High Levels Of Tumor-infiltrating Lymphocytes Research Articles

Tumor infiltrating lymphocytes and programmed death ligand-1 can identify different prognostic profiles in breast cancer.

e12622 Background: Immunologic biomarkers such as tumor infiltrating lymphocytes (TILs) and programmed death ligand-1 (PD-L1) can provide prognostic information in breast cancer (BC) patients. Specifically, a high TILs level has been associated with pathologic complete response and enhanced disease-free survival (DFS). Furthermore, PD-L1 positivity has been associated with worse DFS and overall survival (OS). However, the interaction of both biomarkers as well as its association with survival outcomes in specific BC subtypes is still a subject of ongoing research. Methods: Medical records of women diagnosed with primary BC between 2013 and 2015 in a center in Monterrey, Mexico were reviewed. Eligible patients had at least 1 year of follow-up, stages I-III at diagnosis, and available tissue for TILs and PD-L1 assessment. PD-L1 positivity was defined as the presence of PD-L1 in ≥1% of tumor-infiltrating immune cells using the VENTANA SP142 assay. Classification of TILs into low ( < 30%) and high (≥30%) levels was performed for analytical purposes. Clinicopathological features were compared with Fisher’s exact tests and logistic regression models, as appropriate. The Kaplan-Meier method was used to calculate recurrence-free survival (RFS), and associations between variables were explored with log-rank or Cox regressions. Results: A total of 195 patients were included. Overall, 12.3% of BC biopsy specimens showed positivity to PD-L1 (8.3% [11/132] in HR+/HER2-, 5.9% [2/34] in HER2+, and 38% [11/29] in TNBC; p < 0.001). PD-L1 positivity was significantly associated with ER-negative status (OR 3.1; p = 0.013), high TILs (OR 5.7; p < 0.001), and high Ki67 expression (HR 12.5; p < 0.001). The median follow-up for the entire cohort was 61 months (95%CI 58-63). RFS in the PD-L1- group was significantly superior to the PD-L1+ group (85% vs. 66% at 5 years; p = 0.021). In a multivariate Cox model, PD-L1 positivity (HR 3.3), low TILs (HR 3.4), advanced stage (HR 2.8), and high histological grade (HR 2.5) were all independent prognostic factors for worse RFS. Based on these results, we further classified our cohort into four prognostic profiles based on PD-L1 and TILs status. The low TILs/PD-L1+ group experienced an inferior RFS than low TILs/PD-L1- patients (45% vs. 85% at 5-years; p-value < 0.001), while no difference was observed in the high TILs groups. Notably, when analyzing HR+/HER2- patients only, a similar statistical difference was observed between low TILs/PD-L1+ and low TILs/PD-L1- groups (HR 5.4, p = 0.009). Conclusions: In our cohort, PD-L1 positivity together with low TILs identified a subset of BC patients with a worse RFS. Interestingly, patients with HR+/HER2- BC fared worse in the low TILs/PD-L1+ subcategory than in the low TILs/PD-L1- group. Future studies are warranted to explore the prognostic value of categorizing patients based on TILs and PD-L1 status in different BC subtypes.

Computational features of tumor-infiltrating lymphocyte architecture of residual disease after chemotherapy on H&E images as prognostic of overall and disease-free survival for triple-negative breast cancer.

584 Background: Approximately 30% of all breast cancers are characterized as triple-negative (TNBC). TNBC typically occurs in younger women and is associated with a poorer prognosis relative to other breast cancer subtypes. High levels of tumor-infiltrating lymphocytes (TILs) in residual disease after Neoadjuvant chemotherapy (NACT) have previously been shown to be associated with better prognosis in TNBC. In this work, we sought to evaluate the prognostic value of computationally derived measures of TIL spatial architecture in residual TNBC after NACT. Methods: H&E-stained samples from 92 patients (pts) with TNBC (41 died, 45 had disease recurrence) and residual disease after NACT were retrospectively collected from 2 sites: Instituto Nacional de Enfermedades Neoplásicas (S1) and University Hospitals (S2). 45 pts (16 deaths, 23 recurrences) from S1 formed the training set and 47 pts (25 deaths, 22 recurrences) from S2 formed the independent validation cohort. Samples were digitized at 20x. Computerized algorithms automatically identified 2 types of nuclei (TILs and non-TILs) and built clusters for each nuclei type based on cell proximity. The spatial arrangement of these clusters was then quantified using network graph metrics. The top 5 features, determined by least absolute shrinkage and selection operator, were used to train a Cox regression model that assigned a risk of death and recurrence to each patient on the training set. The percentile 33 risk score was used as a threshold for stratifying pts on the validation set as either low or high risk. For comparison, we also employed a model based on TIL density alone. Survival analysis was used to evaluate the performance of both approaches on disease-free survival (DFS) and overall survival (OS). Results: Pts in S2 (n=47) identified as “high risk” by the model based on spatial architecture of residual TILs had a significantly shorter survival time. The median OS for pts at high risk was 25 months vs. 55 months for low-risk pts. The median DFS for pts at high risk was 32 months vs 51 months for low-risk pts. Univariable analysis showed this model was prognostic for both OS (Hazard Ratio (HR) = 2.57, 95% Confidence Interval (CI): 1.07-6.16, p=0.03) and DFS (HR=2.38, CI: 1.01-5.62, p=0.04). In contrast, the model based on TIL density was not prognostic for OS (HR=1.24, CI: 0.33-4.63, p=0.73) nor DFS (HR=1.19, CI: 0.32-4.34, p=0.78). Conclusions: A computerized image analysis model based on measurements of spatial arrangement of residual TILs and surrounding cells was found to be prognostic in TNBC pts who received NACT. This method appears to be more prognostic than TIL density alone. Additional multisite validation and multivariable analysis is needed to further establish the independent prognostic utility of TIL based image biomarkers in the post-NACT TNBC.

