Assessment of stromal tumor-infiltrating lymphocytes in neoadjuvant chemotherapy for invasive breast carcinoma: Predictive insights across molecular subtypes.

BACKGROUND AND AIMS: Breast cancer, a leading cause of female mortality, has prompted the widespread adoption of Neoadjuvant chemotherapy (NAC) for its potential to minimize metastasis risk and downstaging tumors. Tumor Infiltrating Lymphocytes (TILs) have emerged as key immunological biomarkers, particularly in breast cancer research. This study focuses on evaluating Stromal TILs (sTILs) in pre-NAC core needle biopsies of Invasive Breast Carcinoma, No Special Type (IBC, NST) and correlating it with NAC response. MATERIALS AND METHODS: A retrospective study spanning three years (October 2020 to September 2023) was conducted in a tertiary care hospital, involving 73 patients meeting specific inclusion criteria. Pathological assessments, including hormone receptor status, molecular subtyping, and TILs evaluation, were performed. Logistic regression and statistical analyses were conducted to determine associations between TILs, clinicopathological parameters, and complete response. RESULTS: The study demonstrated excellent discriminatory power of TILs (>10 %) in predicting complete response. Univariate and multivariate logistic regression underscored the independent predictive value of TILs, emphasizing their significance across diverse molecular subtypes. CONCLUSION: This study provides crucial insights into immune response assessment, particularly sTILs, in optimizing breast cancer treatment strategies and patient outcomes during NAC, contributing to the evolving landscape of personalized emphasising oncology.

Longitudinal Assessment of Tumor-Infiltrating Lymphocytes in Primary Breast Cancer Following Neoadjuvant Radiation Therapy.

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have prognostic significance in several cancers, including breast cancer. Despite interest in combining radiation therapy with immunotherapy, little is known about the effect of radiation therapy itself on the tumor-immune microenvironment, including TILs. Here, we interrogated longitudinal dynamics of TILs and systemic lymphocytes in patient samples taken before, during, and after neoadjuvant radiation therapy (NART) from PRADA and Neo-RT breast clinical trials. METHODS AND MATERIALS: We manually scored stromal TILs (sTILs) from longitudinal tumor samples using standardized guidelines as well as deep learning-based scores at cell-level (cTIL) and cell- and tissue-level combination analyses (SuperTIL). In parallel, we interrogated absolute lymphocyte counts from routine blood tests at corresponding time points during treatment. Exploratory analyses studied the relationship between TILs and pathologic complete response (pCR) and long-term outcomes. RESULTS: Patients receiving NART experienced a significant and uniform decrease in sTILs that did not recover at the time of surgery (P < .0001). This lymphodepletive effect was also mirrored in peripheral blood. Our SuperTIL deep learning score showed good concordance with manual sTILs and importantly performed comparably to manual scores in predicting pCR from diagnostic biopsies. The analysis suggested an association between baseline sTILs and pCR, as well as sTILs at surgery and relapse, in patients receiving NART. CONCLUSIONS: This study provides novel insights into TIL dynamics in the context of NART in breast cancer and demonstrates the potential for artificial intelligence to assist routine pathology. We have identified trends that warrant further interrogation and have a bearing on future radioimmunotherapy trials.

A Deep Learning-Based Assessment Pipeline for Intraepithelial and Stromal Tumor-Infiltrating Lymphocytes in High-Grade Serous Ovarian Carcinoma.

Tumor-infiltrating lymphocytes (TILs) are associated with improved survival in patients with epithelial ovarian cancer. However, TIL evaluation has not been used in routine clinical practice because of reproducibility issues. The current study developed two convolutional neural network models to detect TILs and to determine their spatial location in whole slide images, and established a spatial assessment pipeline to objectively quantify intraepithelial and stromal TILs in patients with high-grade serous ovarian carcinoma. The predictions of the established models showed a significant positive correlation with the number of CD8+ T cells and immune gene expressions. Patients with a higher density of intraepithelial TILs had a significantly prolonged overall survival and progression-free survival in multiple cohorts. On the basis of the density of intraepithelial and stromal TILs, patients were classified into three immunophenotypes: immune inflamed, excluded, and desert. The immune-desert subgroup showed the worst prognosis. Gene expression analysis showed that the immune-desert subgroup had lower immune cytolytic activity and T-cell-inflamed gene-expression profile scores, whereas the immune-excluded subgroup had higher expression of interferon-γ and programmed death 1 receptor signaling pathway. The established evaluation method provided detailed and comprehensive quantification of intraepithelial and stromal TILs throughout hematoxylin and eosin-stained slides. It has potential for clinical application for personalized treatment of patients with ovarian cancer.

