Characterization Of Tumor Infiltrating Lymphocytes Research Articles

Characterization of tumor-infiltrating lymphocytes and their spatial distribution in triple-negative breast cancer

BackgroundThe tumor immune microenvironment, particularly tumor-infiltrating lymphocytes (TILs), plays a critical role in disease progression and treatment response in triple-negative breast cancers (TNBCs). This study was aimed to characterize the composition of TILs and investigate their clinicopathological and prognostic significance with a special focus on the spatial distribution of TILs in TNBCs.MethodsWe analyzed TNBC samples through PanCancer Immune Profiling using NanoString nCounter assays to identify immune-related genes that are expressed differentially in relation to TIL levels and evaluated protein expression of selected markers through immunohistochemical staining on tissue microarrays. For a comprehensive assessment of the expression of cytotoxic T lymphocyte (CTL) and natural killer (NK) cell markers, a CTL-NK score was devised based on CD8+, CD56+, CD57+, GNLY+, and GZMB+ TIL levels.ResultsGene expression analysis revealed significant upregulation of CTL and NK cell-associated genes including GNLY, KLRC2, and GZMB in TIL-high TNBCs. Immunohistochemical validation confirmed that TNBCs with higher TILs had a greater amount of CD56+, CD57+, GNLY+, and GZMB+ TILs not only in absolute number but also in proportion relative to CD4+ or CD8+ TILs. High TIL and its subset (CD4+, CD8+, CD56+, CD57+, GNLY+, and GZMB+ TIL) infiltration correlated with favorable clinicopathological features of tumor. In survival analysis, high CTL-NK score was found to be an independent prognostic factor for better disease-free survival (DFS) of the patients. Furthermore, uniformly high TIL infiltration was linked to better DFS, whereas cases with heterogeneous TIL infiltration showed no difference in survival compared to those with uniformly low TIL infiltration.ConclusionOur study showed that CTL and NK cell-associated gene expression and protein levels differ significantly according to TIL levels and that CTL-NK score and distribution of TILs within tumors have a prognostic value. These findings emphasize the importance of CTLs and NK cells as well as the spatial uniformity of TIL infiltration in clinical outcome of TNBC patients, providing valuable insights for refining prognostic assessments and guiding immunotherapeutic strategies.

Effect of Neoadjuvant Chemotherapy on Tumor-Infiltrating Lymphocytes in Resectable Gastric Cancer: Analysis from a Western Academic Center.

Tumor-infiltrating lymphocytes (TILs) are an emerging biomarker predictive of response to immunotherapy across a spectrum of solid organ malignancies. The characterization of TILs in gastric cancer (GC) treated with contemporary, multiagent neoadjuvant chemotherapy (NAC) is understudied. In this retrospective investigation, we analyzed the degree of infiltration, phenotype, and spatial distribution of TILs via immunohistochemistry within resected GC specimens treated with or without NAC at a Western center. We hypothesized that NAC executes immunostimulatory roles, as evidenced by an increased number of anti-tumor TILs in the tumor microenvironment. We found significantly elevated levels of conventional and memory CD8+ T cells, as well as total TILs (CD4+, CD8+, Treg, B cells), within chemotherapy-treated tumors compared with chemotherapy-naïve specimens. We also revealed important associations between survival and pathologic responses with enhanced TIL infiltration. Taken together, our findings advocate for an immunostimulatory role of chemotherapy and underscore the potential synergistic effect of combining chemotherapy with immunotherapy in resectable gastric cancer.

Immune Checkpoint Inhibitors for Solid Tumors in the Adjuvant Setting: Current Progress, Future Directions, and Role in Transplant Oncology.

The rationale for administering immune checkpoint inhibitors (ICIs) in the adjuvant setting is to eradicate micro-metastases and, ultimately, prolong survival. Thus far, clinical trials have demonstrated that 1-year adjuvant courses of ICIs reduce the risk of recurrence in melanoma, urothelial cancer, renal cell carcinoma, non-small cell lung cancer, and esophageal and gastroesophageal junction cancers. Overall survival benefit has been shown in melanoma while survival data are still not mature in other malignancies. Emerging data also show the feasibility of utilizing ICIs in the peri-transplant setting for hepatobiliary malignancies. While ICIs are generally well-tolerated, the development of chronic immune-related adverse events, typically endocrinopathies or neurotoxicities, as well as delayed immune-related adverse events, warrants further scrutiny regarding the optimal duration of adjuvant therapy and requires a thorough risk-benefit determination. The advent of blood-based, dynamic biomarkers such as circulating tumor DNA (ctDNA) can help detect minimal residual disease and identify the subset of patients who would likely benefit from adjuvant treatment. In addition, the characterization of tumor-infiltrating lymphocytes, neutrophil-to-lymphocyte ratio, and ctDNA-adjusted blood tumor mutation burden (bTMB) has also shown promise in predicting response to immunotherapy. Until additional, prospective studies delineate the magnitude of overall survival benefit and validate the use of predictive biomarkers, a tailored, patient-centered approach to adjuvant ICIs that includes extensive patient counseling on potentially irreversible adverse effects should be routinely incorporated into clinical practice.

