High Levels Of Tumor-infiltrating Lymphocytes Research Articles

Cracking the Codes behind Cancer Cells' Immune Evasion.

Immune evasion is a key phenomenon in understanding tumor recurrence, metastasis, and other critical steps in tumor progression. The tumor microenvironment (TME) is in constant flux due to the tumor's ability to release signals that affect it, while immune cells within it can impact cancer cell behavior. Cancer cells undergo several changes, which can change the enrichment of different immune cells and modulate the activity of existing immune cells in the tumor microenvironment. Cancer cells can evade immune surveillance by downregulating antigen presentation or expressing immune checkpoint molecules. High levels of tumor-infiltrating lymphocytes (TILs) correlate with better outcomes, and robust immune responses can control tumor growth. On the contrary, increased enrichment of Tregs, myeloid-derived suppressor cells, and M2-like anti-inflammatory macrophages can hinder effective immune surveillance and predict poor prognosis. Overall, understanding these immune evasion mechanisms guides therapeutic strategies. Researchers aim to modulate the TME to enhance immune surveillance and improve patient outcomes. In this review article, we strive to summarize the composition of the tumor immune microenvironment, factors affecting the tumor immune microenvironment (TIME), and different therapeutic modalities targeting the immune cells. This review is a first-hand reference to understand the basics of immune surveillance and immune evasion.

Prognostic biomarker tumor-infiltrating lymphocytes failed to serve as a predictive biomarker for postoperative radiotherapy in completely resected pN2 non-small cell lung cancer: a retrospective analysis

BackgroundEvidence suggests that radiotherapy is a potent immunomodulator in non-small cell lung cancer (NSCLC). Conversely, it has rarely been demonstrated if immune infiltration can influence radiotherapy efficacy. Herein, we explored the effect of tumor-infiltrating lymphocytes (TILs) on the response to postoperative radiotherapy (PORT) in completely resected stage III-pN2 NSCLC.MethodsThis retrospective study included 244 patients with pathologically confirmed stage III-N2 NSCLC who underwent complete resection at our institution between 2014 and 2020. TILs were assessed with permanent full-face hematoxylin and eosin (H&E) sections and the evaluation of TILs was based on a published guideline. Patients were stratified into the TILlow or TILhigh group with a cutoff value of 50%. Kaplan-Meier method and Log‐rank test were utilized to assess disease-free survival (DFS) and overall survival (OS). Univariate and multivariate Cox regression analysis were conducted to determine prognostic indicators.ResultsAmong 244 patients, a total of 121 patients received PORT whereas 123 did not. TILs level in patients with PORT was significantly higher than that in patients without PORT (p < 0.001). High TILs level was significantly associated with an improved DFS and OS in all the entire chort (DFS, p < 0.001; OS, p = 0.001), PORT chort (DFS, p = 0.003; OS, p = 0.011) and non-PORT chort (DFS, p < 0.001; OS, p = 0.034). There were no significant survival differences between different treatment modalities in the low TILs infiltration (DFS, p = 0.244; OS, p = 0.404) and high TILs infiltration (DFS, p = 0.167; OS, p = 0.958) groups.ConclusionsTILs evaluated with H&E sections could represent a prognostic biomarker in patients with completely resected pN2 NSCLC, and high TILs infiltration was associated with favorable survival outcomes.The predictive value of TILs for PORT still need to be further explored in the future.

Prediction value study of breast cancer tumor infiltrating lymphocyte levels based on ultrasound imaging radiomics.

Construct models based on grayscale ultrasound and radiomics and compare the efficacy of different models in preoperatively predicting the level of tumor-infiltrating lymphocytes in breast cancer. This study retrospectively collected clinical data and preoperative ultrasound images from 185 breast cancer patients confirmed by surgical pathology. Patients were randomly divided into a training set (n=111) and a testing set (n=74) using a 6:4 ratio. Based on a 10% threshold for tumor-infiltrating lymphocytes (TIL) levels, patients were classified into low-level and high-level groups. Radiomic features were extracted and selected using the training set. The evaluation included assessing the relationship between TIL levels and both radiomic features and grayscale ultrasound features. Subsequently, grayscale ultrasound models, radiomic models, and nomograms combining radiomics score (Rad-score) and grayscale ultrasound features were established. The predictive performance of different models was evaluated through receiver operating characteristic (ROC) analysis. Calibration curves assessed the fit of the nomograms, and decision curve analysis (DCA) evaluated the clinical effectiveness of the models. Univariate analyses and multivariate logistic regression analyses revealed that indistinct margin (P<0.001, Odds Ratio [OR]=0.214, 95% Confidence Interval [CI]: 0.103-1.026), posterior acoustic enhancement (P=0.027, OR=2.585, 95% CI: 1.116-5.987), and ipsilateral axillary lymph node enlargement (P=0.001, OR=4.214, 95% CI: 1.798-9.875) were independent predictive factors for high levels of TIL in breast cancer. In comparison to grayscale ultrasound model (Training set: Area under curve [AUC] 0.795; Testing set: AUC 0.720) and radiomics model (Training set: AUC 0.803; Testing set: AUC 0.759), the nomogram demonstrated superior discriminative ability on both the training (AUC 0.884) and testing (AUC 0.820) datasets. Calibration curves indicated high consistency between the nomogram model's predicted probability of breast cancer TIL levels and the actual occurrence probability. DCA revealed that the radiomics model and the nomogram model achieved higher clinical net benefits compared to the grayscale ultrasound model. The nomogram based on preoperative ultrasound radiomics features exhibits robust predictive capacity for the non-invasive evaluation of breast cancer TIL levels, potentially providing a significant basis for individualized treatment decisions in breast cancer.