Immunohistochemistry-based hypoxia-immune prognostic classifier for head-and-neck cancer patients undergoing chemoradiation – Post-hoc analysis from a prospective imaging trial

PurposeAs both tumor hypoxia and an immunosuppressing tumor microenvironment hamper the anti-tumor activity of radiotherapy in head-and-neck squamous cell carcinoma (HNSCC), we aimed to develop an immunohistochemistry-based hypoxia-immune classifier. Methods39 patients receiving definitive chemoradiation for HNSCC within a prospective trial were included in this analysis. Baseline tumor samples were analyzed for the hypoxia marker carbonic anhydrase IX (CAIX) and tumor-infiltrating lymphocytes (TILs) and were correlated with [18F]-misonidazole ([18F]FMISO) PET measurements. The impact of the biomarkers on the locoregional control (LRC) was examined using Cox analyses and concordance index statistics. ResultsLow CAIX (HR = 0.352, 95%CI 0.124–1.001, p = 0.050) and high TIL levels (HR = 0.308, 95%CI 0.114–0.828, p = 0.020) were independent parameters for improved LRC and did not correlate with each other (Spearman’s ρ = 0.034, p = 0.846). Harrell’s C was 0.66 for CAIX and TIL levels alone and 0.71 for the combination. 2-year LRC was 73%, 62% and 11% for the prognostically good (CAIXlow/TILhigh), intermediate (CAIXlow/TILlow or CAIXhigh/TILhigh) and poor groups (CAIXhigh/TILlow), respectively (p = 0.001). Focusing on T lymphocytes, the hypoxia-immune classifier could still stratify between favorable (CAIXlow/CD3 + TILhigh), intermediate (CAIXlow/CD3 + TILlow or CAIXhigh/CD3 + TILhigh) and poor subgroups (CAIXhigh/CD3 + TILlow) with a 2-year LRC of 80%, 59% and 14%, respectively (p = 0.001). There was a positive correlation between baseline CAIX levels and [18F]FMISO SUV in week 2 of chemoradiation (ρ = 0.324, p = 0.050), indicating an association between higher baseline CAIX expression and tumor hypoxia persistence. ConclusionWe developed a clinically feasible hypoxia-immune prognostic classifier for HNSCC patients based on pre-treatment immunohistochemistry. However, external validation is required to determine the prognostic value and the potential usage for personalized radiation oncology.

Immune analysis of lymph nodes in relation to the presence or absence of tumor infiltrating lymphocytes in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with unmet medical needs. Several studies have proved that high levels of tumor infiltrating lymphocytes (TILs) at diagnosis of TNBC confer better prognosis and patients respond better to specific chemotherapies. Nonetheless, current evidence suggests that only 15% of TNBC patients have very high levels of TILs, and another 15% lacks TILs. One possible reason to explain why patients have low TILs at diagnosis is that lymphocytes might be deactivated by an immune checkpoint in local lymph nodes, provoking their retention in there as they are unresponsive to other immune stimuli.We have identified 15 high TILs (≥50%) and 20 low TILs (≤5%) TNBC patients with localised tumour (T1c-T2N0M0) and compared the protein expression of five immune checkpoints in lymph nodes. We have also performed a customised 50-immune gene NanoString expression panel, the NanoString 360 Breast Cancer panel, and whole exome sequencing for mutation and neoantigen load analyses.In low TILs, we observed higher expression of CTLA-4 in local lymph nodes, which could explain why lymphocytes get retained in there and do not migrate to tumour. These patients have also higher neoantigen load and higher expression of B7.H3 and B7.H4 in the tumour. In high TILs, we observed more PD-L1+ tumour cells and more expanded humoral response. These results could provide a strategy to revert low tumour immune infiltration at diagnosis of TNBC, improving their prognosis.

Potential Impacts of Interleukin-17A Promoter Polymorphisms on the EGFR Mutation Status and Progression of Non-Small Cell Lung Cancer in Taiwan.