Fertility potential and safety assessment of residual ovarian cortex in young women diagnosed with epithelial borderline and early-stage malignant ovarian tumors.

OBJECTIVE: To establish the safety and quality of ovarian cortex surrounding epithelial ovarian tumors in women eligible for fertility-sparing surgery by identifying occult malignant lesions and characterizing the ovarian follicle pool. METHODS: Multicentric retrospective study of 48 subjects (15-45 years), diagnosed with borderline ovarian tumors (BOTs) or early-stage epithelial ovarian cancers (EOCs) and eligible for fertility-sparing surgery. Histological samples of ovarian cortex surrounding tumors were analyzed to characterize the follicle pool, find any occult malignant lesion using tumor-specific markers (cytokeratin 7 and mucin 1), and quantify tumor-infiltrating lymphocytes (TILs) by CD3 and tumor associated macrophages (TAMs) by CD68. RESULTS: Occult ovarian lesions were observed in 6 out of 45 cases investigated (14.6%), including one mucinous stage-I BOT (1/14), one serous stage-I BOT (1/13), 3 advanced-stage serous BOTs (3/11) and one early-stage serous EOC (1/7). Notably, follicle density was significantly lower in subjects diagnosed with ovarian tumors compared to controls (p < 0.001) and at a younger age. Significantly higher follicle atresia was encountered in the ovarian tumor group then in controls (20.1 ± 8.8% vs 9.2 ± 9.4%, p < 0.001) at all ages. Both TILs and TAMs were found in ovarian tumors irrespective of histotype, but no link was established with the status of the ovarian reserve. CONCLUSIONS: Personalized counseling for fertility preservation is required in the event of BOTs and early-stage EOCs. Fertility-sparing surgery and adjuvant gamete preservation should be considered, balancing the oncological risks according to tumor stage and histotype and fertility potential, especially at a younger age.

Cost-effectiveness of treating advanced melanoma with tumor-infiltrating lymphocytes based on an international randomized phase 3 clinical trial.

INTRODUCTION: In a multicenter, open-label randomized phase 3 clinical trial conducted in the Netherlands and Denmark, treatment with ex vivo-expanded tumor-infiltrating lymphocytes (TIL-NKI/CCIT) from autologous melanoma tumor compared with ipilimumab improved progression-free survival in patients with unresectable stage IIIC-IV melanoma after failure of first-line or second-line treatment. Based on this trial, we conducted a cost-utility analysis. METHODS: A Markov decision model was constructed to estimate expected costs (expressed in 2021€) and outcomes (quality-adjusted life years (QALYs)) of TIL-NKI/CCIT versus ipilimumab in the Netherlands. The Danish setting was assessed in a scenario analysis. A modified societal perspective was applied over a lifetime horizon. TIL-NKI/CCIT production costs were estimated via activity-based costing. Through sensitivity analyses, uncertainties and their impact on the incremental cost-effectiveness ratio (ICER) were assessed. RESULTS: Mean total undiscounted lifetime benefits were 4.47 life years (LYs) and 3.52 QALYs for TIL-NKI/CCIT and 3.33 LYs and 2.46 QALYs for ipilimumab. Total lifetime undiscounted costs in the Netherlands were €347,168 for TIL-NKI/CCIT (including €67,547 for production costs) compared with €433,634 for ipilimumab. Undiscounted lifetime cost in the Danish scenario were €337,309 and €436,135, respectively. This resulted in a dominant situation for TIL-NKI/CCIT compared with ipilimumab in both countries, meaning incremental QALYs were gained at lower costs. Survival probabilities, and utility in progressive disease affected the ICER most. CONCLUSION: Based on the data of a randomized phase 3 trial, treatment with TIL-NKI/CCIT in patients with unresectable stage IIIC-IV melanoma is cost-effective and cost-saving, both in the current Dutch and Danish setting. These findings led to inclusion of TIL-NKI/CCIT as insured care and treatment guidelines. Publicly funded development of the TIL-NKI/CCIT cell therapy shows realistic promise to further explore development of effective personalized treatment while warranting economic sustainability of healthcare systems.

The assessment of tumor-infiltrating lymphocytes in invasive apocrine carcinoma of the breast in relation to the HER2 status.