Abstract PO5-17-01: Pre-treatment tumor infiltrating lymphocytes are associated with higher likelihood of pathologic complete response among black women with TNBC receiving neoadjuvant chemoimmunotherapy

Abstract Background: High risk, early-stage triple negative breast cancer (TNBC) is a heterogenous disease that is associated with early recurrence and high mortality, particularly among black women as compared to their white counterparts. Pathologic complete response (pCR) to neoadjuvant chemotherapy (NACT) is a strong-predictor of long-term disease-free survival in TNBC, and the KEYNOTE-522 trial demonstrated that the addition of immunotherapy (pembrolizumab) to NACT resulted in a higher likelihood of pathologic complete response (pCR) in high-risk early-stage TNBC. Following these findings, the standard of care has been to administer the K522 regimen (K522R) in the neoadjuvant setting for patients with high risk early-stage TNBC. However, our understanding of the clinical and tumor characteristics to prognosticate response to K522 are lacking, and additional real-world data is needed to better understand if these variables differ among race. Methods: We evaluated 76 patients who were treated with the K522R at our institution. There were slides available from 29 patients’ pre-treatment biopsies for pathology review. In the available slides, tumor infiltrating lymphocytes (TIL) were evaluated as the percentage of lymphocytes in the stroma within the tumor area. For the remaining cases that did not have available slides, these variables were retrieved from pathology reports and patients’ charts. Patient characteristics including age, race and body mass index (BMI) were acquired via retrospective chart review. These clinicopathologic variables were correlated with pCR, which was defined by the absence of invasive carcinoma in the breast and axillary lymph nodes at the time of surgery. Binary logistic regression analysis was performed to correlate the above variables with pCR versus residual disease (RD). Results: Seventy-six pre-treatment biopsy specimens treated with the K522R were evaluated. The patient population spanned a broad spectrum of age, body habitus, and race, with mean age 55, mean BMI 28.3, and predominantly black (over 60 percent) population. At the time of analysis, 64 of the 76 patients had completed the K522R and subsequently undergone surgery at our institution. Of the 64 patients who had undergone definitive surgery, 31 (48.4%) achieved pCR. In univariate analysis, pre-treatment TIL was significantly associated with attainment of pCR, with TIL of 28.5% (± 19.1) in patients who achieved pCR versus 9.73% (± 9.40) in those who had RD (95% CI 1.03-1.21, p=0.014*). When TIL was further stratified by race, mean percentage of TIL was significantly higher among black women who achieved pCR (27.5% ± 4.7) compared to black women with RD (12.2% ± 3.05) (95% CI 0.021 – 0.197, p=0.016*). Additionally, mean percentage of TIL was higher overall among black women when compared to the white patients in our cohort, though this finding was not statistically significant (p=0.252). Conclusion: Our real-world data demonstrates high levels of pre-treatment TIL are significantly associated with achievement of pCR in patients with TNBC treated with the K522 regimen. When TIL was further stratified by race, black patients were found to have higher TIL than white patients. Within the black population, a statistically significant difference in TIL percentage was found in those achieving pCR compared to those with RD. It is well understood that black women often have poorer breast cancer outcomes than white women, and our finding of higher TIL percentage among this population, along with the significant association between TIL and attainment of pCR with chemoimmunotherapy, could have important implications for optimization of therapy. Further exploration of this phenomenon in larger, diverse breast cancer populations is warranted. Citation Format: Sarah Wood, Ji-Hoon Lee, Yuan Gao, Xiaoxian Li, Jane Meisel. Pre-treatment tumor infiltrating lymphocytes are associated with higher likelihood of pathologic complete response among black women with TNBC receiving neoadjuvant chemoimmunotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-17-01.

Abstract PO3-14-06: Immunologic features and association with prognosis in hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancer (MBC) treated with chemotherapy (CT) or CDK4/6-inhibitors (CDK4/6i) + endocrine therapy (ET)