Abstract P1-08-26: Morphologic characterization of tumor-infiltrating lymphocytes and its relation with pathological response in a series of breast cancer patients treated with primary chemotherapy

Abstract Introduction: Tumor-Infiltrating Lymphocytes (TILS) is a well-known predictor of response to primary chemotherapy1,2 and a prognostic factor for improved survival in different breast cancer subtypes3. We present data on the association of the level of TILs and morphologic characteristics of such infiltrate with pCR. Material and methods: A series of 477 breast cancer patients (479 tumors) treated with primary chemotherapy at Catalan Institut of Oncolgy - H.U. Bellvitge between January 2009 and December 2016. Chemotherapy consisted on anthracyclines and taxanes (plus trastuzumab if Her-2 positive disease). Levels of percentage of TILs using hematoxylin-eosin-stained sections of diagnostic core-needle biopsy were evaluated according to international guidelines in a prospectively defined retrospective analysis. Characterization of TILS consisted on identification of plasma cells, intraepithelial infiltrate vs stromal infiltrate and homogeneous vs heterogeneous infiltrate. A sub-classification using levels of TILS and heterogeneity was done for statistical purposes. Levels of TILS and their morphological characteristics were examined for their associations with pCR adjusted for predictive clinic-pathological factors, by univariate and multivariate logistic regression, statistical significance set at 0.05. A ROC curve was performed to look for a cut-point of TILs to predict PCR. Results: The mean value of TILs was 23.79% (SD, 24%). TILs were significantly higher in ductal carcinomas (39.1% vs 0%, p=0.007), grade 3 (55.2 vs 17.7%p<0.001), ki 67 >30 (48.8% vs 24.5%, p<0.001), HER2 (56.6%) and triple negative tumors (TNBC)(56.6%) vs luminal (20%), p<0.001). The level of TILS was set in 20% to predict pCR by a ROC curve (S=62.3% E=71%). Characterization of TILs found plasma cells in 41.1% of the samples, intraepithelial infiltrate in 11.2% and homogeneous infiltrate in 60.1%. Homogeneously high infiltrate was found in 20% of the samples. In the univariate analysis pCR was higher in samples with TILs > 20% (15.2% vs 41.5%, OR: 3.96 [95%CI, 2.57-6.10]; P < 0.001); plasma cells (OR 6.61 [95%CI, 1.51-28.8]; P=0.01), intraepithelial TILS (OR: 10.34 [95%CI, 2.22-48.01]; P =0.003), homogeneous high infiltrate (OR: 13.6 [95%CI, 3.04-60.77]; P =0.001). In luminal tumors, TILs over 20% predicted pCR (OR 12.3 [95%CI, 4.0-37.7]; P < 0.001) as well as in TNBC (OR 4.32 [95%CI, 1.77-10.53]; P=0 .001) but not in those cases with HER2 positive tumors (luminalB HER2 + HER2) (OR 1.65 [95%CI, 0.88-3.07]; P=0.118). In the multivariate analyses, levels of TILs > 20% were associated with higher pCR rates (adjusted odds ratio, 2.44 [95%CI, 1.48-4.01]; P < .001). Conclusions: The presence of TILs over 20% at diagnosis is an independent, positive, predictive marker of pCR in early breast cancer treated with neoadjuvant chemotherapy. Interestingly, the predictive information added by TILs >20% was higher in luminal and triple negative tumors compared to HER2 positive cases. Careful morphological characterization of TILS may add valuable predictive information and can be done in current pathologic laboratories with a well-trained breast cancer pathologist.

Abstract P3-19-04: Minimal increases in tumor infiltrating lymphocytes despite excellent tumor responses after pre-operative accelerated partial breast irradiation in early stage ER+ breast cancer patients

Abstract Purpose/Objectives: Local immune activity as measured by tumor infiltrating lymphocytes (TILs) has been correlated with improved outcomes in triple negative breast cancer (TNBC) and HER2+ disease. Furthermore, increased TILs after pre-operative systemic therapy for TNBC are also correlated with improved outcomes. However, the role of TILs in predicting outcomes in ER/PR+ breast cancer is less well-established. Radiation therapy (RT) can enhance immune cell infiltration in various tumors. However, breast cancer RT is typically given post-operatively, limiting the ability to determine whether RT enhances the %TILs in breast tumors. Here we report %TILs and tumor responses after pre-operative accelerated partial breast irradiation (APBI) as part of a clinical trial to determine whether RT enhances %TILs and whether this correlates with RT response. Materials/Methods: A breast pathologist assessed blindly the % stromal TILs and % tumor cellularity in biopsy and post-RT surgical specimens for patients treated on an in-house pre-operative APBI trial in patients with ER/PR+ breast cancer (NCT02728076). Patients received APBI to 30 Gy in 5 fractions on non-consecutive days, followed by surgery after no less than 5 weeks. Correlation between %TILs and pathologic response was assessed. Results: Thirty-five patients were enrolled and completed treatment. Median age was 65 (50-80). All patients were clinically node-negative and ER/PR+; one was HER2+. Median MRI tumor size was 0.9 cm (0.4-2.1 cm); 54% were grade 2 and 46% grade 1. 23/35 patients had 21- or 70-gene recurrence scores (RS), with 1 high RS; 2 patients received adjuvant chemotherapy and 33/35 received endocrine therapy after surgery. Most patients demonstrated robust tumor responses to RT, with 16/35 (46%) having &lt5% cellularity after RT. With a median cellularity at diagnosis of 40%, 27/35 (77%) had a >20% decrease in cellularity, with only 3 patients having no decrease (range 0-59%). All tumors had low levels of TILs (median 5%, range 1-20%). There was minimal change in %TILs from biopsy to surgery, with 10/35 (28%) patients having increase of TILs &gt4%, 2/35 with an increase of 10%, 2/35 having a decrease of 10% and the remainder with no %TIL increase. There were no correlations between %TILs (at diagnosis or post-RT) and tumor response to RT or RS. Conclusions: Pre-operative APBI in ER/PR+ breast cancers did not significantly increase the %TILs after 5 weeks, despite leading to significant tumor responses as measured by % cellularity. Low %TILs were seen, with no more than a 10% TIL increase after RT. These findings suggest this RT dose and fractionation schedule causes only mild immune changes, as measured by TILs, in this mostly favorable cohort of ER/PR+ cancers. Alternatively, the timing of surgery may have missed maximum TIL levels. Further characterization of TILs along with further studies in similar cohorts and in other breast cancer subtypes are warranted and will be performed to examine the role of pre-operative RT on breast cancer immune responses. Citation Format: Adam Currey, Julie M Jorns, Nina Desai, Tracy R Kelly, Joseph Bovi, Amanda L Kong, Anubha Wadhwa, Eric Paulson, Carmen Bergom. Minimal increases in tumor infiltrating lymphocytes despite excellent tumor responses after pre-operative accelerated partial breast irradiation in early stage ER+ breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-19-04.

