman, Amber Ortiz, Maria Wang, Clifton Drew, Kao-Tai Tsai, Mathieu Marella, Brian Fox, Garth McGrath, Matthew Trotter, Fadi Towfic, Ian Cushman, Alexander Ratushny, Brian Lu, Daniel Pierce, Jim Cassidy. Spatial organization of pancreatic ductal adenocarcinoma (PDAC)–associated immune cells from the Adjuvant Pancreatic Adenocarcinoma Clinical Trial (APACT) study [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A43.

Relationship Between Tumor Immune Markers and Fluorine-18-α-Methyltyrosine ([18F]FAMT) Uptake in Patients with Lung Cancer.

3-[18F]Fluoro-α-methyl-L-tyrosine ([18F]FAMT) is an amino acid positron emission tomography (PET) tracer specific for cancer detection by assessment of tumor amino acid metabolism. Little is known on whether or not the uptake of [18F]FAMT within cancer cells is associated with the expression of programmed death ligand-1 (PD-L1), a predictor of anti-PD-1 antibody efficacy. We conducted a clinicopathological study to assess the expression of PD-L1 and the presence of tumor-infiltrating lymphocytes (TILs) in patients with non-small cell lung cancer (NSCLC) diagnosed by PET. A total of 75 patients with NSCLC who underwent [18F]FAMT and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) PET were enrolled in the study. Tumor specimens were stained by immunohistochemistry for glucose transporter 1 (Glut1), PD-L1 (using different antibody clones including E1L3N and 28-8), CD3, CD4, and CD8. The uptake of [18F]FAMT was correlated with clinicopathological variables. High uptake of [18F]FAMT was significantly associated with disease staging, initial treatment (surgical resection or chemotherapy), and the expression of PD-L1 (E1L3N). The value of the maximum standardized uptake value (SUVmax) for [18F]FAMT was significantly correlated with PD-L1 (E1L3N) expression, Glut1, and the SUVmax for [18F]FDG in patients with histological results of adenocarcinoma (AC) and advanced disease. A validation cohort for anti-PD-L1 using clone 28-8 showed a statistically significant correlation between SUVmax for [18F]FAMT and the expression of PD-L1 (28-8) and between the expression of PD-L1 (E1L3N) and PD-L1 (28-8). The uptake of [18F]FAMT on PET imaging was significantly correlated with PD-L1 expression in NSCLC, especially in patients with AC and advanced disease.

DNMT1 and DNMT3B genetic polymorphisms affect the clinical course and outcome of melanoma patients.

The aberrant DNA methylation plays a critical role in a number of different malignancies, including melanoma. DNA methylation is catalyzed by DNA methyltransferases (DNMTs), involved in methylation maintenance (DNMT1) and de novo DNA methylation (DNMT3A and DNMT3B). The current study investigated the association of genetic variants in the DNMT1 and DNMT3B with the clinicopathologic features and the clinical course of melanoma patients. In the present study, DNMT1 (rs2228612, rs2228611, and rs2114724) and DNMT3B (rs406193 and rs2424932) polymorphisms were examined in 123 melanoma patients. Single nucleotide polymorphisms were assessed using TaqMan SNPs Genotyping Assays according to the manufacturer's protocols. The carriers of the variant genotype of DNMT1 rs2228612 had poorer overall survival and recurrence-free survival, (P = 0.000 and 0.000, respectively), and an increased risk for adverse outcome [hazard ratio (HR) = 6.620, 95% confidence interval (CI): 2.214-19.791, P = 0.001]. DNMT1 rs2228612 was also associated with ulceration (P = 0.045), nodal status (P = 0.030), progression (P = 0. 007), and stage of disease (P = 0.003). Univariate analysis indicated that tumor-infiltrating lymphocytes could be a marker of good prognosis in melanoma patients (HR = 0.323, 95% CI: 0.127-0.855, P = 0.025), whereas the genotype distribution of the DNMT3B rs406193 polymorphism correlated significantly with the presence of tumor-infiltrating lymphocytes (P = 0.012). The multivariate analysis showed that the DNMT1 rs2228612 polymorphism (HR = 12.126, 95% CI: 2.345-62.715, P = 0.003) is an independent predictor of poor overall survival in melanoma patients. As expected, disease progression was also found to be an independent prognostic factor in melanoma patients (HR = 37.888, 95% CI: 3.615-397.062, P = 0.002). DNMT1 rs2228612 was found to be an independent predictor of poor overall survival in melanoma patients. DNMTs polymorphisms could serve as a potential target for novel therapeutic approaches.