Non-small cell lung cancer (NSCLC) is a typical inflammation-associated cancer, and lung adenocarcinoma (LUAD) is the most common histopathological subtype. Epidermal growth factor receptor (EGFR) mutations are the most common driver mutations of LUAD, and they have been identified as important therapeutic targets by EGFR tyrosine kinase inhibitors. Interleukin (IL)-17A secreted by T-helper 17 lymphocytes is a proinflammatory cytokine that plays an important role in cancer pathogenesis. The present study was designed to investigate the possible associations among IL-17A genetic polymorphisms, EGFR mutation status, and the clinicopathologic development of LUAD in a Taiwanese population. Our study population consisted of 277 LUAD patients harboring the wild-type (WT) EGFR or a mutant (MT) EGFR. Four single-nucleotide polymorphisms (SNPs) of IL-17A in the peripheral blood, including rs8193036(C > T), rs8193037(G > A), rs2275913(G > A), and rs3748067(C > T) loci, were genotyped using a TaqMan allelic discrimination assay. Our results showed that none of these IL-17A SNPs were correlated with the risk of developing mutant EGFR. However, patients with a smoking habit who carried the GA genotype of IL-17A rs8193037 had a significantly lower susceptibility to EGFR mutations (adjusted odds ratio (AOR): 0.225; 95% confidence interval (CI): 0.056~0.900, p = 0.035). Moreover, compared to individuals carrying the CC genotype of rs8193036 at IL-17A, T-allele carriers (CT + TT) were at higher risk of developing more-advanced stages (stage III or IV; p = 0.020). In the WT EGFR subgroup analysis, IL-17A rs8193036 T-allele carriers had higher risks of developing an advanced tumor stage (p = 0.016) and lymphatic invasion (p = 0.049). Further analyses of clinical datasets revealed correlations of IL-17 receptor A (IL-17RA) and IL-17RC expressions with a poor prognosis of LUAD patients with a smoking history or with higher levels of tumor-infiltrating lymphocytes. In conclusion, our results suggested that two functional promoter polymorphisms of IL-17A, i.e., rs8193036 and rs8193037, were associated with the EGFR mutation status and progression in LUAD patients, indicating that these two genetic variants might act as possible markers for predicting patients’ clinical prognoses.

Ubiquitin-Specific Protease 6 Functions as a Tumor Suppressor in Ewing Sarcoma through Immune Activation.

Ewing sarcoma is the second most common pediatric bone cancer, with a 5-year survival rate for metastatic disease of only 20%. Recent work indicates that survival is strongly correlated with high levels of tumor-infiltrating lymphocytes (TIL), whose abundance is associated with IFN-inducible chemokines CXCL10 and CCL5. However, the tumor-intrinsic factors that drive chemokine production and TIL recruitment have not been fully elucidated. We previously showed that ubiquitin-specific protease 6 (USP6) directly deubiquitinates and stabilizes Jak1, thereby inducing an IFN signature in Ewing sarcoma cells. Here, we show that this gene set comprises chemokines associated with immunostimulatory, antitumorigenic functions, including CXCL10 and CCL5. USP6 synergistically enhanced chemokine production in response to exogenous IFN by inducing surface upregulation of IFNAR1 and IFNGR1. USP6-expressing Ewing sarcoma cells stimulated migration of primary human monocytes and T lymphocytes and triggered activation of natural killer (NK) cells in vitro. USP6 inhibited Ewing sarcoma xenograft growth in nude but not NSG mice and was accompanied by increased intratumoral chemokine production and infiltration and activation of NK cells, dendritic cells, and macrophages, consistent with a requirement for innate immune cells in mediating the antitumorigenic effects of USP6. High USP6 expression in patients with Ewing sarcoma was associated with chemokine production, immune infiltration, and improved survival. This work reveals a previously unrecognized tumor-suppressive function for USP6, which engenders an immunostimulatory microenvironment through pleiotropic effects on multiple immune lineages. This further raises the possibility that USP6 activity may be harnessed to create a "hot" tumor microenvironment in immunotherapy. SIGNIFICANCE: This study reveals a novel tumor-suppressive function for USP6 by inducing an immunostimulatory microenvironment, suggesting that USP6 activity may be exploited to enhance immunotherapy regimens.

Metaplastic breast cancer: an old histotype but a current therapeutic problem.

Metaplastic breast cancer (MPBC) is a rare and aggressive tumor type in greatneed of satisfactory therapies. Although most cases of MPBC are 'triple negative', they are nonetheless related to worse outcomes compared with other triple-negative invasive tumors. MPBC presents high levels of genetic and molecular heterogeneity, suggesting that novel targeted therapies can be exploited. Overexpression of PD-L1 and high levels of tumor-infiltrating lymphocytes havealso been observed in these tumors, suggesting a role for immunotherapy. We present an updated literature revision on clinical, histopathological and molecular features of MPBC and their significance to prognosis and therapy options. We discuss emerging efforts to improve and personalize prognostic and therapeutic approaches, exploiting the molecular signature of MPBC with targeted therapies and immunotherapies.

Clinicopathological characteristics and prognostic marker of triple-negative breast cancer in older women

Triple-negative breast cancer (TNBC) lacks an effective treatment target and is usually treated with chemotherapy. Treatment of older patients with TNBC, however, should be decided carefully because of the side effects of chemotherapy in this population. Some forms of TNBC are associated with a favorable prognosis and do not require chemotherapy. To optimize the treatment of older patients with TNBC, it is important to know the clinicopathological characteristics and a prognostic marker. In this study, classic clinicopathological factors, immunohistochemical characteristics (androgen receptor [AR], cytokeratin 5/6 [CK5/6], epidermal growth factor receptor), tumor-infiltrating lymphocytes (TILs), and the clinical outcome based on the status of each biomarker were compared among a consecutive series of female patients with TNBC aged ≥75 years (n = 75) and among those aged 55–64 years matched for the pathological stage (n = 47) who underwent surgery without neoadjuvant therapy. TNBC with special histology (particularly apocrine carcinoma, pleomorphic invasive lobular carcinoma, and metaplastic carcinoma) was more frequent in the older group than in the younger group (35/75, 57% versus 11/47, 23%, P = 0.010). The AR positivity rate was higher in older patients than in younger patients, whereas TILs and CK5/6 exhibited the opposite results. In multivariate analyses, AR positivity was an independent predictor of a favorable outcome in older patients (lower recurrence rate), whereas the high level of TILs was favorable in younger patients (lower recurrence and mortality rates). AR positivity or apocrine morphology was frequent and predicts a favorable clinical outcome in older patients with TNBC, suggesting the importance of AR examination in this population.