Abstract Purpose: Little is known regarding the prognostic and predictive role of tumor immunological features in HR+/HER2- MBC treated with CT or CDK4/6i+ET. Methods: The Italian KENDO phase 2 trial randomized CT-naïve patients with HR+/HER2-neg. MBC with aggressive characteristics to receive CDK4/6i+ET (arm A) or CT+/-ET (arm B). Primary endpoint was progression-free survival (PFS). A tumor sample from the primary or metastatic tumor (archived or newly-obtained) was mandatory for inclusion. In this correlative biomarker analysis, tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLS) were detected on baseline tumor samples by hematoxylin&amp;eosin staining, while immune genomic signatures were assessed with the nCounter BC360 panel. Immune pattern at immunohistochemistry was defined as inflamed (IF), excluded (IE) or desert (ID). TILs and signatures were dichotomized (high vs. low) with the maximally-selected rank statistics (MSRS) method. Survival analyses were conducted with the Kaplan-Meier (KM) method and differences were tested with log-rank test. Hazard ratios (HR) with 95% confidence intervals (CI) were estimated with Cox regression. Significance was set at p≤0.05. Since the trial was stopped earlier due to slow accrual all analyses were exploratory. Results: Forty-nine patients were randomized, 17 (34.7%) in arm A and 32 (65.3%) in arm B. No significant clinicopathological differences were observed at baseline between the two arms, except for tumors in arm B showing more TLS (50.0% vs. 12.5%, p=0.013). Median PFS (mPFS) with CT+/-ET was numerically shorter than mPFS with CDK4/6i+ET (11.2 vs. 19.9 months, HR: 1.41, 95%CI: 0.75-2.64, p=0.289). The median OS (mOS) for arm A was not estimable (NE) vs. 30.6 months in arm B (p=0.283). In arm A, median TILs levels were 3% (interquartile range [IQR]: 1-5%) and TLS, with/without germinal centers, were present in 2 cases (12.5%). These were also the only IF tumors in the arm, whereas the rest (87.5%) were ID. In arm B, 7 (25.0%) tumors were IF, 19 (87.5%) ID and 2 (7.1%) IE. Median TILs were 4.5% (IQR: 2-12.8%) and TLS were present in 50% cases. In arm A, ID tumors showed lower mPFS than IF (15.8 vs. 27.5 months), with concordant trend at the OS KM curve. In arm B there was no clear difference in PFS, but IF tumors did not reach mOS, differently from ID (28.7 months) and IE (28.1 months). High vs. low TILs levels were significantly associated with better PFS (p=0.003) and OS (p=0.005) in arm A. High levels of a TGFβ gene expression signature were significantly associated with better PFS (p=0.020) and OS (p=0.005) in arm A and PFS (p=0.03) in arm B; higher levels of a cytokine/chemokine signature were associatedwith better PFS (p=0.02) in arm A, and higher levels of a mastcells signature was associated with worse OS in both arms (p=0.02 and p=0.03). In arm B, higher levels of a macrophage (p &amp;lt; 0.001) and antigen presentation signatures (p=0.04) were associated with worse and better OS, respectively, whereas higher levels of an immune infiltration and a cytotoxic cells signature were associated with better PFS (p=0.03 both). TLS presence was numerically associated to longer mPFS (23.5 vs. 15.8 months) in arm A, with consistent OS KM curve’s trend, and longer mOS (44.5 vs. 28.1 months) in arm B. Conclusions: The KENDO trial further supports CDK4/6i+ET use in aggressive CT-naïve HR+/HER2- MBC. Biomarkers of immune activation such as IF tumors, higher TILs and presence of TLS pointed towards better survival outcomes. Genomic immunological features also showed prognostic effect, with differences according to treatment arm and/or the immune process or cell line tracked. Further research on larger cohorts is needed to confirm these preliminary findings. Citation Format: Francesco Schettini, Michela Palleschi, Francesca Mannozzi, Fara Brasó-Maristany, Lorenzo Cecconetto, Patricia Galván, Marita Mariotti, Alessia Ferrari, Emanuela Scarpi, Anna Miserocchi, Oriana Nanni, Esther Sanfeliu, Aleix Prat, Andrea Rocca, Ugo De Giorgi. Immunologic features and association with prognosis in hormone receptor-positive/HER2-negative (HR+/HER2-) metastatic breast cancer (MBC) treated with chemotherapy (CT) or CDK4/6-inhibitors (CDK4/6i) + endocrine therapy (ET) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-14-06.

Risk factors for head and neck squamous cell carcinoma in disease pathogenesis and prognosis: the importance of smoking cessation and the concept of harm reduction

Introduction. Cigarette smoking along with alcohol consumption, infection with human papilloma (HPV) and the Epstein–Barr viruses, is one of the main risk factors for head and neck squamous cell carcinoma. It can also contribute to virus-associated carcinogenesis and affect the clinical and prognostic features of the tumor. Continued smoking at diagnosis of head and neck squamous cell carcinoma is a negative prognostic factor and contributes to reduced patient survival rates. Aim. To analyze the smoking status and the effect of etiopathogenesis on molecular and clinical features in patients with oropharyngeal squamous cell carcinoma (OpSCC). Materials and methods. In patients with OPSCC, the smoking status and the level of motivation to its cessation were clarified. Immunohistochemical analysis of tumor sections from paraffin blocks from the ThermoScentific immunohis-tosteiner was performed using monoclonal antibodies to estrogen receptors α (ERα), programmed death-ligand 1 (pD-L1), Bcl-2, p53 and p16. The percentage of tumor-infiltrating lymphocytes (TILs) in the stromal component of the tumor (hematoxylin and eosin staining) was also analyzed. Results. Of 113 patients with OpSCC, 80 % of patients were current smokers, including 75 % of patients with Hpv-positive tumor status. men with OpSCC were statistically significantly more likely to smoke than women (p = 0.001), as were men with Hpv-positive tumor (p = 0.015). Smoking patients were characterized by a larger size of the primary tumor (T3–4), including those with Hpv (p = 0.015). positive Hpv status of the tumor is associated with autoimmune diseases (autoimmune thyroiditis, psoriasis) (p ˂ 0.05). A high level of TILs is characteristic of smokers, including those with p16+-status (p ˂0.05), but not for all patients with this status. The positive expression of ERα is associated with p16+-status, and the latter is associated with the absence of p53 expression (p ˂0.05). During conservative therapy, tumor regression is associated with the primary tumor size T1–2, p16 + status, female sex, age less than 55 years, TILs level ≥ 57.5 %, pD-L1 expression levels on tumor cells ≥2.2 %, Bcl-2 ≥5 % and ERα ≥65 % (p ˂ 0.05). At the same time, quality of good predictive model for tumor regression showed a TILs level of ≥57.5 % (area under curve, AuC) 0.796; sensitivity 78.6 %; specificity 75 %) and ERα (AuC 0.725; sensitivity 65 %; specificity 83 %). The level of motivation to quit smoking ranged from 1 to 7 points according the 10-point motivation scale in 89 % of respondents. Only 10 patients were ready to quit smoking immediately (10 points on the motivation scale). Conclusion. Molecular heterogeneity of OPSCC was revealed, including in the group of p16+-tumors. Correlation of clinical and molecular parameters with response to conservative therapy can be used in planning of primary therapy and earlier use of surgical treatment. The level of motivation to quit smoking in patients with squamous cell carcinoma of the head and neck may be increased by drawing attention to the problem at each doctor’s appointment as well as by informing about how to quit smoking and therapeutic approaches. In unmotivated patients, the concept of reducing the harm of smoking can be used involving the replacement of cigarettes with electronic tobacco heating systems, eliminating the carcinogenic effect of its combustion products.