In the current study, we assessed the prevalence and molecular features of HER2-low phenotype in the apocrine carcinomas of the breast (ApoCa) and its relationship with tumor-infiltrating lymphocytes (TILs). A cohort of 64 well-characterized therapy-naïve ApoCa was used. The TIL distribution was assessed using the hematoxylin and eosin whole slide/scanned images following the international TILs working group recommendations. Next-generation sequencing (NGS) was performed in a subset of HER2-low ApoCa. All patients were women, with a mean age of 62 years. Forty-three carcinomas were pure apocrine carcinoma (PAC; ER-/AR+), and the remaining 21 were classified as apocrine-like carcinomas (ALCs; ER+/-, AR+/-). HER2/neu was positive (score 3+ by IHC and/or amplified by FISH) in 20/43 (47%) PAC and 4/21 (19%) ALC. The prevalence of HER2-low expression (scores 1+ or 2+ without HER2 amplification) in ApoCa was 39% without significant differences between PAC and ALC (P = 0.14); however, the HER2-low phenotype was more prevalent in triple-negative PAC than in ALC (P < 0.001). Levels of TILs were low (≤10%) in 74% of ApoCa (median 5%, range 0%-50%). TIL levels were significantly higher in ALC than in PAC (P = 0.02). HER2 status had no impact on TIL distribution (P = 0.45). The genomic profile of HER2-low ApoCa was similar to other subtypes of ApoCa. ApoCa has predominantly low TIL, particularly PAC. The prevalence of the HER2-low phenotype in ApoCa is high, which should have therapeutic and clinical implications given the recently approved therapies with antibody-drug conjugates (ADCs) for HER2-low breast cancers.

Systematic assessment of tumor necrosis at baseline in cervical cancer - An independent factor associated with poor outcome.

Cervical cancer (CC) is a leading challenge in oncology worldwide, with high prevalence and mortality rates in young adults, most prominent in low to middle-income countries with marginal screening facilities. From the prospectively collected BioRAIDS (NCT02428842) cohort of primary squamous CC conducted in 7 European countries, a central pathology review was carried out on 294 patients' tumors. The focus was on identification of tumor-stromal characteristics such as CD8+, CD45+, CD68+ staining cells, PD-L1 expression, tumor infiltrating lymphocytes (TILs) together with the degree of tumor necrosis. Both (FIGO-2018) stage (I-II/III-IV) as well as tumor necrosis were highly significantly associated with Progression-free Survival (PFS); with tumor necrosis scoring as most potent independent factor in a multivariable analysis (p < 0.001). Tumor necrosis can be assessed in the very first diagnostic biopsyand our data suggest that this rapid, simple and cost-effective biomarker, should be routinely assessed prior to treatment decisions.

Cost-effectiveness analysis of tumor-infiltrating lymphocytes biomarkers guiding chemotherapy de-escalation in early triple-negative breast cancer.

BACKGROUND: To accelerate the clinical translation of tumor-infiltrating lymphocytes (TILs) biomarkers for guiding chemotherapy de-escalation in early-stage triple-negative breast cancer (TNBC), cost-effectiveness evidence is essential but has not been investigated. We intend to evaluate the cost-effectiveness of using TILs to guiding chemotherapy de-escalation in patients with early-stage TNBC from the perspective of the Chinese health service system. METHODS: The hybrid decision-tree-Markov model was designed to compare the cost-effectiveness of cytotoxic chemotherapy guided by whether TILs assay was performed in 50-year-old female patients with early-stage TNBC over a lifetime horizon. In Strategy (1), if TILs testing was performed, patients with TILs values exceeding 30% could be spared from chemotherapy. In Strategy (2), where no TILs testing was performed, all patients were administered chemotherapy following China's clinical practices. Based on the algorithm built by Guyot, the individual patient data were reconstructed from the published Kaplan-Meier curves, and the survival functions were calculated by parametric methods. Cost estimates were valued in Chinese yuan (as per rates in 2022). RESULTS: In 50-year-old female patients with early-stage TNBC, Strategy (1), which employs TILs testing to guide cytotoxic chemotherapy yielded an additional 0.47 quality-adjusted life years (QALYs) and saved 40,976 yuan, with an incremental cost-effectiveness ratio (ICER) of -87,182.98 yuan per QALY gained compared with Strategy (2). This indicates that compared with Strategy (2), Strategy (1) is the dominant scheme. The results were sensitive to utility parameters, discount rates, and treatment costs after relapse. At a willingness-to-pay threshold of 85,700 yuan (based on GDP per capita) per QALY, the probability of TILs being cost-effective was almost 100%. CONCLUSIONS: The application of biomarkers (TILs) to guide decisions for chemotherapy de-escalation is a cost-effective strategy for early-stage TNBC patients and deserves to be widely promoted in clinical practice.