Abstract P4-07-11: Prognostic significance of changes in tumor infiltrating lymphocytes and neutrophil-to-lymphocyte ratio after neoadjuvant chemotherapy in early breast cancer patients

Abstract Introduction. There are still 30-40% of patients with early breast cancer (BC) that relapse after neoadjuvant chemotherapy (NAC). New prognostic biomarkers are needed. Current evidence shows that both markers of antitumor local and systemic immune response, such as tumor infiltrating lymphocytes (TIL) and neutrophil-to-lymphocyte ratio (NLR) have prognostic value in early BC. Most works have evaluated baseline levels, but their variation after NAC might be related to treatment resistance and should be explored.The aim of this study was to simultaneously investigate the prognostic significance of the pre-post-NAC variation of TIL and NLR in patients with early BC.Methods. Retrospective, single-center cohort of 121 patients with early BC treated with NAC between 2001 and 2010. Pre- and post-NAC TIL (CD3+) were assessed by tumor tissue microarrays performed in both diagnostic core-needle biopsy and surgical excision specimens, respectively. Pre- and post-NAC NLR were calculated based on blood tests collected within 1 month of cancer diagnosis and at the moment of surgery, respectively. The change on TIL and NLR after NAC was estimated by calculating the absolute difference of post- and pre- values of these parameters (Δpost-pre). We examined the association between the absolute difference of TIL and NLR (both divided into deciles) and survival outcomes by Cox regression. To represent in Kaplan-Meier curves the combined effect of the change of TIL and NLR in OS, we divided patients into 4 groups, depending on the sign of Δpost-pre values of TIL and NLR: 1) no TIL nor NLR increment after NAC (ΔTIL & ΔNRLpost-pre≤0); 2) no TIL but NLR increment (ΔTILpost-pre≤0, ΔNRLpost-pre>0); 3) TIL but no NLR increment (ΔTILpost-pre>0, ΔNRLpost-pre≤0); and 4) TIL and NLR increment following NAC (ΔTIL & ΔNRLpost-pre>0).Results. 47 (40%) out of 121 patients had pre- and post-NAC values of both NLR and TIL. Patients’ baseline characteristics are shown in Table 1. The median follow-up of this group was 11.5 years (IQR: 10.5 - 12.5 years). At the final follow-up date, overall survival (OS) was 87.2 %. In the univariable analysis, the increase in TIL and NLR after NAC among deciles showed a negative prognostic value for OS (TIL: HR 1.54, 95%CI 1.05-2.26, p=0.026; HR 1.32 95%CI 0.98-1.79, p=0.070). In the multivariable analysis, adjusted by cancer staging after NAC, both the increase of TIL and NLR after NAC among deciles add independent predictive value (TIL: HR 1.53, 95%CI 1.00-2.34, p=0.049; RNL: HR 1.42 95%CI 0.95-2.12, p=0.09).The subpopulation of patients with no TIL increment after NAC (Groups 1&2, N=23) showed the best prognosis, with no deaths at 10 years. Among patients with TIL increment (Groups 3&4, N=22), those with no NLR increment (Group 3, N=5) showed an intermediate prognosis (20% deaths at 10 years), whereas patients with concurrent NLR increment (Group 4, N=17) had the lowest OS (29.4% deaths at 10 years). Differences observed between groups were statistically significant (Log Rank p=0.042).Conclusion. The integrated characterization of TIL and NLR variations after NAC identifies different prognostic subgroups in early BC patients. In our cohort, both the increment of TIL and/or NLR after NAC are associated to a worse prognosis. Future validation of these findings in large, multicenter cohorts might allow treatment optimization by means of new strategies such as immunotherapy. Table 1.Patients’ baseline characteristicsN=47Age (median, range)56 (21, 78)Menstrual statusPostmenopausal25 (53,2%)Premenopausal22 (46,8%)Histologic subtypeInvasive ductal carcinoma42 (91,5%)Other subtypes4 (8,5%)Histologic gradeGrade 12 (4,3%)Grade 218 (38,3%)Grade 323 (48,9%)Unknown4 (8,6%)cTcT221 (44,7%)cT324 (51,1%)cT4a-d2 (4,2%)cNcN018 (38,3%)cN114 (29,8%)cN26 (12,8%)cN39 (19,1%)Molecular subtypeHR+ HER2-22 (46,8%)HER2+ HR+8 (17,0%)HER2+ HR-6 (12,8%)Triple negative10 (21,3%)Breast surgeryConservative26 (55,3%)Mastectomy21 (44,7%)Axillary surgerySentinel lymph node biopsy12 (25,5%)Axillary lymph node dissection35 (74,5%)Relapse typeMetastasis7 (14,9%)Local/contralateral1 (0,83%)Deaths6 (12,8%) Citation Format: Esmeralda Garcia-Torralba, Beatriz Álvarez-Abril, Carlos Bravo-Pérez, Esther Navarro Manzano, Pilar de la Morena Barrio, Alejandra Ivars Rubio, Elisa García-Garre, Gema Marín Zafra, Francisco Ayala de la Peña, Elena García-Martínez. Prognostic significance of changes in tumor infiltrating lymphocytes and neutrophil-to-lymphocyte ratio after neoadjuvant chemotherapy in early breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-07-11.