Tumor infiltrating lymphocytes (TILs) in triple negative breast cancer

Tumor infiltrating lymphocytes (TILs) are a promising inexpensive prognostic and predictive biomarker in breast cancer. High levels of TILs are associated with improved survival and higher probability to achieve pathological complete response in triple-negative breast cancer (TNBC). To assess the level of TILs in TNBC samples and analyze the association between the level of TILs and the main pathological parameters, to identify their impact on long-term results. The study included information on 140 patients with I-III stage TNBC and estrogen receptors <10%. Tumor tissue samples at baseline biopsies were evaluated the histological type, HER2 expression, estrogen expression levels, Ki-67 and TILs. The pathological response was evaluated according to the ypTNM, Miller-Payne, and RCB classifications. The average level of TILs in biopsy specimens before NACT was 29.3±23.1%. Low levels of TILs (<10%) were defined in 21% of cases, intermediate levels (≥10% to ≤40%) in 55% of cases, and high levels (>40%) in 24% of cases. Using the two-tiered system, low TILs (≤40%) were defined in 76% and high TILs (>40%) in 24% of cases. The level of TILs was correlated with histological grade (R=0.187; p=0.027) and estrogen receptor expression level (R=0.211; p=0.012). There were no significant differences depending on the level of TILs and other pathological parameters. Three-year event-free survival (EFS) in patients with high TILs levels was 95% versus 65% in the low TILs group (p=0.037). Stromal TILs are an important prognostic biomarker in TNBC. Using a cutoff of 40%, high TILs are significantly associated with longer EFS.

How to incorporate new agents into precise medicine for cholangiocarcinoma?

Cholangiocarcinoma, a rare and aggressive form of cancer originating from the bile ducts in the liver, poses a significant challenge for treatment. However, the emergence of precision medicine has brought newfound hope for more effective therapies. Several precision medicine approaches have demonstrated promise in the treatment of cholangiocarcinoma. One such approach is targeted therapy, which involves utilizing drugs that specifically target the genetic mutations or alterations present in the tumor cells. In the case of cholangiocarcinoma, mutations in the IDH1 and IDH2 genes are frequently observed. Immunotherapy is another precision medicine approach being explored for the treatment of cholangiocarcinoma. Immune checkpoint inhibitors like pembrolizumab and nivolumab can be used to bolster the body's immune response against cancer cells. While the response to immunotherapy can vary among individuals, studies have shown promising results, particularly in patients with high levels of tumor-infiltrating lymphocytes or microsatellite instability. Moreover, molecular profiling of cholangiocarcinoma tumors can play a crucial role in identifying potential targets for precision medicine. Through advanced next-generation sequencing techniques, specific gene alterations or dysregulations in pathways can be identified, potentially guiding treatment decisions. This personalized approach enables tailored treatment plans based on the unique genetic characteristics of each patient's tumor. In conclusion, the advent of precision medicine has opened up new avenues for the treatment of cholangiocarcinoma. Targeted therapy and immunotherapy have exhibited promising results, and further molecular profiling is expected to uncover additional therapeutic options. Such advancements represent a significant step forward in the quest to enhance outcomes for individuals affected by cholangiocarcinoma.

Abstract C078: Pembrolizumab response in melanoma patients: an analysis of the tumor microenvironment

Abstract Background: Melanoma has been associated with poor prognosis and limited treatment options. Immunotherapy, particularly the use of immune checkpoint inhibitors, has revolutionized the treatment landscape for advanced melanoma. Pembrolizumab, a PD-1 inhibitor, has shown promising results; however, the underlying mechanisms of its efficacy and the influence of the tumor microenvironment (TME) on treatment response remain incompletely understood. This study aims to investigate the response to pembrolizumab in melanoma patients and analyse the TME using a multiplexed immunofluorescence (mIF) spatial tissue analysis to gain insights into potential predictive factors and therapeutic targets. Methods: A retrospective analysis was conducted on formalin-fixed paraffin-embedded (FFPE) tumour samples from baseline melanoma patient samples, classified as responders (n = 6) or non-responders (n = 4) based on the response at 3 months post pembrolizumab treatment. Analysis was done using the mIF platform MultiOmyx™, a proprietary, immunofluorescence (IF) multiplexing assay that enables visualization and characterization of up to 60 biomarkers on a single FFPE section. Image segmentation, cell phenotyping, and spatial analytics were performed using our AI-powered image analytics software, NeoLYTX™. Results: Analysis of the 10 FFPE samples from metastatic melanoma patients treated with pembrolizumab revealed significant differences in immune cell populations between non-responders and responders. Patients who responded favorably to pembrolizumab exhibited increased infiltration of cytotoxic T lymphocytes (CTLs) and tumor-associated macrophages within the TME. This finding suggests that an immune-inflamed microenvironment, characterized by high levels of tumor-infiltrating lymphocytes may be indicative of a robust antitumor immune response. Moreover, immune checkpoint molecules PD-1, CTLA-4, LAG3, and TIM3 were also significantly increased in responding patients, suggesting potential therapeutic targets to enhance pembrolizumab response, including combination strategies targeting multiple immune checkpoints. When spatially characterizing T cell activation and exhaustion status in different regions of the TME we found an increase in checkpoint molecules among intra-tumoral CTLs as compared to intra-stromal CTLs. In particular, the fraction of CTLs with quadruple-positive CTLA4+LAG3+PD1+TIM3+ expression was doubled. This suggests an immunosuppressive TME in which CTLs are rapidly exhausted upon reaching the tumor. Work is now ongoing to characterize how this cell population spatially interacts with other immune and tumor cells in the TME. Conclusions: This study provides valuable insights into the response to pembrolizumab in melanoma patients and highlights the crucial role of the TME in determining treatment outcomes. Identification of immune cell subsets, and immunosuppressive mechanisms associated with pembrolizumab response may guide the development of novel therapeutic approaches to improve patient outcomes in advanced melanoma. Citation Format: Anna Juncker-Jensen, Juliana Wortman, Nigel Beaton, Kristina Beeler, Tobias Treiber, Mitchell Levesque, Julia Martinez Gomez, Jakob Vowinckel, Harry Nunns. Pembrolizumab response in melanoma patients: an analysis of the tumor microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C078.