Approaching the tumor microenvironment in patients with advanced hepatocellular carcinoma using needle biopsy samples.

334 Background: Currently, combined immunotherapy of atezolizumab (anti-PD-L1 antibody) plus bevacizumab (a humanized anti-VEGF monoclonal antibody) is the standard first-line treatment in patients with advanced hepatocellular carcinoma (HCC). At the threshold of this new era, there is limited information about tumor microenvironment (TME) in advanced HCC. Several studies on TME in HCC have analyzed samples obtained via hepatic resection. In general, hepatic resection is indicated for patients with limited size and number of intrahepatic nodules, i.e., early stage HCC. In contrast, most patients who have an indication for systemic therapy have developed macroscopic vascular invasion (MVI) or/and extrahepatic metastasis, namely in advanced stage HCC. Progression from an early stage HCC to an advanced stage HCC involves a lengthy clinical course, therefore, the TME at the time of initial diagnosis may differ from that at the time of systemic therapy indication. The present study was aimed to analyze the TME by using needle biopsy samples obtained prior to initiation of systemic therapy in patients with advanced HCC. Methods: Between March 2019 and May 2020, 80 patients underwent liver tumor biopsy at the time of indication for systemic chemotherapy. HCC was confirmed via pathological examination in 70 patients and their samples were analyzed. Microsatellite instability (MSI) was evaluated using polymerase chain reaction. Programed death-ligand 1 (PD-L1) expression and the levels of tumor-infiltrating lymphocytes (TIL) were evaluated using immunohistochemical staining. PD-L1 expression was defined as per the tumor proportion score (TPS; the number of PD-L1-positive cells/total number of tumor cells) and was classified as low (TPS < 1%) or high (TPS >1%). Levels of TIL were defined as the mean number of CD8-positive lymphocytes in the tumor per 1 mm2 and classified as low or high using the median value. Results: Out of the 70 tumors, one was MSI-high and 69 were MSI-negative. The PD-L1 expression was < 1% in 50 samples, 1%–10% in 12, 11%–20% in 7, and 21%–30% in 1. The median level of TIL was 266/mm2. PD-L1highTILhighwas present in 20.0%, PD-L1lowTILlowin 38.5%, PD-L1highTILlowin 8.6%, and PD-L1lowTILhighin 32.9%. In the MSI-high tumor, PD-L1 expression was < 1% and the level of TIL was 142/mm2. High PD-L1 expression and high levels of TIL were associated with hepatitis C virus infection, high alpha-fetoprotein levels, and presence of MVI respectively. We are currently performing RNA-sequencing in order to obtain more details about TME in patients with advanced HCC. Conclusions: MSI-high advanced HCC was detected in 1.4% patients and was not necessarily associated with a “hot” immune microenvironment. PD-L1 expression and levels of TIL were associated with some clinical parameters. In the present study, we also reported the changes in the TME over time.

Response to Neoadjuvant Chemotherapy in Locally Advanced Cervical Cancer: The Role of Immune-related Factors.

Treatment of locally advanced cervical cancer (LACC) consists of concomitant chemoradiation or neoadjuvant chemotherapy (NACT) plus radical surgery (RS). This study analyzed the prognostic role of neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), tumor infiltrating lymphocytes (TILs), and PD-L1 expression in LACC patients, treated with NACT+RS. We prospectively analyzed 37 LACC patients treated from December 2016 to September 2019. Patients were submitted to pelvic examination, biopsy and imaging. In 65% of cases, a nodal involvement was present at pre-treatment MRI. All cancers showed the presence of stromal TILs and PD-L1 staining of inflammatory cells. No significant correlations were found between clinicopathological parameters and the number of TILs and PDL-1 at baseline. After NACT, 29 patients (78%) were submitted to RS; 28% of patients showed pathological complete response, 62% partial response and 10% stable disease. Seven (24%) patients reported a positive node. Patients with high levels of stromal TILs and low NLR and PLR showed a significantly better response to NACT. No significant correlation was observed between PD-L1 expression and response to NACT. The number of TILs, the expression of PDL1, and NLR and PLR ratios correlate significantly with the response of LACC patients to NACT.