Tumor infiltrating lymphocytes (TILs) in endometrioid and clear cell ovarian carcinoma: Characterization, association with mismatch repair system deficiency and endometriosis, and prognostic implications.

e17549 Background: In contrast to high-grade serous ovarian cancer, relationship between tumor infiltrating lymphocytes (TILs) with endometrioid (EC) and clear cell ovarian carcinomas (CC) are less known. However, these tumors are thought to arise from a transformed endometriosis, a disease with a marked inflammatory response, and are also more related to Lynch syndrome. The characterization of TILs in these tumors could lead to stratify patients according to their prognosis and to identify candidates for immumotherapy in case of relapse. Methods: We conducted a retrospective observational study of 121 patients with EC and CC, diagnosed and treated in La Paz University Hospital, from November 1992 until December 2013. An expert pathologist reviewed the histopathological diagnosis, using the immunohistochemical algorithm with WT1, p53, Napsin A and progesterone receptor, and the presence of endometriosis. Clinicopathological data, genetic testing and personal and familial background were collected. Tissue-microarrays (TMAs) were used to analyze CD8+, CD4+ and CD3+ intra-epithelial TILs, according to the score designed by the Ovarian Tumor Tissue Analysis Consortium, peritumoral and endometriosis lymphocytes, ARID1A, MLH1, PMS2, MSH2 and MSH6. Software SPSS18 version was used for descriptive and statistical analysis. Results: Mean age was 54.3 years, and the median follow-up 10.02 years. 48.8% were CC and 51.2% EC, of which, 55.9% were well-differentiated, 32.2% moderately and 11.9% poorly. 58.8% of patients were diagnosed in early stages and 41.2% in advanced ones. 9.1% had family history of ovarian, colorectal or endometrial cancer and 16.5% of breast cancer. 37.2% of patients had a relapse (median progression-free survival of 31 months) and 41.6% died (32.6% related to the tumor). 55.4% of the patients had endometriosis (53.2% were EC and 57.6% CC). 8.2% had lost of MLH1, PMS2, MSH2 or MSH6 and 42% lost ARID1A expression. Moderate or high levels of CD3+, CD8+ and CD4+ TILs were seen in the 57.9%, 53.7% and 1.1%, respectively. There was no relationship between TILs and the lost of MLH1, PMS2, MSH2 and MSH6. Moderate or higher levels of CD3+ TILs were related with better overall survival (OS) only in early stages (p = 0.045). Increased CD8+ TILs in EC patients were associated with a trend towards an improved OS in comparison with those patients with lower or negative CD8+ TILs. Moderate o higher levels of CD8+ TILs were also associated, without statistical significance, with the presence of endometriosis and ARID1A. Conclusions: In our study CD3+ TILs correlated to a better OS, but only in early stages of CC and EC. CD8+ TILs were also associated with a tendency to better OS in EC. We did not find statistically significant association between TILs and Mismatch Repair System deficiency, ARID1A or endometriosis.

A phase 2, two-stage study of avelumab and axitinib in patients with mismatch repair proficient (MMR-P) recurrent or persistent endometrial cancer (EC).

TPS5609 Background: Despite significant strides in understanding the molecular pathogenesis of EC, there remain few effective therapies for recurrent disease. Deeper insight into the roles of disordered tumor vasculature and HIF1α- and VEGF-mediated immunosuppressive effects on myeloid-derived suppressor cells, T-cells, and PD-L1 expression contributed to the development of new targeted regimens. Activity of pembrolizumab and lenvatinib was demonstrated in a phase 2 trial in MMR-P EC (NCT02501096). By inhibiting VEGF receptor (VEGFR) and PD-L1 signaling, immunologically “cold” tumors may become inflamed. However, there are concerns regarding the toxicity of pembrolizumab/lenvatinib and alternatives are sought. The combination of the anti-PD-L1 antibody avelumab with axitinib, an inhibitor of VEGFR 1-3 and PDGFR with more potent IC50 inhibitory activity than lenvatinib, has also shown synergistic activity and is FDA approved as first line treatment for patients with renal cell cancer. We therefore hypothesized that this combination would be well tolerated and efficacious in recurrent MMR-P EC. Methods: This is an investigator-initiated, phase 2, two-stage single cohort trial evaluating avelumab with axitinib in recurrent or persistent EC. Participants must have MMR-P EC of any histology and have received at least one chemotherapeutic regimen, with no upper limit on the number of prior lines received. Prior use of immune checkpoint (IC) inhibitors is excluded. Treatment consists of avelumab 800mg IV every 2 weeks and axitinib 5mg orally twice daily. Co-primary endpoints are progression-free survival at 6 months (PFS6) and objective response rate by RECIST 1.1. Translational objectives include characterization of tumor-infiltrating lymphocytes, infiltrating myeloid cells, expression of IC markers, and whole exome sequencing to evaluate mutations in genes related to DNA repair and immunologic response. This is a two-stage design in the method of Sill et al, with 16 participants anticipated in stage 1 and 19 participants in stage 2, for a total of 35 participants. Accrual is ongoing. Clinical trial information: NCT02912572.