Establishment of humanized tumor microenvironment mouse models based on the injection of peripheral blood mononuclear cells and IFN-γ to evaluate the efficacy of PD-L1/PD-1-targeted immunotherapy

ABSTRACTProgrammed death ligand-1 (PD-L1) expression and the presence of tumor-infiltrating lymphocytes (TILs) in tumor microenvironment were common in chronic inflammatory tumor types and frequently responded to the PD-L1 pathway immune checkpoint blockade in the clinic. Animal models to optimize such immunotherapeutics comprise an important strategy but often fail to predict the efficacy of clinical approaches. To address this, we aimed to establish new mouse models. In this study, we found that the expression of PD-L1was present at the beginning stage but a gradual decline over time in the in vitro cell culture and also in the mouse model. Based upon this finding, we established the IFN-γ-(human peripheral blood mononuclear cell) PBMC-CDX (cell line-derived xenograft) and IFN-γ-PBMC-PDX (patient-derived xenograft) mouse models, which recapitulate human tumor and human immune system interactions. IFN-γ was injected peritumorally to maintain the positivity of PD-L1 in the tumor microenvironment. Under this circumstance, the PD-1 molecule on the human T lymphocyte surface is in contact with the PD-L1 molecule on the human tumor cells and, thus, the formatin of the PD-L1/PD-1 pathway in the tumor microenvironment.Treatment with anti-PD-1 monoclonal antibody (mAb) significantly inhibited the growth of both CDX and PDX tumors, but not non-human NCG models (without allogeneic human PBMCs and IFN-γ) . These experimental data provide an important and promising platform for the development of drugs and the evaluation of the drug efficacy of immunotherapies with anti-PD-1 mAb as well as the basis of preclinical mAb drug research.

PD-L1 Expression and Tumor-Infiltrating Lymphocytes in Thymic Epithelial Neoplasms.

Thymic epithelial tumors (TETs) are rare malignant mediastinal tumors that are difficult to diagnose and treat. The programmed death 1 (PD-1) receptor and its ligand (PD-L1) are expressed in various malignant tumors and have emerged as potential immunotherapeutic targets. However, the immunobiology of TETs is poorly understood. We evaluated PD-L1 expression and the presence of tumor-infiltrating lymphocytes (CD8 and CD3 expression) in surgical TET specimens from 39 patients via immunohistochemistry and determined their relation to clinicopathological parameters. Cases with membranous reactivity of the PD-L1 antibody in ≥1% of tumor cells were considered positive. Positive PD-L1 expression was observed in 53.9% of cases. Histologically, PD-L1 expression was positive in 2/6 type A, 2/6 type AB, 3/9 type B1, 4/4 type B2, 5/6 type B3, and 5/8 type C TET cases. Thus, the number of cases with PD-L1 expression and the percent expression of PD-L1 were significantly higher in more aggressive thymomas (type B2 or B3). CD3+ and CD8+ tumor-infiltrating lymphocytes were diffusely and abundantly distributed in all cases. These data suggest that a PD-1/PD-L1 blockade is a promising treatment for TETs, with more beneficial treatment effects for aggressive thymomas such as type B2 or B3.

Tumor infiltrating lymphocytes in malignant melanoma - allies or foes?

This is an overview of current problematics regarding the role of tumor infiltrating lymphocytes (TILs) in malignant melanomas. Various and often conflicting data have been published, correlating tumor type, stage, prognosis, as well as sex and age of patients. This is partly due to heterogeneity in scaling systems and unstandardized TILs grading but also due to changes of tumor-host interactions. Melanomas are an immunologically heterogeneous group with variability of TILs, where distinct gene expression patterns were found in tumors with absent, and/or non- brisk TIL grade versus brisk TIL grade. However, the presence of TILs alone appears to be inadequate for implicating them as immunologically functional. Further characterisation of TIL phenotype and function is warranted. This especially concerns, evaluation of TILs of the suppressor phenotype but rather than as a prognostic factor, more for prediction of targeted immunotherapy.

Potential clinical application of tumor-infiltrating lymphocyte therapy for ovarian epithelial cancer prior or post-resistance to chemotherapy.

Immunotherapy has become a powerful treatment option for several solid tumor types. The presence of tumor-infiltrating lymphocytes (TIL) is correlated with better prognosis in ovarian cancer, pointing at the possibility to benefit from harnessing their anti-tumor activity. This preclinical study explores the feasibility of adoptive cell therapy (ACT) with TIL using an improved culture method. TIL from high-grade serous ovarian cancer were cultured using a combination of IL-2 with agonistic antibodies targeting 4-1BB and CD3. The cells were phenotyped using flow cytometry in the fresh tissue and after expansion. Tumor reactivity was assessed against HLA-matched ovarian cancer cell lines via IFN-γ ELISPOT. Ovarian cancer is highly infiltrated with CD8+ TIL that are preferentially and robustly expanded with the addition of the agonistic antibodies. With a 95% success rate, the TIL are grown to ≥ 100 × 106 cells in 2-3weeks without over differentiation. In addition, the CD8+ TIL grown with this method showed HLA-restricted tumor recognition. These results indicate the viability of TIL ACT for refractory ovarian cancer by allowing for the large expansion of anti-tumor TIL in a short time and consistent manner.