Automated Assessment of the Area of Infiltration by CD8+ Cells in Gastric Carcinoma and Areas of Normal Mucosa as a Significant Prognostic Factor.

In 139 patients with verified gastric cancer, the infiltration of the postoperative material with CD8+ cells was analyzed. Automated morphometric analysis of immunostained slides was performed separately in different specimen sites (tumor center, invasive edge, and peritumoral mucosa). The mean area of infiltrating CD8+ cells in the tumor center and in the invasive edge was not predictive, while in the peritumoral mucosa it provided a new negative predictive factor (hazard ratio 2.10; confidence interval 0.87-4.92, Cox regression) reliably associated with the TNM stage (hazard ratio 1.91; confidence interval 0.91-4.61, Cox regression).

Pitfalls in machine learning-based assessment of tumor-infiltrating lymphocytes in breast cancer: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer

The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Assessment of Intratumoural and Stromal Infiltrating Lymphocytes In The Various Subtypes of Breast Carcinoma Patients who have Received Neoadjuvant Chemotherapy.

BACKGROUND: Breast cancer comprises a highly heterogeneous subset of tumours that respond well to Neoadjuvant Chemotherapy (NAC). Tumour Infiltrating Lymphocytes (TIL) act as a means to an end by shedding light on the treatment response as well as predictive factors to the clinicopathological features for the same. Therefore, this article attempts to shift the attention to the relevance of TIL in the aforementioned aspects by bringing to notice the contrasting traits displayed by them in the different immunohistochemical subtypes of breast carcinoma. MATERIALS AND METHODS: 75 triple-negative breast cancer (TNBC) patients, 25 human epidermal growth factor receptor (HER2BC) positive patients and 77 hormone receptor (HRBC) positive breast cancer patients were included in this study who received NAC before surgical excision of the tumour which was then stained using routine Haematoxylin and Eosin techniques. Standardised guidelines were used to evaluate TIL in the stroma and the tumour. RESULTS: In TNBC, a significant association between Intratumoural (IT) TIL (p=0.0288) and Intrastromal (IS) TIL (p=0.0250) with pathological complete response (pCR). IS TIL and age at operation (p=0.0494) showed significant values but no correlation was found with IT TIL. In HER2BC, IS TIL revealed a significant association with the tumour response(p=0.0229). A strong association was found between IT TIL and the age of menopause(p=0.0441). In HRBC, no significant associations were found between IT and IS TIL scores and the clinicopathological features. CONCLUSION: The predictive factors of TIL and complete response post-neoadjuvant chemotherapy can be a strong indicative factor for immunohistochemical markers. It also helps throw light on further studies which can be carried out to determine the clinicopathological features and TIL correlation in the various subtypes of breast carcinoma.

Patient-derived melanoma organoid models facilitate the assessment of immunotherapies.

BACKGROUND: Only a minority of melanoma patients experience durable responses to immunotherapies due to inter- and intra-tumoral heterogeneity in melanoma. As a result, there is a pressing need for suitable preclinical models to investigate resistance mechanisms and enhance treatment efficacy. METHODS: Here, we report two different methods for generating melanoma patient-derived organoids (MPDOs), one is embedded in collagen gel, and the other is inlaid in Matrigel. MPDOs in Matrigel are used for assessing the therapeutic effects of anti-PD-1 antibodies (αPD-1), autochthonous tumor infiltrating lymphocytes (TILs), and small molecule compounds. MPDOs in collagen gel are used for evaluating the chemotaxis and migratory capacity of TILs. FINDING: The MPDOs in collagen gel and Matrigel have similar morphology and immune cell composition to their parental melanoma tissues. MPDOs show inter- and intra-tumoral heterogeneity and contain diverse immune cells such as CD4+, CD8+ T, Treg, CD14+ monocytic, CD15+, and CD11b+ myeloid cells. The tumor microenvironment (TME) in MPDOs is highly immunosuppressive, and the lymphoid and myeloid lineages express similar levels of PD-1, PD-L1, and CTLA-4 as their parental melanoma tissues. Anti-PD-1 antibodies (αPD-1) reinvigorate CD8+ T cells and induce melanoma cell death in the MPDOs. TILs expanded by IL-2 and αPD-1 show significantly lower expression of TIM-3, better migratory capacity and infiltration of autochthonous MPDOs, and more effective killing of melanoma cells than TILs expanded by IL-2 alone or IL-2 with αCD3. A small molecule screen discovers that Navitoclax increases the cytotoxicity of TIL therapy. INTERPRETATION: MPDOs may be used to test immune checkpoint inhibitors and cellular and targeted therapies. FUNDING: This work was supported by the NIH grants CA114046, CA261608, CA258113, and the Tara Miller Melanoma Foundation.