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 <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.

Deep-Learning-Based Characterization of Tumor-Infiltrating Lymphocytes in Breast Cancers From Histopathology Images and Multiomics Data.

PURPOSETumor-infiltrating lymphocytes (TILs) and their spatial characterizations on whole-slide images (WSIs) of histopathology sections have become crucial in diagnosis, prognosis, and treatment response prediction for different cancers. However, fully automatic assessment of TILs on WSIs currently remains a great challenge because of the heterogeneity and large size of WSIs. We present an automatic pipeline based on a cascade-training U-net to generate high-resolution TIL maps on WSIs.METHODSWe present global cell-level TIL maps and 43 quantitative TIL spatial image features for 1,000 WSIs of The Cancer Genome Atlas patients with breast cancer. For more specific analysis, all the patients were divided into three subtypes, namely, estrogen receptor (ER)–positive, ER-negative, and triple-negative groups. The associations between TIL scores and gene expression and somatic mutation were examined separately in three breast cancer subtypes. Both univariate and multivariate survival analyses were performed on 43 TIL image features to examine the prognostic value of TIL spatial patterns in different breast cancer subtypes.RESULTSThe TIL score was in strong association with immune response pathway and genes (eg, programmed death-1 and CLTA4). Different breast cancer subtypes showed TIL score in association with mutations from different genes suggesting that different genetic alterations may lead to similar phenotypes. Spatial TIL features that represent density and distribution of TIL clusters were important indicators of the patient outcomes.CONCLUSIONOur pipeline can facilitate computational pathology-based discovery in cancer immunology and research on immunotherapy. Our analysis results are available for the research community to generate new hypotheses and insights on breast cancer immunology and development.

The Ovarian Cancer Tumor Immune Microenvironment (TIME) as Target for Therapy: A Focus on Innate Immunity Cells as Therapeutic Effectors.

Ovarian cancer (OvCA) accounts for one of the leading causes of death from gynecologic malignancy. Despite progress in therapy improvements in OvCA, most patients develop a recurrence after first-line treatments, dependent on the tumor and non-tumor complexity/heterogeneity of the neoplasm and its surrounding tumor microenvironment (TME). The TME has gained greater attention in the design of specific therapies within the new era of immunotherapy. It is now clear that the immune contexture in OvCA, here referred as tumor immune microenvironment (TIME), acts as a crucial orchestrator of OvCA progression, thus representing a necessary target for combined therapies. Currently, several advancements of antitumor immune responses in OvCA are based on the characterization of tumor-infiltrating lymphocytes, which have been shown to correlate with a significantly improved clinical outcome. Here, we reviewed the literature on selected TIME components of OvCA, such as macrophages, neutrophils, γδ T lymphocytes, and natural killer (NK) cells; these cells can have a role in either supporting or limiting OvCA, depending on the TIME stimuli. We also reviewed and discussed the major (immune)-therapeutic approaches currently employed to target and/or potentiate macrophages, neutrophils, γδ T lymphocytes, and NK cells in the OvCA context.

Abstract A15: Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy

Abstract The natural occurrence of immune cells in the tumor microenvironment of ovarian cancer (OC) facilitates the ex vivo generation and expansion of tumor-infiltrating lymphocytes (TIL) for infusion in the context of adoptive T-cell transfer. Yet, the lack of tumor-reactive TILs and the suppressive microenvironment have hindered the translation of TIL therapy into meaningful clinical responses in OC patients. Therefore, generating a proinflammatory tumor microenvironment with oncolytic adenoviruses, in order to induce a steady and potent antitumor activity, may prove an effective add-on to TIL therapy for OC. Besides their well-established efficacy in OC, oncolytic adenoviruses genetically modified to carry the interleukin-2 (IL-2) and tumor necrosis factor alpha (TNFa) transgenes may further support and promote T-cell mediated antitumor immunity. In this study, we assessed the capability of an oncolytic virus coding for IL-2 and TNFa (Ad5/3-E2F-D24-hIL-2-IRES-TNFa; TILT-123) to enhance the reactivity of TILs towards OC in a suppressive microenvironmental context. Fresh tumor tissue from regional metastatic or primary sites was obtained from stage III-IV OC patients. Part of the samples were diced in small fragments for the generation and rapid expansion of TILs under clinically relevant in vitro conditions. Phenotypic characterization of TILs was performed by flow cytometry after rapid expansion. Following TIL production and characterization, TILs were co-cultured with T cell-depleted autologous OC single-cell suspensions in the presence or absence of oncolytic virus. TIL reactivity was determined by quantifying Interferon (IFN)-gamma in culture supernatants through ELISA. The remainder of the tumor sample was dissociated overnight and cultured in the presence or absence of oncolytic virus. Supernatants were harvested at different days post-infection for detection of relevant cytokines. Following a 14-day rapid expansion period, the final TIL product was composed mostly of CD8+ T cells (56%), CD4+ T cells (27%) and a small fraction of CD3+CD56+ NK-like T cells (2%). When TILs were cocultured with T-cell depleted OC single-cell suspensions, treatment with oncolytic virus coding for IL-2 and TNFa caused a significant increase in the release of IFNg by TILs as compared to conditions where no virus or the backbone oncolytic virus (without cytokine transgenes) was added (p<0.05). Microenvironmental changes by the cytokine-encoding oncolytic adenovirus were evidenced by increased release of proinflammatory cytokines such as, TNFa, IL-2 and IFNg in 7-day cell cultures of ex vivo single-cell suspensions derived from OC patients. In summary, tailoring the tumor microenvironment with IL-2 and TNFa using oncolytic viruses sharpens the TIL reactivity in an immune-suppressive OC microenvironmental context. Hence, this approach may enable effective responses in OC patients treated with TIL therapy. This evidence supports further clinical validation. Citation Format: João Manuel Santos, Camilla Hëinio, Victor Cervera-Carrascon, Mikko Siurala, Dafne Quixabeira, Tanja De Gruijl, Heini Lassus, Anna Kanerva, Akseli Hemminki. Rewiring the tumor microenvironment with an oncolytic virus to enhance and support tumor-infiltrating lymphocytes for ovarian cancer immunotherapy [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 A15.

