BACKGROUND: The expansion of tumor-infiltrating lymphocytes (TIL) for adoptive cellular therapy is under investigation in many solid tumors of adulthood. Marrow-infiltrating lymphocytes (MIL) have demonstrated antitumor reactivity preclinically. Successful expansion of TIL/MIL has not been reported across pediatric solid tumor histologies. The objective of this study was to demonstrate successful expansion of TIL from pediatric solid tumors for translation in an adoptive cell therapy (ACT) treatment strategy.
METHODS: A prospective study of TIL/MIL expansion was performed on solid tumors of pediatric patients undergoing standard-of-care procedures. TIL/MIL expansions were performed in the presence of high-dose interleukin 2. To demonstrate a full-scale expansion to clinically-relevant cell doses for TIL therapy, initial TIL culture was followed by a rapid expansion protocol for select patients. Expanded specimens were analyzed for phenotype by flow cytometry and for anti-tumor reactivity by the interferon-gamma release assay.
RESULTS: Eighteen tumor samples were obtained. Initial TIL cultures were successfully generated from 14/18 samples (77.7%). A median of 5.52 × 107 (range: 2.5 × 106-3.23 × 108) cells were produced from initial cultures, with 46.9% expressing a CD3 phenotype (46.9%). Eight samples underwent rapid expansion, demonstrating a median 458-fold expansion and a CD3 phenotype of 98%. Initial MIL cultures were successfully generated from five samples, with a predominantly CD3 phenotype (45.2%). Sufficient tumor tissue was only available for seven TIL samples to be tested for reactivity; none demonstrated responsiveness to autologous tumor.
CONCLUSIONS: TIL and MIL expansion from pediatric solid tumors was successful, including the full-scale expansion process. This data supports translation to an ACT-TIL treatment strategy in the pediatric population and thus a Phase I trial of ACT-TIL in pediatric high-risk solid tumors is planned.

OBJECTIVE: To investigate the correlation between programmed death ligand-1 (PD-L1) expression and tumor-infiltrating lymphocytes (TILs) and evaluate the prognostic value of PD-L1 and TILs in Chinese triple-negative breast cancer (TNBC) patients with different molecular subtype METHODS: This retrospective study was conducted at 2020. Specifically, the pre-chemotherapy clinical data and non-stained tissue blocks of 465 TNBC patients visited the Fudan University Shanghai Cancer Center (FUSCC) between 2008 and 2014 were collected, with their blocks sliced and stained using PD-L1(SP142), and the outcome of subsequent chemotherapy obtained in 2020. The relapse-free survival (RFS) of the study population was calculated. The baseline PD-L1 expression status correlations with TILs and molecular subtypes were assessed using Spearman\'s rank correlation analysis and the Kruskal-Wallis test. Kaplan-Meier survival analyses were undertaken to evaluate the prognosis value of TILs and PD-L1 expression.
RESULTS: PD-L1 expression status on IC was moderately and positively correlated with stromal tumor-infiltrating lymphocytes (sTILs) (rs = 0.502, P <0.001) and iTILs (rs = 0.410, P < 0.001), respectively. PD-L1 expression status and TILs showed significant differences among molecular subtypes (P < 0.001), with the highest proportion of PD-L1+ and high TILs patients observed in the immunomodulatory (IM) subtype. TILs were significantly associated with RFS. Moreover, sTILs could act as an independent predictor of RFS (RR 0.953, 95 % CI 0.920 ∼ 0.987, P = 0.007), while PD-L1 expression status did not show the same prognostic significance.
CONCLUSIONS: The incorporation of pre-treatment TILs and PD-L1 expression status as valuable tools for optimizing patient selection for immunotherapy and managing the risks associated with chemotherapy in Chinese TNBC patients.
METHODS: The data sets generated and analyzed during the current study are available from the corresponding author.