Relationships between pathological factors and long-term outcomes in patients enrolled in two prospective randomized controlled trials comparing the efficacy of oral tegafur\u2013uracil with CMF (N\xb7SAS-BC\xa001 trial and CUBC trial)

PurposeTo evaluate the efficacies of cyclophosphamide, methotrexate, and fluorouracil (CMF) and tegafur–uracil (UFT) as adjuvant therapy in patients with resected stage I–IIIA breast cancer by immunohistochemistry (IHC)-based subtype and to determine the relationships between clinicopathological factors and long-term outcomes.MethodsA pooled analysis of the randomized controlled N·SAS-BC 01 and CUBC studies was conducted. Expression of hormone receptors (HRs; estrogen and progesterone receptors), human epidermal growth factor receptor 2 (HER2), and Ki67were assessed by IHC. Tumor-infiltrating lymphocytes (TILs) and nuclear/histological grades were determined by hematoxylin and eosin staining. Relapse-free survival (RFS) and overall survival (OS) were estimated by Kaplan–Meier analysis and hazard ratios were determined by Cox model adjusted for baseline tumor size and nodal status.ResultsA total of 689 patients (342 CMF and 347 UFT) were included in the analyses with a median follow-up of 11.1 years. There was no significant difference in RFS or OS between the two cohorts (RFS: 0.96 [95% confidence interval: 0.71–1.30], log-rank test p = 0.80; OS: 0.93 [0.64–1.35], p = 0.70). There was no difference in RFS or OS between the two cohorts for HR+/HER2− and HR+/HER2+ subtypes. RFS was significantly longer in patients treated with UFT compared with CMF in patients with HR−/HER2+ subtype (0.30 [0.10–0.88], p = 0.03). A high TILs level was associated with a better OS compared with low TILs level (p = 0.02).ConclusionsThis long-term follow-up study showed that RFS and OS were similar in patients with luminal-type breast cancer treated with CMF and UFT.

Correlative Analyses Between Immune Markers, Tumor Hypoxia And Outcomes Of Head-And-Neck Cancer Patients Undergoing Chemoradiation: Results From A Prospective Trial

Tumor-infiltrating lymphocytes (TILs) influence the response of patients with head-and-neck squamous cell carcinomas (HNSCCs) to chemoradiation and are modulated by the tumor microenvironment and tumor-associated hypoxia. This analysis aimed to assess the prognostic value of TILs and their association with the dynamic tumor hypoxia during chemoradiation in a prospective trial of HNSCC patients. 49 patients with locally advanced HNSCCs were enrolled in this prospective trial prior to undergoing definitive chemoradiation. Patients received tumor biopsies and [18F]-FDG-PET imaging at baseline and [18F]-FMISO hypoxia PET at baseline and in weeks 2 and 5 into treatment. Total lymphocytes, CD3-, CD4-, CD8-positive lymphocytes and expression of PD-1 and PD-L1 were quantified by immunohistochemistry in tumoral and stromal areas of the biopsies and correlated with the tissue hypoxia markers HIF1α, CAIX, CD34 and the longitudinal hypoxia PET/CT data. Potential correlations of immune markers with locoregional control and patient survival were assessed. Patients with high TIL numbers demonstrated increased locoregional control (p = 0.007) and progression-free survival (p = 0.003) compared to patients with lower levels in the biopsies. High numbers of CD3-positive T lymphocytes correlated with improved progression-free (p = 0.018) and overall survival (p = 0.037). In contrast, there was a trend towards impaired progression-free survival for high numbers of CD4-positive regulatory T lymphocytes (p = 0.091), while neither CD8-positive cytotoxic T lymphocytes nor PD-1 or PD-L1 expression correlated with patient outcomes. High levels of TILs were linked to an increased density of CD34-positive microvessels, but not increased levels of hypoxia markers HIF1α or CAIX. No significant correlations could be observed between TILs and dynamic hypoxia PET imaging parameters. In contrast, high expression of intratumoral and stromal PD-1 was associated with low initial tumor hypoxia levels (p = 0.023 for intratumoral PD-1, p = 0.004 for stromal PD-1) as well as a further decrease in the PET hypoxia signal between treatment initiation and week 5 (p = 0.033 for intratumoral PD-1, p = 0.001 for stromal PD-1). In this trial, infiltration of CD3-positive, but not CD4-positive or CD8-positive lymphocytes correlated with an improved locoregional control and increased survival of HNSCC patients after chemoradiation. While no clear associations were found between lymphocyte counts and tumor hypoxia, tumoral PD-1 expression was linked to low initial hypoxia levels and further reductions during treatment. The interplay between tumor hypoxia and tumor-associated immune patterns warrants further investigations to predict treatment response of HNSCC patients.

Immunological Network in Head and Neck Squamous Cell Carcinoma-A Prognostic Tool Beyond HPV Status.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous disease that affects more than 800,000 patients worldwide each year. The variability of HNSCC is associated with differences in the carcinogenesis processes that are caused by two major etiological agents, namely, alcohol/tobacco, and human papillomavirus (HPV). Compared to non-virally induced carcinomas, the oropharyngeal tumors associated with HPV infection show markedly better clinical outcomes and are characterized by an immunologically “hot” landscape with high levels of tumor-infiltrating lymphocytes. However, the standard of care remains the same for both HPV-positive and HPV-negative HNSCC. Surprisingly, treatment de-escalation trials have not shown any clinical benefit in patients with HPV-positive tumors to date, most likely due to insufficient patient stratification. The in-depth analysis of the immune response, which places an emphasis on tumor-infiltrating immune cells, is a widely accepted prognostic tool that might significantly improve both the stratification of HNSCC patients in de-escalation trials and the development of novel immunotherapeutic approaches.