Abstract 2706: Comprehensive immune profiling of primary tumor-infiltrating lymphocytes isolated from primary non-small cell lung cancer specimens

Abstract In the past years, multiple inhibitors of the immune checkpoint receptor (ICR) PD-1 or its ligand PD-L1 have been approved in the U.S. in a selected, yet growing number of indications including non-small cell lung cancer (NSCLC). In patients with advanced NSCLC, checkpoint inhibition significantly prolongs overall survival and, due to its overall responsiveness, additional novel immunotherapeutic combinations are being investigated. Rational design of future combination treatment with immunomodulatory agents like PD-1 inhibitors will greatly benefit from a detailed understanding of the immune contexture and the functional state of tumor infiltrating lymphocytes (TILs) within NSCLC. To this end, we characterized the immune infiltrate in 13 primary NSCLC resections using multiple orthogonal techniques: (1) surface expression of multiple ICRs on various T cell subsets as well as the functional markers granzyme B and Ki-67 were assessed by flow cytometry; (2) the spatial localization of immune infiltrates was analyzed by 7-color multiplex immunohistochemistry (mIHC); (3) gene expression profiling enabled in depth molecular characterization of TILs and indicated their functional status. Flow cytometry revealed differential contributions of ICR expression in various T cell compartments. Of note, around 30% (median=29.1%; 95% CI, 20.3-38.5%) of CD8+ T cells co-expressed CD39 and CD103, which represents a recently described population of tumor-reactive CD8+ T cells. Furthermore, approximately 90% of these double-positive CD8+ T cells expressed PD-1 (median=91.3%; 95% CI, 78.3-96.4%) and about 50% were granzyme B-positive (median=51.8%; 95% CI, 32.5-63.95%), indicative of functionally active effector status. Interestingly, more than 90% of PD-1+ CD8+ T cells co-expressed at least one more ICR (TIM-3 and/or LAG-3) (median=90.6%; 95% CI, 70.3-95%), suggesting ongoing development of T cell exhaustion in response to chronic T cell activation. In contrast, the CD4+ T cell compartment was dominated by PD-1 single-positive cells and contained a median of 15.4% (95% CI, 11.7-20%) regulatory T cells.The overall composition of the immune infiltrates in these patients were corroborated by mIHC, revealing strong immune infiltration within the tumor stroma. Furthermore, immune cell deconvolution of bulk RNA-seq data from FFPE tissues revealed a high concordance with mIHC. In conclusion, we established a pipeline for high content immune profiling of human TIL enabling correlative analyses of proteomic and transcriptomic data. The high proportion of markers indicative of TIL with anti-tumor effector potential was in line with the responsiveness of NSCLC to immunotherapy. Integration of additional gene expression data, mutational burden, and T cell clonality will deepen our understanding of the NSCLC tumor microenvironment. Citation Format: Yoshinobu Koguchi, William Miller, Brian D. Piening, Venkatesh Rajamanickam, Brady Bernard, Zhaoyu Sun, Yaping Wu, Johanna K. Kaufmann, William L. Redmond. Comprehensive immune profiling of primary tumor-infiltrating lymphocytes isolated from primary non-small cell lung cancer specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2706.