Predictive factors for response to neoadjuvant chemotherapy: inflammatory and immune markers in triple-negative breast cancer.

Tumor-infiltrating lymphocytes (TILs) predict response to neoadjuvant chemotherapy (NAC) in triple-negative breast cancer (TNBC) patients. However, the TIL level can be determined at a few facilities. By contrast, neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) are easily and objectively determined from the results of full blood counts. We conducted a retrospective study to investigate whether TILs, NLR, and PLR predict NAC efficacy and whether NLR and PLR could be surrogate markers for TILs in TNBC. Of the 266 patients diagnosed with TNBC between 2013 and 2019, 66 who underwent radical surgery after sequential administration of anthracycline and taxane as NAC were included in the study. TILs, NLR, and PLR were evaluated as predictors of pathologic complete response (pCR) using cutoff values determined from receiver operating characteristic curves. The cutoff values of TILs, NLR, and PLR were 20%, 2.6, and 180, respectively. High TIL level was associated with low NLR (P = 0.01) and low PLR (P = 0.01). High TIL level (odds ratio [OR] 4.28 [95% CI 1.40-13.1]; P = 0.01), low NLR (OR 5.51 [95% CI 1.60-18.9]; P = 0.01), and low PLR (OR 3.29 [95% CI 1.13-9.57]; P = 0.03) were associated with pCR. Low NLR predicted pCR independently (OR 6.59 [95% CI 1.45-30.0]; P = 0.01). TILs, NLR, and PLR predicted NAC efficacy against TNBC. TIL level was associated with NLR and PLR. NLR was an independent predictive factor and may be a useful surrogate marker for TILs when predicting pCR.

Prognostic relevance of tumor‑infiltrating lymphocytes in residual tumor tissue from patients with triple‑negative breast cancer following neoadjuvant chemotherapy: A systematic review and meta‑analysis.

Further adjuvant chemotherapy treatment can provide benefits to certain patients with triple-negative breast cancer (TNBC) that fail to achieve pathological complete response (pCR) after the administration of a neoadjuvant chemotherapy (NAC) regimen. However, biomarkers suitable for identifying patients likely to experience poor prognostic outcomes after undergoing additional adjuvant chemotherapy are currently lacking. Accordingly, the present meta-analysis was conducted to explore the relationship between tumor-infiltrating lymphocytes (TILs) or TIL subtypes (CD4+ or CD8+) in residual tumor (RT) tissue following NAC and TNBC patient prognosis. Relevant studies published through March 2023 were identified in Pubmed, The Cochrane Library, Embase and Web of Science databases. After excluding irrelevant studies, data were extracted from the remaining reports, while study quality was analyzed with the Newcastle-Ottawa Scale. Subsequent analyses were performed with Stata 14.0 and Review Manager 5.3. In total, seven relevant studies incorporating 1,202 patients were identified, all of which were retrospective cohort studies. Pooled analyses demonstrated that those patients exhibiting higher levels of RT TIL infiltration following NAC exhibited significantly improved recurrence-free, metastasis-free and event-free survival (RFS/MFS/EFS) compared with patients with lower RT TIL infiltration levels, together with an improved distant recurrence-free interval (DRFI) [hazard ratio (HR)=0.52; 95% confidence interval (CI)=0.39-0.69; P<0.00001]. In addition, patients exhibiting high RT TIL infiltration exhibited improved overall survival (OS) and breast cancer-specific survival (BCSS; HR=0.49; 95% CI=0.38-0.65; P<0.00001). Additional subgroup analyses revealed that patients with higher TIL infiltration levels or TIL subtype (CD4+ or CD8+) infiltration exhibited improved RFS/MFS/EFS/DRFI as compared with patients with lower levels of overall TIL or TIL subtype (CD4+ or CD8+) infiltration in RT tissue (HR=0.35, 95% CI=0.20-0.59, P<0.0001; HR=0.49, 95% CI=0.33-0.71, P=0.0002). Consistently, the OS/BCSS of patients exhibiting high levels of overall TIL or TIL subtype (CD4+ or CD8+) infiltration was increased compared with patients with lower levels of such infiltration (HR=0.33, 95% CI=0.19-0.59, P=0.0002; HR=0.55, 95% CI=0.41-0.76, P=0.0002). These data thus demonstrate that levels of overall TIL infiltration or infiltration by CD4+ or CD8+ TILs in RT following NAC can be used as a biomarker to reliably predict prognostic outcomes in patients with TNBC, in addition to highlighting possible targets that may guide the further immunotherapeutic management of these patients.

High expression of MORC2 predicts worse neoadjuvant chemotherapy efficacy in triple negative breast cancer.