Association between levels of tumor-infiltrating lymphocytes in different subtypes of primary breast tumors and prognostic outcomes: a meta-analysis

BackgroundTo investigate the impact of the elevation of tumor-infiltrating lymphocytes (TILs) in different molecular subtypes of primary breast cancer, i.e. each 10% increment of TILs and high-level TILs (TILs≥50%) in tumor, on overall survival (OS) and pathological complete response (pCR) and to compare the presentation of high-level TILs across these molecular subtypes.MethodsCitation retrieval was performed in the PubMed, Cochrane Library, Embase and Web of Science databases. All statistical calculations were performed by the software of StataSE version 12.0.ResultsTwenty-two eligible clinical trials including 15,676 unique patients were included for meta-analysis. Each 10% increment of TILs significantly improved OS in human epidermal growth factor receptor 2 (HER2)-overexpression (pooled Hazard ratio (HR), 0.92; 95% CI, 0.89–0.95) and triple-negative (TN) (pooled HR, 0.90; 95% CI, 0.89–0.92) breast tumors but not in luminal tumor subtype (pooled HR, 1.06; 95% CI, 0.99–1.13). It was also associated with an increased pCR rate in breast cancers (pooled Odds ratio (OR), 1.27; 95% CI, 1.19–13.5). High-level TILs were significantly related with a higher pCR rate (pooled OR, 2.73; 95% CI, 2.40–3.01) than low-level TILs. The HER2-amplified (pooled OR, 3.14; 95% CI, 1.95–5.06) and TN (pooled OR, 4.09; 95% CI, 2.71–6.19) phenotypes of breast cancers expressed significantly more high-level TILs than the luminal tumor subtype, although the presentation of those between the former two subsets was not significantly different (pooled OR, 1.30; 95%CI, 0.83–2.04).ConclusionsThe elevation of TILs in breast tumors predicts favorable prognostic outcomes, particularly in the HER2-overexpression and TN subtypes.

Abstract LB-359: Gene expression profile and tumor infiltrating lymphocytes in Hispanic women with breast cancer

Abstract Breast cancer (BC) is the most commonly diagnosed cancer among women and is currently classified into four molecular subtypes. These subtypes have been characterized by their distinct immune gene profiles, where only triple-negative (TN) and HER2+ subtypes are recognized for being tumor immunogenic cancers. The main objective of this pilot study is to understand whether the immune gene expression profile from breast tumors can be associated with tumor infiltrating lymphocytes (TILs) levels in Hispanic women. Breast tumors were obtained through the Puerto Rico Biobank at Ponce Health Sciences University and in collaboration with Moffitt Cancer Center. Pathology reports were also obtained to collect information about tumor characteristics and molecular subtype classification. Fresh frozen (FF) (n=40) and FFPE (n=27) breast tumors were cut into slide sections and stained with H&E to confirm the presence of tumor cells. The percentage of TILs was measured by staining additional tumor sections for CD3+ T cells. TILs levels were scored by analyzing the infiltration of CD3+ T cells and were further classified into: low (≤40%) or high (>40%). After microdissection of the breast tumors by a pathologist, total RNA was extracted to assess the expression changes of 770 immune genes (Pancancer Immune Panel, NanoString Technologies). The RNA quality was tested to ensure that it met the required specification for genomic analysis. Data was analyzed using the “randomForest” R package followed by proportion tests of the clinical data using contingency table analysis. All the samples were infiltrating ductal carcinomas with BC subtype distribution as follows: luminal A (n=50), luminal B (n=8), HER2+ (n=4), and TN (n=5). No significant differences were found after comparing the proportions of TILs between FF and FFPE tumor samples (p=1.00). Differentially expressed genes (DEGs) were obtained separately for FF and FFPE tumor with high and low TIL levels. The random forest analyses showed high predictive performance (~80% accuracy) to classify samples stratified by TIL levels in terms of these DEGs expressions. Sixteen (16) DEGs were found consistenly in FF and FFPE samples when stratifiying by TIL levels. The expression profiles of these DEGs show consistently higher expression in samples with high TILs levels. Among those DEGs are CLTA4 (p<0.01, adj: FF 0.2030, FF: 0.0206), CXCL9 (p<0.01, adj: FF: 0.203, FFPE: 0.229), CCL17 (p<0.01, adj: FF: 0.151, FFPE: 0.203), and TIGIT (p<0.01, adj: FF: 0.157, FFPE: 0.203). CLTA4 codifies for an immune checkpoint receptor while CXCL9 and CCL17 are known enhancers of antitumor immunity in BC. Another immune checkpoint molecule, was recently reported to be produced by BC cells to suppress the TH1 response. Our results suggest that the tumor cells can activate transcription factors that can affect the immune response in Hispanic women. Supported by grant: #4U54CA163071-05. Citation Format: Jarline Encarnacion, Carmen Ortiz-Sanchez, Julie Dutil, Wandaliz Torres-Gracia, Steven Eschrich, Shari Pilon-Thomas, Jaime L. Matta. Gene expression profile and tumor infiltrating lymphocytes in Hispanic women with breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-359.