Can the plasma PD-1 levels predict the presence and efficiency of tumor-infiltrating lymphocytes in metastatic melanoma patients?

e14035 Background: The immune response to melanoma has been shown to be locally affected by presence of tumor-infiltrating lymphocytes (TILs), generally divided into brisk (infiltrating the entire base of the invasive tumor), non-brisk (infiltrating only focally) and absent. Several studies showed that greater presence of TILs, especially brisk, in primary melanoma is associated with a better prognosis and a higher survival rate. Since recent studies revealed an association between PD-1/PD-L1 expression levels and tumor response, the aim of our study was to investigate the correlation between plasma PD-1 and presence/absence/class of TILs in metastatic melanoma patients. Methods: The plasma PD-1 levels were analyzed in 28 patients with metastatic melanoma using a specific ELISA assay. The characterization of TILs in tumor tissue was performed by immunohistochemistry. Statistical analysis was assessed using t-Student and ANOVA tests. Survival curves were estimated by using the Kaplan-Meier method and log-rank test to evaluate significant differences among them. Results: 16 out of 28 patients showed the presence of TILs in primary tumor, 10 of which brisk and 6 nonbrisk. The plasma PD-1 levels were analyzed in relation to the presence/absence of TILs (p = 0.022), brisk TILs versus nonbrisk/absent TILs (p = 0.014), and brisk vs nonbrisk vs absent TILs (p = 0.032). In particular, low plasma PD-1 levels have been shown to be associated with brisk TILs in primary melanoma, intermediate values with nonbrisk TILs, and high expression with absent TILs. Although the low number of samples did not allow us to obtain a statistically significant association between the plasma PD-1 expression levels and overall survival (OS) depending on the absence or presence of TILs (brisk/nonbrisk), however the median survival of patients having brisk TILs was five months higher than other 2 groups of patients with absent and nonbrisk TILs, respectively. Conclusions: This work highlights, for the first time, the potential ability of using the plasma PD-1 levels to predict prognosis also in patients with metastatic melanoma at diagnosis for which it is not possible to identify the primary tumor.

PD-L1 and PD-1 and characterization of tumor-infiltrating lymphocytes in high grade sarcomas of soft tissue – prognostic implications and rationale for immunotherapy

ABSTRACTTherapies targeting programmed death 1-(PD-1) or its ligand (PD-L1), promoting antitumor T-cell activity have been successfully introduced into clinical practice. Clinical response correlates with PD-L1 expression by tumor cells or immune cells within the tumor microenvironment. The PD-L1/PD-1 axis and tumor microenvironment has been rarely studied in high-grade sarcomas of soft tissue (hSTS), a group of rare, genetically heterogenous and clinically aggressive tumors. We examined PD-L1 protein and CD274/PD-L1 gene copy number variations in 128 primary resected, therapy-naive hSTS using immunohistochemistry and fluorescence-in-situ hybridization. Frequency of tumoral PD-L1 expression varied widely in different disease subentities, with highest rates of positivity (40%) seen in undifferentiated pleomorphic sarcomas (UPS) and rare positivity detected in synovial sarcomas (6%). Amplification of the CD274/PD-L1 gene occurred in 14% of UPS and was rare in other subtypes. PD-L1 protein expression was significantly more frequent in CD274/PD-L1 amplified cases (p = 0.015). The subgroup of UPS was further characterized regarding the interaction between PD-L1 and the immunologic tumor microenvironment. High density of CD3+ and CD8+ tumor infiltrating lymphocytes (TILs) was significantly correlated with the presence of PD-L1 expression and seen more frequently in tumors with lower TNM stage (p = 0.024). Both, PD-L1 expression and high density lymphocytic infiltration were independent prognostic factors for a favorable overall (p = 0.001, HR 6.105 (2.041–8.258)), disease-specific (p = 0.003, HR 10.536 (2.186–50.774)) and disease-free survival (p = 0.020, HR 3.317 (1.209–9.106); values for CD8) in this particular subgroup of hSTS, whereas PD-L1 expression in TILs or CD274/PD-L1 gene amplification were not associated with outcome. These findings represent novel insights into the immune landscape of soft tissue sarcomas, in particular UPS and strengthen the rationale for immunotherapy, including targeting the PD-1/PD-L1 axis in these tumors.

Abstract P2-05-20: Tumor infiltrating lymphocytes in recurrent HER2+ and triple negative breast cancer: Prognostic value according to tumor phenotype