Tumor infiltrating lymphocytes (TILs) are closely related to the patients' prognosis. Recently, Microrchidia 2 (MORC2) has been documented as a prognostic and predictive biomarker in triple negative breast cancer (TNBC). To compare whether MORC2 is a better predictor than TILs, as well as clinicopathological parameters, in predicting the efficacy of neoadjuvant chemotherapy (NAC) in TNBC, we detected the expression of MORC2 on neoplastic cells through immunohistochemistry and quantified the stromal TILs through Hematoxylin-eosin staining on core biopsies from 50 locally advanced TNBC patients who underwent standard NAC. Among all the 50 patients, 28 (56%) cases had residual tumors, while the other 22 (44%) achieved pathologic complete response (pCR). In these studied patients, age and T-stage showed no correlation with pCR rate, while percentage of TILs, nodal involvement and expression of MORC2 on tumor cells showed significant association with pCR rate. Positive nodal involvement was correlation with worse pathologic response at multivariate analysis (P = .0036), and high TILs levels (≥50%) was positively associated with better NAC efficacy at univariate analysis (P = .002). Whereas high expression of MORC2 was statistically associated with worse pCR rate both at univariate (P < .001) and multivariate (P = .036) analysis. Our results indicate that MORC2 expression has a better predictive role in predicting the efficacy of NAC than TILs in TNBC patients.

The Distinctive Features behind the Aggressiveness of Oral and Cutaneous Squamous Cell Carcinomas

Squamous cell carcinomas arise from stratified squamous epithelia. Here, a comparative analysis based on recent studies defining the genetic alterations and composition of the stroma of oral and cutaneous squamous cell carcinomas (OSCC and CSCC, respectively) was performed. Both carcinomas share some but not all histological and genetic features. This review was focused on how mutations in tumor suppressor genes and protooncogenes cooperate to determine the differentiation, aggressiveness, and metastatic potential of OSCC and CSCC. In fact, driver mutations in tumor suppressor genes are more frequently observed in OSCC than CSCC. These include mutations in TP53 (encoding pP53 protein), CDKN2A (encoding cyclin dependent kinase inhibitor 2A), FAT1 (encoding FAT atypical cadherin 1), and KMT2D (encoding lysine methyltransferase 2D), with the exception of NOTCH (encoding Notch receptor 1), whose mutation frequency is lower in OSCC compared to CSCC. Finally, we describe the differential composition of the tumor microenvironment and how this influences the aggressiveness of each tumor type. Although both OSCC and CSCC tumors are highly infiltrated by immune cells, high levels of tumor-infiltrating lymphocytes (TILs) have been more frequently reported as predictors of better outcomes in OSCC than CSCC. In conclusion, OSCC and CSCC partially share genetic alterations and possess different causal factors triggering their development. The tumor microenvironment plays a key role determining the outcome of the disease.

Effects of tumor-infiltrating lymphocytes on nonresponse rate of neoadjuvant chemotherapy in patients with invasive breast cancer

High level of tumor-infiltrating lymphocytes (TILs) can predict the rate of total pathological complete remission (tpCR) of breast cancer patients who receive neoadjuvant chemotherapy (NACT). This study focused on evaluating the data of patients whose primary tumor and/or lymph node metastasis show nonresponse (NR) to NACT, trying to provide a basis for the clinical decision which patients will develop NACT resistance. The study included breast cancers from 991 patients who received NACT. ROC curve analysis confirmed that TILs showed significant predictive value for NR of hormone receptor (HR)+HER2− and triple-negative breast cancer (TNBC). Among HR+HER2− breast cancer, TILs ≥ 10% was an independent predictor for low NR rate. Furthermore, positive correlation of TILs with Ki67 index and Miller-Payne grade, and negative correlation with ER and PR H-scores were only identified in this subgroup. In TNBC, TILs ≥ 17.5% was an independent predictor for low NR rate. The predictive value of low TILs on NR may facilitate to screen patients with HR+HER2− or TNBC who may not benefit from NACT. HR+HER2− breast cancer with low levels of TILs should be carefully treated with neoadjuvant chemotherapy, and other alternatives such as neoadjuvant endocrine therapy can be considered.

The role of innate immune system in modulating CHK1 inhibitor (CHK1i) response in BRCA wild-type (BRCAwt), platinum-resistant high-grade serous ovarian cancer (PR-HGSOC): Exploratory analysis from a phase II study of CHK1i prexasertib.

5557 Background: Immune checkpoint blockade, e.g., anti-PD-1/PD-L1, demonstrated limited efficacy in HGSOC despite high levels of tumor-infiltrating lymphocytes, suggesting the innate, rather than adaptive, immune system may play a prominent role in anticancer response. Preclinical studies suggest CHK1i modulates the innate immune milieu. We previously showed clinical activity of prexasertib in BRCAwt, PR-HGSOC patients (pts). Here, we investigated the association of innate immunogenic features with CHK1i response. Methods: Prexasertib was given at 105 mg/m2 IV every 14 days in a 28-day cycle. Baseline (BL) fresh core biopsies were collected from pts enrolled in the biopsy (Bi) cohort for RNA sequencing. Computational immunogenomic analysis (CIBERSORT) was used for immune cell fractions quantification. Blood samples (BS) were obtained at BL and cycle 1 day 15 (C1D15) from pts enrolled in both Bi and non-Bi (nBi) cohorts for multiparametric flow cytometry (FC) analysis to evaluate dynamic changes in immune cell subsets. Mann-Whitney and Wilcoxon tests were used to compare unpaired and paired data, respectively. Results: 49 BRCAwt, PR-HGSOC pts were enrolled in the Bi cohort (n=25) and nBi cohort (n=24). 39 were evaluable per RECIST v1.1. 18/39 pts (46.2%) had a clinical benefit (CB), defined as progression-free survival≥6 months. Transcriptomic profiles of BL biopsies (n=15) met the quality control and assurance (Gene Similarity Index, Principal Component Analysis) for further in silico analysis. CIBERSORT analysis did not exhibit any differences in the immune cell fractions between CB and non-CB (NCB) groups at BL. FC analysis on paired BS (BL and C1D15) showed an increase of immunosuppressive monocytic myeloid-derived suppressor cells (M-MDSCs, p&lt;0.001) and classical monocytes (CM, p=0.003) in the NCB group (18 paired BS). In the CB group (19 paired BS), CD141+ and CD1c+ dendritic cells (DCs) express higher rates of CD83, a marker of maturity and functionality (p&lt;0.001 and p&lt;0.001, respectively), despite no increase in the absolute number of DCs. At C1D15, a higher rate of M-MDSCs (median 7.8% vs. 5.52%; p=0.03) and CM (median 40.7% vs. 31.7%; p=0.03) were found in the NCB group (n=18) compared to the CB group (n=19); also, CM showed lower expression of HLA-DR in the NCB group (median 19.25% vs. 27.8%; p=0.02). Conclusions: Collectively, our data suggest the possible involvement of innate immunity in modulating CHK1i response. Increasing peripheral immunosuppressive cells and lower immunocompetence of the innate immune system may contribute to CHK1i resistance, whereas the functionality of DCs may be involved in the response. Enhancing innate immunity may represent a strategy to increase CHK1i response in this hard-to-treat population. Clinical trial information: NCT02203513 .