Abstract 2754: Inhibition of nitric oxide synthase and cyclooxygenase potentiate immune responses that control aggressive mammary tumor in a murine model

Abstract Progression of aggressive, triple negative breast cancers (TNBC) is driven by inflammation and the presence of tumor infiltrating lymphocytes (TIL) improves survival. Recent studies show that TNBCs show high levels of TILs but these cells are exhausted as evident from decreased IFNγ, Ki67 and granzyme B. High expression of the inflammation-associated protein, inducible nitric oxide synthase (NOS2) and co-expression with high cyclooxygenase 2 (COX2) means poor prognosis in ER- breast cancer. Interestingly, NOS2 and COX2 are upregulated in different cells in the tumor microenvironment (TME). Thus, we hypothesized that the tandem use of NOS2 and COX2 inhibitors would curtail tumor progression by targeting different cell types within the TME simultaneously. As COX2 has also been reported to regulate tumor infiltration of myeloid cells, this could regulate both the lymphoid and myeloid arms of the immune system for better response. We investigated the effects of NOS2 and COX2 on tumorigenesis and pulmonary metastasis in a murine model of TNBC by injecting 4T1 cells into the fourth mammary fat pad of wild type (WT) and NOS2 knockout (NOS2-/-) BALB/c mice followed by treatment with COX inhibitor, indomethacin. Changes in infiltrating immune cells was analyzed using flow cytometry and confocal microscopy. We measured the levels of serum cytokines using bead-based, LegendPlex assay. We found that NOS2-/- mice developed fewer pulmonary metastatic lesions than WT mice while indomethacin directly reduced primary tumor growth in both WT and NOS2-/- mice. Indomethacin treated NOS2-/- mice showed better median survival (60 days compared 51 in WT mice). Two NOS2-/-, indomethacin treated mice showed complete remission and long term (5-months) tumor growth delay after 4T1 re-challenge, indicating immune activation in mice lacking NOS2 and COX2 function. Flow cytometry of primary tumors showed higher infiltration of activated CD4, CD8 cells, macrophages and reduction of Tregs in treated mice. Microscopy revealed a physical ‘walling -off' of TILs by CD11b+ cells in untreated tumors that was abrogated upon indomethacin treatment. As many TNBC patients succumb to pulmonary metastasis and because we found reduced metastasis in NOS2-/- mice, we studied the immune cells in metastatic lungs. Lung niche of NOS2-/- mice had more activated T-cells, bone marrow-derived macrophages and neutrophils that are not conducive to establishment of metastasis. Serum of NOS2-/- mice had elevated IL-6 that drove increased IL17A subsequently contributing to reduced metastasis and this effect was improved upon indomethacin treatment. IL6 is also a potential inhibitor of Tregs thus further improving immune surveillance. COX inhibition can treat aggressive TNBC directly by reducing cancer cell proliferation and indirectly by immune activation. Tandem NOS2 inhibition can render the pulmonary microenvironment unfavorable for metastatic spread and induce a favorable systemic cytokine storm that further improves outcome. Citation Format: Veena Somasundaram, Debashree Basudhar, Christopher McGinity, Robert Y. Cheng, Lisa A. Ridnour, Stephen J. Lockett, David A. Wink. Inhibition of nitric oxide synthase and cyclooxygenase potentiate immune responses that control aggressive mammary tumor in a murine model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2754.

The presence of tumour-infiltrating lymphocytes (TILs) and the ratios between different subsets serve as prognostic factors in advanced hypopharyngeal squamous cell carcinoma

BackgroundCancer cells induce the infiltration of various immune cells that are located or distributed in different sites and play multiple roles, which have recently been proposed to predict clinical outcomes. We therefore studied the prognostic significance of the presence of tumour-infiltrating lymphocytes (TILs) and the ratios between different types of immune cells in hypopharyngeal squamous cell carcinoma (HPSCC).MethodsWe retrospectively analysed 132 consecutive patients diagnosed with advanced HPSCC in 2013–2017. Tumoural parenchyma was immunohistochemically counted manually for the number of CD8, CD4 and Foxp3 cells. The ratios of CD8/Foxp3 and CD8/CD4 ratios were calculated for each specimen and analyzed with respect to patient clinicopathological variables and prognosis.ResultsHPSCC patients with high levels of TILs showed evident correlations with well differentiated tumors (P < 0.05). Moreover, Foxp3+ TIL is also associated with overall staging group and T category (P = 0.048 and P = 0.046, respectively). Kaplan-Meier analysis showed that high CD8 and FoxP3 infiltration correlated with favourable overall survival (OS, P = 0.019 and P = 0.001), disease-free survival (DFS, P = 0.045 and P = 0.028) and distant metastasis-free survival (DMFS, P = 0.034 and P = 0.009), respectively, but only Foxp3 displayed prognostic significance for DMFS in multivariate analysis (MVA). In the lymphocyte ratio analysis, CD8/Foxp3 appeared to play a pivotal role, and patients with a high CD8/Foxp3 ratio had a superior 3-year DFS and DMFS compared with those a low CD8/Foxp3 ratio in both univariate analysis (UVA) and MVA (P = 0.015 and P = 0.011). A high CD8/CD4 ratio was associated with better DFS and local relapse-free survival (LRFS) in UVA, and was an independent prognostic factor for improved LRFS in MVA (P = 0.040).ConclusionAlthough high TILs levels were determined to be prognostically significant in advanced HPSCC, the ratios of these subsets may be more informative. Particularly, a higher ratio of CD8/Foxp3 accurately predicts prognosis for improved DFS and DMFS, and an increased CD8/CD4 ratio is an independent predictor for favourable LRFS.

High levels of tumor-infiltrating lymphocytes showed better clinical outcomes in FOLFOX-treated gastric cancer patients.