Acral Melanoma Is Infiltrated with cDC1s and Functional Exhausted CD8 T Cells Similar to the Cutaneous Melanoma of Sun-Exposed Skin.

Acral melanoma (AM) is the most common melanoma in non-Caucasian populations, yet it remains largely understudied. As AM lacks the UV-radiation mutational signatures that characterize other cutaneous melanomas, it is considered devoid of immunogenicity and is rarely included in clinical trials assessing novel immunotherapeutic regimes aiming to recover the antitumor function of immune cells. We studied a Mexican cohort of melanoma patients from the Mexican Institute of Social Security (IMSS) (n = 38) and found an overrepresentation of AM (73.9%). We developed a multiparametric immunofluorescence technique coupled with a machine learning image analysis to evaluate the presence of conventional type 1 dendritic cells (cDC1) and CD8 T cells in the stroma of melanoma, two of the most relevant immune cell types for antitumor responses. We observed that both cell types infiltrate AM at similar and even higher levels than other cutaneous melanomas. Both melanoma types harbored programmed cell death protein 1 (PD-1+) CD8 T cells and PD-1 ligand (PD-L1+) cDC1s. Despite this, CD8 T cells appeared to preserve their effector function and expanding capacity as they expressed interferon-γ (IFN-γ) and KI-67. The density of cDC1s and CD8 T cells significantly decreased in advanced stage III and IV melanomas, supporting these cells' capacity to control tumor progression. These data also argue that AM could respond to anti-PD-1-PD-L1 immunotherapy.

Clinical significance of overall assessment of tumor-infiltrating lymphocytes in oropharyngeal cancer: A meta-analysis.

BACKGROUND: The overall assessment of tumor-infiltrating lymphocytes (TILs) evaluated using hematoxylin and eosin (HE) staining has been recently studied in oropharyngeal squamous cell carcinoma (OPSCC). METHODS: We conducted a systematic review of Scopus, Ovid Medline, PubMed, Web of Science, and Cochrane Library to retrieve studies assessing TILs in HE-stained sections of OPSCC. We used fixed-effect models and random-effect models to estimate the pooled hazard ratios (HRs) and confidence intervals (CIs) for disease-free survival (DFS), overall survival (OS) and disease-specific survival (DSS). RESULTS: Eleven studies were identified that had analyzed the prognostic significance of TILs in OPSCC using HE-stained specimens. Our meta-analyses showed that a high infiltration of TILs was significantly associated with improved DFS (HR 0.39, 95%CI 0.24-0.65, P = 0.0003), OS (HR 0.38, 95%CI 0.29-0.50, P < 0.0001), and DSS (HR 0.32, 95%CI 0.19-0.53, P < 0.0001). CONCLUSION: Findings of our meta-analysis support a growing body of evidence indicating that assessment of TILs in OPSCC using HE-stained sections has reliable prognostic value. The clinical significance of such assessment of TILs has been reported repeatedly in many studies on OPSCC. The assessment is cost-effective, feasible, easy to transfer from lab to clinic, and therefore can be incorporated in daily practice.

Regression in cutaneous melanoma: histological assessment, immune mechanisms and clinical implications.