Tumor-infiltrating lymphocytes (TILs) for prediction of response to platinum-based neoadjuvant chemotherapy (NACT) in triple-negative breast cancer (TNBC).

e12620 Background: According to several previous trials the rate of pathological complete response (pCR) for patients with TNBC is increased when carboplatin is added to NACT. However, it is largely unknown which group of patients would truly benefit from platinum-based NACT and for whom chemotherapy could be de-escalated. Several clinical trials have evaluated the stromal TILs as an important prognostic and predictive biomarker in breast cancer. We assessed the pCR rates and the outcomes in patients with TNBC randomized to receive NACT ± Carboplatin regarding TILs status. Methods: The clinical data were obtained from 132 patients diagnosed with TNBC (ER&amp;PgR &lt; 10%) and treated with either anthracycline-taxane NACT (n = 68) or сarboplatin-based chemotherapy (n = 64) from 2017 to 2022 at N.N. Petrov National Medicine Research Center of Oncology. TILs were evaluated in 132 formalin-fixed paraffin-embedded tumor tissue samples stained with H&amp;E at baseline biopsies according to guidelines from the International TILs Working Group. Classification of TILs into low ( &lt; 10%), intermediate (≤10% to &lt; 40%) and high (≥ 40%) levels was performed. Data are presented using the methods of descriptive statistics. The differences between groups were assessed using the Сhi-square test. P-values below 0.05 were considered statistically significant. Results: The proportion of patients who achieved a pCR was higher in the carboplatin group 66% than in patients without carboplatin 35% (OR 3,5; 95% CI 1,71-7,17; p = 0,0006). There were 25 (18.9%), 77 (58.3%) and 30 (22.7%) patients with low ( &lt; 10%), intermediate (≤10% to &lt; 40%) and high (≥ 40%) levels of TILs, respectively. In total, the tumors with high TILs had a pCR rate of 63.3%, compared with 46.1% for low TILs (OR 2,0; 95% CI 0,87-4,67; p = 0,097). No significant statistical difference in pCR rates was shown between carboplatin-based and standard NACT groups with high TILs - 71% and 54%, respectively (OR 2,06; 95% CI 0,45-9,30; p = 0,346). When analyzing a group with low TILs infiltration, the pCR was more frequent in the platinum-based 64% compared to 31% in the standard NACT group (OR 3,9; 95% CI 1,72-9,00; p = 0,0011). Conclusions: These data may support the de-escalation of carboplatin-based NACT in patients diagnosed with TNBC with high TILs infiltration. Further investigations are warranted to explore the predictive value of TILs in platinum-based NACT. [Table: see text]

Hope and Hype around Immunotherapy in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) holds a poor prognosis compared to other breast cancer subtypes, and the development of new effective treatment strategies is an unmet medical need. TNBC has traditionally been considered not amenable to treatment with targeted agents due to a lack of actionable targets. Therefore, chemotherapy has remained the mainstay of systemic treatment for many decades. The advent of immunotherapy raised very hopeful expectations in TNBC, possibly due to higher levels of tumor-infiltrating lymphocytes, PD-L1 expression and tumor mutational burden compared to other breast cancer subtypes, that predict an effective anti-tumor immune-engagement. The results of clinical trials testing immunotherapy in TNBC led to the approval of the combination of immune checkpoint inhibitors and chemotherapy in both early and advanced settings. However, some open questions about the use of immunotherapy in TNBC still exist. These include a deeper understanding of the heterogeneity of the disease, identification of reliable predictive biomarkers of response, determination of the most appropriate chemotherapy backbone and appropriate management of potential long-term immune-related adverse events. In this review we aim to examine the available evidence on the use of immunotherapy strategies in both early and advanced TNBC, to critically discuss some of the limitations encountered in clinical research and to summarize data on novel promising immunotherapeutic strategies beyond PD-(L)1 blockade that have been investigated in the most recent trials.

An increase in tumor-infiltrating lymphocytes after treatment is significantly associated with a poor response to neoadjuvant endocrine therapy for estrogen receptor-positive/HER2-negative breast cancers