Background: Tumor-infiltrating lymphocytes (TILs) and postoperative chemotherapeutics interact in the tumor micro-environment. This interaction has not been well investigated in gastric cancer. Materials & methods: A total of129 patients were divided into high or low TILs based on the median number of positive CD3+ and FoxP3+ T cells, which was assessed by immunocytochemistry. Results: Cox regression analysis showed that the stage III disease with shorter overall survival was significant. The analysis showed that high numbers of CD3+ or FoxP3+ T cells have better clinical outcomes in FOLFOX-treated patients. Conclusion: High CD3+ and FoxP3+ T-cell infiltration was associated with better clinical outcomes in patients with gastric cancer treated with FOLFOX, suggesting TILs incorporated into algorithms to improve the therapeutic efficacy of optimal chemotherapy.

Immunological Differences Between Immune-Rich Estrogen Receptor-Positive and Immune-Rich Triple-Negative Breast Cancers.

PURPOSEA subset of estrogen receptor–positive (ER-positive) breast cancer (BC) contains high levels of tumor-infiltrating lymphocytes (TILs), similar to triple-negative BC (TNBC). The majority of immuno-oncology trials target TNBCs because of the greater proportion of TIL-rich TNBCs. The extent to which the immune microenvironments of immune-rich ER-positive BC and TNBC differ is unknown.PATIENTS AND METHODSRNA sequencing data from The Cancer Genome Atlas (TCGA; n = 697 ER-positive BCs; n = 191 TNBCs) were used for discovery; microarray expression data from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n = 1,186 ER-positive BCs; n = 297 TNBCs) was used for validation. Patients in the top 25th percentile of a previously published total TIL metagene score distribution were considered immune rich. We compared expression of immune cell markers, immune function metagenes, and immuno-oncology therapeutic targets among immune-rich subtypes.RESULTSRelative fractions of resting mast cells (TCGA Padj = .009; METABRIC Padj = 4.09E-15), CD8+ T cells (TCGA Padj = .015; METABRIC Padj = 0.390), and M2-like macrophages (TCGA Padj= 4.68E-05; METABRIC Padj = .435) were higher in immune-rich ER-positive BCs, but M0-like macrophages (TCGA Padj = 0.015; METABRIC Padj = .004) and M1-like macrophages (TCGA Padj = 9.39E-08; METABRIC Padj = 6.24E-11) were higher in immune-rich TNBCs. Ninety-one immune-related genes (eg, CXCL14, CSF3R, TGF-B3, LRRC32/GARP, TGFB-R2) and a transforming growth factor β (TGF-β) response metagene were significantly overexpressed in immune-rich ER-positive BCs, whereas 41 immune-related genes (eg, IFNG, PD-L1, CTLA4, MAGEA4) were overexpressed in immune-rich TNBCs in both discovery and validation data sets. TGF-β pathway member genes correlated negatively with expression of immune activation markers (IFNG, granzyme-B, perforin) and positively with M2-like macrophages (IL4, IL10, and MMP9) and regulatory T-cell (FOXP3) markers in both subtypes.CONCLUSIONDifferent immunotherapy strategies may be optimal in immune-rich ER-positive BC and TNBC. Drugs targeting the TGF-β pathway and M2-like macrophages are promising strategies in immune-rich ER-positive BCs to augment antitumor immunity.

Differences in immune-related gene expressions and tumor-infiltrating lymphocytes according to chemotherapeutic response in ovarian high-grade serous carcinoma

BackgroundHigh-grade serous carcinoma (HGSC) of the ovary is the most common subtype of epithelial ovarian cancer (EOC) and has an overall poor prognosis. There is increasing awareness of the importance of immune cell populations and tumor-infiltrating lymphocytes (TILs) in various immune pathways in the tumor microenvironment. The present study evaluated immune-related gene expressions and TIL levels, as well as associated chemotherapeutic responses, to elucidate the correlation between gene expression and TIL levels in HGSC.Materials and methodsFresh tissue samples from 12 HGSC patients were included in this study. Depending on their response to adjuvant chemotherapy, the patients were divided into two groups: chemosensitive (CS) or chemoresistant (CR). The expression levels of 770 genes were analyzed using the nCounter® PanCancer Immune Profiling Panel of the NanoString nCounter® Analysis System. Quantitative real-time polymerase chain reaction (qPCR) was performed to validate the NanoString data obtained. The TIL levels in representative sections were examined via hematoxylin and eosin staining. Gene and TIL levels were subsequently correlated with the chemotherapeutic response.ResultsSeveral genes were differentially expressed in the two study groups. Eleven representative genes were selected for further evaluation. Of those, 9 genes (IRF1, CXCL9, LTB, CCL5, IL-8, GZMA, PSMB9, CD38, and VCAM1) were significantly overexpressed in the CS group; whereas expressions of 2 genes (CD24 and CD164) were increased in the CR group. Results of qPCR were consistent with those of the NanoString nCounter® analysis. Stromal TIL levels were significantly associated with adjuvant chemotherapeutic response (p = 0.001).ConclusionsSignificant differences between the CS and CR groups were observed in the expression levels of immune-related genes. Immune-related gene expressions were significantly higher in the CS group, which also had higher levels of TILs. We, therefore, suggest that, in patients with HGSC, immune-related gene expressions and TIL levels may be associated with chemotherapeutic sensitivity.