Tumour regression is an immunologically driven process that results in complete or partial disappearance of tumour cells. This can be observed in histological sections as replacement of tumour cells with fibrosis, angiogenesis, and a variable inflammatory infiltrate. In primary cutaneous melanoma, the prognostic significance of regression has been debated for decades, in part because inconsistent histological criteria are used in prognostication studies. It is broadly accepted that CD8+ T lymphocytes are the primary effectors of the anti-tumour response, but the interplay between melanoma and the immune system is complex, dynamic, and incompletely understood. Sustained progress in unravelling the pathogenesis of melanoma regression has led to the identification of therapeutic targets, culminating in the development of immune checkpoint inhibitors for the management of advanced disease. Modern techniques allow for high-resolution spatial analyses of the tumour microenvironment. Such studies may lead to better understanding of the immune drivers of melanoma regression, thereby facilitating the search for new prognostic and predictive biomarkers to assist clinical decision-making.

Association of Machine Learning-Based Assessment of Tumor-Infiltrating Lymphocytes on Standard Histologic Images With Outcomes of Immunotherapy in Patients With NSCLC.

Importance: Currently, predictive biomarkers for response to immune checkpoint inhibitor (ICI) therapy in lung cancer are limited. Identifying such biomarkers would be useful to refine patient selection and guide precision therapy. Objective: To develop a machine-learning (ML)-based tumor-infiltrating lymphocytes (TILs) scoring approach, and to evaluate TIL association with clinical outcomes in patients with advanced non-small cell lung cancer (NSCLC). Design, Setting, and Participants: This multicenter retrospective discovery-validation cohort study included 685 ICI-treated patients with NSCLC with median follow-up of 38.1 and 43.3 months for the discovery (n = 446) and validation (n = 239) cohorts, respectively. Patients were treated between February 2014 and September 2021. We developed an ML automated method to count tumor, stroma, and TIL cells in whole-slide hematoxylin-eosin-stained images of NSCLC tumors. Tumor mutational burden (TMB) and programmed death ligand-1 (PD-L1) expression were assessed separately, and clinical response to ICI therapy was determined by medical record review. Data analysis was performed from June 2021 to April 2022. Exposures: All patients received anti-PD-(L)1 monotherapy. Main Outcomes and Measures: Objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were determined by blinded medical record review. The area under curve (AUC) of TIL levels, TMB, and PD-L1 in predicting ICI response were calculated using ORR. Results: Overall, there were 248 (56%) women in the discovery cohort and 97 (41%) in the validation cohort. In a multivariable analysis, high TIL level (≥250 cells/mm2) was independently associated with ICI response in both the discovery (PFS: HR, 0.71; P = .006; OS: HR, 0.74; P = .03) and validation (PFS: HR = 0.80; P = .01; OS: HR = 0.75; P = .001) cohorts. Survival benefit was seen in both first- and subsequent-line ICI treatments in patients with NSCLC. In the discovery cohort, the combined models of TILs/PD-L1 or TMB/PD-L1 had additional specificity in differentiating ICI responders compared with PD-L1 alone. In the PD-L1 negative (<1%) subgroup, TIL levels had superior classification accuracy for ICI response (AUC = 0.77) compared with TMB (AUC = 0.65). Conclusions and Relevance: In these cohorts, TIL levels were robustly and independently associated with response to ICI treatment. Patient TIL assessment is relatively easily incorporated into the workflow of pathology laboratories at minimal additional cost, and may enhance precision therapy.

Assessment of radiographic and prognostic characteristics of programmed death-ligand 1 expression in high-grade gliomas.

PURPOSE: Gliomas are characterized by immunosuppressive features. Programmed death-ligand 1 (PD-L1) is overexpressed and plays an important role in the immunosuppressive tumor microenvironments of gliomas. However, the radiographical and prognostic significance of PD-L1 expression remains unclear. METHODS: Using tissue microarrays, we evaluated PD-L1 expression and the presence of tumor-infiltrating CD4+ and CD8+T cells and CD204+macrophages using immunohistochemical analysis. Contrast enhancement area and fluid-attenuated inversion recovery (FLAIR) hyperintensity area were evaluated by two-dimensional analysis. Kaplan-Meier analysis was performed to evaluate the overall survival time in 44 patients with isocitrate dehydrogenase (IDH)-wildtype glioblastoma. RESULTS: We evaluated 71 patients with newly diagnosed high-grade gliomas who were treated between October 1998 and April 2012. PD-L1 expression was observed in 15 patients (21.1%). A significant association of PD-L1 expression with the CD4+ and CD8+ T cell densities, but not with CD204+ macrophage densities, was observed (p = 0.025, p = 0.0098, and p = 0.19, respectively). The FLAIR-to-enhancement ratio was significantly higher in PD-L1+ tumors than in PD-L1- tumors (p = 0.0037). PD-L1 expression did not show a significant association with the median survival time (PD-L1 + vs. PD-L1-: 19.2 vs 14.9 months; p = 0.39). CONCLUSION: PD-L1 expression was associated with CD4+ and CD8+ T cell infiltration, indicating a significant interplay between PD-L1 and immune cells. The positive correlation of PD-L1 expression with an increased FLAIR-to-enhancement ratio suggested that radiographical characteristics could reflect the immunological status. Our results did not support the prognostic impact of PD-L1 in patients with IDH-wildtype glioblastomas.