Abstract Background: Tumor infiltrating lymphocytes (TILs) have emerged as a prognostic and potential predictive factor in early triple negative (TN) and HER2+ breast cancer (BC). The prognostic role of TILs in advanced disease is largely unknown. Methods: 109 HER2+ and TNBC patients with available tumor tissue from regional/distant BC recurrence (collected between 2001 and 2015) were identified from a prospectively maintained database at the Istituto Oncologico Veneto of Padova (Italy). Ipsilateral in-breast relapse/second primaries and contralateral BC were excluded from the definition of recurrence. StrTILs were assessed according to consensus guidelines (Salgado, 2014) on hematoxylin and eosin stained slides from BC recurrence samples and, when available, matched primaries. Post-progression survival was calculated as the time from first BC recurrence to last follow up or death. Results: StrTILs were evaluable on recurrent BC for 72 cases (HER2+ n=43, TN n=29), after exclusion of lymphnode metastases. Median time to recurrence from initial BC diagnosis was longer for HER2+ than TN cases: 37 months (95%CI 23-51) and 18 months (95%CI 13-23), respectively. Accordingly, median time to biopsy of recurrence from initial BC diagnosis was 43 months (95%CI 35-51) for HER2+ patients and 20 months (95%CI 9-31) for TN patients. Site of biopsy was visceral metastasis in 54% and soft tissue metastasis in 46% of cases (similar for HER2+ and TN). Median StrTILs level on recurrence was 5% (Q1 2,5%, Q3 10%), without differences according to TN or HER2+ phenotype (Student's t-test p=0.5). In the whole cohort, post-progression survival did not differ for patients with high (>10%) vs low (<10%) StrTILs on recurrence (HR 0.83 95% CI 0.38-1.80, p=0.64). In the TN subgroup, high StrTILs on recurrence were associated to a better post-progression survival (median not reached vs 12.7 months for StrTILs >10% and <10%, respectively, HR 0.03 95%CI 0.00-3.64, log-rank p=0.019). To the opposite, in the HER2+ subgroup, high StrTILs were associated to worse post-progression survival compared to low StrTILs (median 27.7 vs 41.1 months for high vs low StrTILs, HR 2.93 95%CI 1.17-7.31, log-rank p=0.016). Test for interaction between tumor phenotype and StrTILs was p=0.15. Similar results were obtained when including only those patients maintaining a concordant TN or HER2+ phenotype on both primary tumor and recurrence (n=59). StrTILs were assessed on matched primary tumors for 43 patients. Overall, no significant StrTILs variation between primary tumor and recurrence was observed (mean change -4.5%, Wilcoxon p=0.5). Mean change was -2.5% and -7% in HER2+ and TN cases (Wilcoxon p=0.63 and p=0.15, respectively). For TN patients with StrTILs <10% on recurrence, a significant reduction from the primary tumor was observed (mean StrTILs 16% and 4% on primary and recurrent BC, respectively, Wilcoxon p 0.008). Conclusions: Levels of StrTILs on recurrent BC seem to have an opposite effect on prognosis of metastatic BC patients according to tumor phenotype. Immunohistochemical characterization of TILs is ongoing, data will be available for the meeting. Citation Format: Dieci MV, Giaratano T, Miglietta F, Griguolo G, Orvieto E, Falci C, Giorgi CA, Mioranza E, Tasca G, Cappellesso R, Ghiotto C, Conte P, Guarneri V. Tumor infiltrating lymphocytes in recurrent HER2+ and triple negative breast cancer: Prognostic value according to tumor phenotype [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-20.

Expansion and characterization of tumor-infiltrating lymphocytes from human sarcoma

Meeting abstracts Adoptive Cell Transfer (ACT) using Tumor Infiltrating Lymphocytes (TIL) has previously been shown at our institution and others to be an effective treatment for metastatic melanoma, resulting in a 38% response rate [[1][1]]. We applied this therapy to other solid tumors that have

Defining the role of CD2 in disease progression and overall survival among patients with completely resected stage-II to -III cutaneous melanoma

Accurate assessment of prognosis remains clinically challenging in stage II to III cutaneous melanoma. Studies have implicated CD2 in immune surveillance, T-cell activation, and antitumor immunity, but its role in melanoma progression warrants further investigation. We sought to investigate the prognostic role of CD2 in primary cutaneous melanoma. Patients with American Joint Committee on Cancer stage II and III cutaneous melanoma were identified by retrospective review of dermatopathology databases from 2001 to 2010 at Mount Sinai Medical Center and Geisinger Medical Center. Additional patients were provided by New York University Medical Center based on retrospective review and tissue availability. Immunohistochemistry was performed on tumors from 90 patients with known recurrence status and documented follow-up. Primary tumors from patients who developed recurrent disease had fewer CD2(+) cells (P = .0003). In multivariable analyses including standard clinicopathologic predictors, CD2 was an independent predictor of disease recurrence (P = .008) and overall survival (P = .007). CD2 count correlated with characterization of tumor-infiltrating lymphocytes (P = .0004). Among the intermediate prognosis group of patients with nonbrisk tumor-infiltrating lymphocytes, CD2 count was predictive of disease recurrence (P = .0006) and overall survival (P = .0318). Our retrospective design may have resulted in incomplete representation of patients lacking documented follow-up. CD2 may be an independent predictor of disease recurrence and overall survival among patients with primary cutaneous melanoma.

Infiltration of Lynch Colorectal Cancers by Activated Immune Cells Associates with Early Staging of the Primary Tumor and Absence of Lymph Node Metastases

Lynch syndrome colorectal cancers often lose human leukocyte antigen (HLA) class I expression. The outgrowth of clones with immune evasive phenotypes is thought to be positively selected by the action of cytotoxic T cells that target HLA class I-positive cancer cells. To investigate this hypothesis, we related the type and density of tumor lymphocytic infiltrate in Lynch colorectal cancers with their HLA class I phenotype and clinicopathologic stage. HLA class I expression was assessed by means of immunohistochemistry. Characterization of tumor-infiltrating lymphocytes was carried out by using a triple immunofluorescence procedure that allowed the simultaneous detection of CD3-, CD8-, and granzyme B (GZMB)-positive cells. Additional markers were also used for further characterization of an elusive CD3(-)/CD8(-)/GZMB(+) cell population. We discovered that high tumor infiltration by activated CD8(+) T cells correlated with aberrant HLA class I expression and associated with early tumor stages (P < 0.05). CD8(+) T cells were most abundant in HLA class I heterogeneous tumors (P = 0.02) and frequent in HLA class I-negative cases (P = 0.04) when compared with HLA class I-positive carcinomas. An elusive immune cell population (CD45(+)/CD8(-)/CD56(-)/GZMB(+)) was characteristic for HLA class I-negative tumors lacking lymph node metastases (P < 0.01). The immune system assumes an important role in counteracting the progression of Lynch colorectal cancers and in selecting abnormal HLA class I phenotypes. Our findings support the development of clinical strategies that explore the natural antitumor immune responses occurring in Lynch syndrome carriers.