BackgroundThe reason for the poor prognosis of estrogen receptor (ER) + /human epidermal growth factor receptor 2 (HER2)− breast cancer patients with high levels of tumor-infiltrating lymphocytes (TILs) is poorly understood. The association between TILs and response to neoadjuvant endocrine therapy (NET) was examined.MethodsWe recruited 170 patients with ER + /HER2− breast cancer who were treated with preoperative endocrine monotherapy. TILs were evaluated before and after NET, and their changes were noted. Furthermore, T cell subtypes were examined using CD8 and FOXP3 immunohistochemical analyses. Neutrophil and lymphocyte counts in the peripheral blood were analyzed with reference to TIL levels or changes. Responders were defined as Ki67 expression levels ≤ 2.7% after treatment.ResultsPost-treatment (p = 0.016), but not pre-treatment (p = 0.464), TIL levels were significantly associated with the response to NET. TIL levels increased significantly after treatment among non-responders (p = 0.001). FOXP3 + T cell counts increased significantly after treatment in patients with increased TILs (p = 0.035), but not in those without increased TILs (p = 0.281). Neutrophil counts decreased significantly after treatment in patients without increased TILs (p = 0.026), but not in patients with increased TILs (p = 0.312).ConclusionAn increase in TILs after NET was significantly associated with a poor response to NET. Given that FOXP3 + T-cell counts increased, and neutrophil counts did not decrease in patients with increased TILs after NET, the induction of an immunosuppressive microenvironment was speculated to play a role in the inferior efficacy. These data might partially indicate the involvement of the immune response in the efficacy of endocrine therapy.

PD-L1 and HLA-class I expression status and their therapeutic implication in oesophageal small-cell carcinoma.

Oesophageal small-cell carcinoma is a rare and highly aggressive subtype of oesophageal cancer with a dismal prognosis. To explore the potential applicability of immunotherapy, we investigated the expression status of programmed death ligand 1 (PD-L1) and human leukocyte antigen (HLA)-class I and the degree of tumour-infiltrating lymphocytes (TILs) in oesophageal small-cell carcinoma. PD-L1 and HLA-class I expression levels were evaluated in 10 pure small-cell carcinomas and five mixed neuroendocrine-non-neuroendocrine neoplasms (MiNENs). The combined positive score (CPS) and tumour proportion score (TPS) were used for PD-L1 assessment. Immunohistochemistry for mismatch repair (MMR) proteins was also performed. PD-L1 immunohistochemistry demonstrated CPS ≥1 in nine (60%), CPS ≥10 in five (33%), and TPS ≥1 in five (33%) cases. Overall survival was significantly longer in patients with CPS ≥1 than in those with CPS <1. HLA-class I deficiency (>50% tumour cells) was noted in five cases (33%), with no significant correlation with PD-L1 expression status. Among the five MiNENs, HLA-class I expression was decreased in the small-cell carcinoma component of three cases. HLA-class I deficiency was significantly associated with higher TNM stage and reduced TIL levels. MMR deficiency was not observed in any case. Given that a significant subset (40%) exhibited PD-L1 CPS ≥1 with preserved HLA-class I expression and high levels of TIL, the PD-1/PD-L1 pathway is a potential therapeutic target for oesophageal small-cell carcinoma.

Abstract 4638: Integrative spatial analysis of paired IHC and H&amp;E images identifies Foxp3 enriched tumor-infiltrating lymphocytes associated with disease-free survival in human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma

Abstract Background: Recent studies support that high levels of tumor-infiltrating lymphocytes (TILs) were associated with better prognosis in HPV(+) oropharyngeal squamous cell carcinoma (OPSCC). While these studies provide a potential role for TILs as a prognostic biomarker, the analyses were relied on manual quantification performed by pathologists, resulting in inter-observer variability. Deep Learning (DL)-based whole slide hematoxylin and eosin (H&amp;E) image analyses may overcome these challenges. However, this analysis only provides the presence of cell types without consideration for the functional roles of each cell within the tumor immune microenvironment (TME). In this work, we develop a computational pipeline to integrate paired H&amp;E and immunohistochemistry (IHC) images to functionally characterize TILs and investigate their prognostic utility. Methods: We analyzed 88 patients: 42 stage I, 39 stage II, and 7 stage III. Our data contain both H&amp;E and IHCs examining FoxP3, CD3, PD-L1, CD20, etc. on serial sections of the tissue. In-house DL-based H&amp;E analysis used to identify TILs, tumors, and stroma in each tumor, then performed registration between adjacent H&amp;E and IHC images from the same tissue. The patients were then classified into three basic immune phenotypes: immune inflamed (IN; high TILs in the tumor region), immune excluded (EX; TILs are mostly localized in stroma), and immune desert (ID; few/no TILs) based on TIL enrichment in the TME. To functionally characterize TILs, we quantified protein expression from the adjacent IHCs. For example, we further classified each patient into different subtypes based on enriched protein expression (e.g., FoxP3 high IN, FoxP3 low IN). We used the Kaplan-Meier method and multivariate Cox analysis to evaluate the prognostic value of different subtypes enriched with different proteins to predict disease-free survival (DFS). Results: The IN group with 43 patients was significantly associated with good prognosis. Interestingly, further stratification of the IN subgroup based on Foxp3 quantification on TIL regions (i.e., high FoxP3 IN and low FoxP3 IN) showed that high protein expression of FoxP3 in TILs in the IN subgroup is significantly associated with a better prognosis compared to other immune subgroups (HR, 0.16; p-value, 0.003). Multivariate analysis, including other clinical covariates showed that the immune subtypes associated with high FoxP3 are independently associated with DFS. These results demonstrate that DL-based integrative IHC and H&amp;E image analysis could be used to identify subgroups with distinct clinical outcomes. Furthermore, our results reveal unknown roles for Foxp3 expression in the TILs in HPV(+) OPSCC as a prognostic biomarker, a finding which should be evaluated in a larger cohort. Citation Format: Sumanth Reddy Nakkireddy, Inyeop Jang, Minji Kim, Linda X. Yin, Michael Rivera, Joaquin J. Garcia MD, Kathleen R. Bartemes, David M. Routman, Eric J. Moore, Daniel J. Ma, Kathryn M. Van Abel, Tae Hyun Hwang. Integrative spatial analysis of paired IHC and H&amp;E images identifies Foxp3 enriched tumor-infiltrating lymphocytes associated with disease-free survival in human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4638.