Objective assessment of tumor infiltrating lymphocytes as a prognostic marker in melanoma using machine learning algorithms.

BACKGROUND: The prognostic value of tumor-infiltrating lymphocytes (TILs) assessed by machine learning algorithms in melanoma patients has been previously demonstrated but has not been widely adopted in the clinic. We evaluated the prognostic value of objective automated electronic TILs (eTILs) quantification to define a subset of melanoma patients with a low risk of relapse after surgical treatment. METHODS: We analyzed data for 785 patients from 5 independent cohorts from multiple institutions to validate our previous finding that automated TIL score is prognostic in clinically-localized primary melanoma patients. Using serial tissue sections of the Yale TMA-76 melanoma cohort, both immunofluorescence and Hematoxylin-and-Eosin (H&E) staining were performed to understand the molecular characteristics of each TIL phenotype and their associations with survival outcomes. FINDINGS: Five previously-described TIL variables were each significantly associated with overall survival (p<0.0001). Assessing the receiver operating characteristic (ROC) curves by comparing the clinical impact of two models suggests that etTILs (electronic total TILs) (AUC: 0.793, specificity: 0.627, sensitivity: 0.938) outperformed eTILs (AUC: 0.77, specificity: 0.51, sensitivity: 0.938). We also found that the specific molecular subtype of cells representing TILs includes predominantly cells that are CD3+ and CD8+ or CD4+ T cells. INTERPRETATION: eTIL% and etTILs scores are robust prognostic markers in patients with primary melanoma and may identify a subgroup of stage II patients at high risk of recurrence who may benefit from adjuvant therapy. We also show the molecular correlates behind these scores. Our data support the need for prospective testing of this algorithm in a clinical trial. FUNDING: This work was also supported by a sponsored research agreements from Navigate Biopharma and NextCure and by grants from the NIH including the Yale SPORE in in Skin Cancer, P50 CA121974, the Yale SPORE in Lung Cancer, P50 CA196530, NYU SPORE in Skin Cancer P50CA225450 and the Yale Cancer Center Support Grant, P30CA016359.

Evaluation of PD-L1 (E1L3N, 22C3) expression in venous tumor thrombus is superior to its assessment in renal tumor in predicting overall survival in renal cell carcinoma.

BACKROUND: Renal cell carcinoma (RCC) frequently invades renal vein forming neoplastic thrombus. The expression of immune checkpoint receptors in RCC was addressed in multiple studies, but little is known about the expression and prognostic significance of programmed death ligand-1 in tumor thrombus. MATERIAL AND METHODS: The study aimed to evaluate the expression of PD-L1 within venous tumor thrombus and primary tumor using 2 independent antibody clones (22c3 and E1L3N) and to assess its value in predicting overall survival (OS) in the subgroup of patients with RCC and renal vein thrombus. RESULTS: Eighty-two patients with RCC and venous tumor thrombus that underwent nephrectomy were enrolled. The expression of PD-L1 was assessed utilizing tissue microarrays separately on tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs). The frequency of PD-L1 expression on TCs and TILs varied between tumor and thrombus compartments and was dependent on the antibody clone used. The expression of PD-L1 on TCs and/or TILs was associated with worse OS irrespectively of the antibody and the analyzed compartment. Nevertheless, the best prognostic performance was noted for the combined assessment of PD-L1 expression on TCs and TILs in venous tumor thrombus with the use of 22c3 antibody. The multivariable Cox regression model predicting OS incorporated PD-L1 22c3 in venous tumor thrombus (hazards ratio [HR] = 3.64, 95% confidence interval [CI] = 1.63-8.14, P = 0.002) and nodal status (HR = 2.88, 95% CI = 1.18-7.03, P = 0.02). CONCLUSIONS: PD-L1 may become a valuable tool for prognostic purposes in this specific subgroup of patients and be incorporated into the respective models qualifying for adjuvant treatment.