T cell immunoglobulin and mucin domain-3 (TIM-3), a crucial immune checkpoint following PD1 and CTLA4, is widely found in several immune cells. Nonetheless, its performance in recent clinical trials appears disappointing. Ovarian cancer (OC), a malignant tumor with a high mortality rate in gynecology, faces significant hurdles in immunotherapy. The broad presence of TIM-3 offers a new opportunity for immunotherapy in OC. This study reviews the role of TIM-3 in OC and assesses its potential as a target for immunotherapy. The regulatory effects of TIM-3 on the immune microenvironment in OC are discussed, with a focus on preclinical studies that demonstrate TIM-3\'s modulation of various immune cells in OC. Additionally, the potential therapeutic advantages and challenges of targeting TIM-3 in OC are examined.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown remarkable results in melanoma, but only modest clinical benefits in other cancers, even after TIL have been genetically modified to improve their tumor homing, cytotoxic potential or overcome cell exhaustion. The required ex vivo TIL expansion process may induce changes in the T cell clonal composition, which could likely compromise the tumor reactivity of TIL preparations and ultimately the success of TIL therapy. A promising approach based on the production of bispecific T cell-engagers (TCE) by engineered T cells (STAb-T therapy) improves the efficacy of current T cell redirection strategies against tumor-associated antigens in hematological tumors. We studied the TCRβ repertoire in non-small cell lung cancer (NSCLC) tumors and in ex vivo expanded TIL from two unrelated patients. We generated TIL secreting anti-epidermal growth factor receptor (EGFR) × anti-CD3 TCE (TILSTAb) and tested their antitumor efficacy in vitro and in vivo using a NSCLC patient-derived xenograft (PDX) model in which tumor fragments and TIL from the same patient were transplanted into hIL-2 NOG mice. We confirmed that the standard TIL expansion protocol promotes the loss of tumor-dominant T cell clones and the overgrowth of virus-reactive TCR clonotypes that were marginally detectable in primary tumors. We demonstrated the antitumor activity of TILSTAb both in vitro and in vivo when administered intratumorally and systemically in an autologous immune-humanized PDX EGFR+ NSCLC mouse model, where tumor regression was mediated by TCE-redirected CD4+ TIL bearing non-tumor dominant clonotypes.

Lung cancer continues to contribute to the highest percentage of cancer-related deaths worldwide. Advancements in the treatment of non-small cell lung cancer like immune checkpoint inhibitors have dramatically improved survival and long-term disease response, even in curative and perioperative settings. Unfortunately, resistance develops either as an initial response to treatment or more commonly as a progression after the initial response. Several modalities have been utilized to combat this. This review will focus on the various combination treatments with immune checkpoint inhibitors including the addition of chemotherapy, various immunotherapies, radiation, antibody-drug conjugates, bispecific antibodies, neoantigen vaccines, and tumor-infiltrating lymphocytes. We discuss the status of these agents when used in combination with immune checkpoint inhibitors with an emphasis on lung cancer. The early toxicity signals, tolerability, and feasibility of implementation are also reviewed. We conclude with a discussion of the next steps in treatment.

Neoadjuvant chemotherapy (NAC) is now the standard of care for patients with locally advanced breast cancer (BC). TIL scoring is prognostic and adds predictive value to the residual cancer burden evaluation after NAC. However, NAC induces changes in the tumor, and the reliability of TIL scoring in post-NAC samples has not yet been studied. H&E- and dual CD3/CD20 chromogenic IHC-stained tissues were scored for stromal and intra-tumoral TIL by two experienced pathologists on pre- and post-treatment BC tissues. Digital TIL scoring was performed using the HALO® image analysis software (version 2.2). In patients with residual disease, we show a good inter-pathologist correlation for stromal TIL on H&E-stained tissues (CCC value 0.73). A good correlation for scoring with both staining methods (CCC 0.81) and the digital TIL scoring (CCC 0.77) was also observed. Overall concordance for TIL scoring in patients with a complete response was however poor. This study reveals there is good reliability for TIL scoring in patients with detectable residual tumors after NAC treatment, which is comparable to the scoring of untreated breast cancer patients. Based on the good consistency observed with digital TIL scoring, the development of a validated algorithm in the future might be advantageous.

This study used artificial intelligence (AI)-based analysis to investigate the immune microenvironment in endometrial cancer (EC). We aimed to evaluate the potential of AI-based immune metrics as prognostic biomarkers. In total, 296 cases with EC were classified into 4 molecular subtypes: polymerase epsilon ultramutated (POLEmut), mismatch repair deficiency (MMRd), p53 abnormal (p53abn), and no specific molecular profile (NSMP). AI-based methods were used to evaluate the following immune metrics: total tumor-infiltrating lymphocytes (TIL), intratumoral TIL, stromal TIL, and tumor cells using Lunit SCOPE IO, as well as CD4+, CD8+, and FOXP3+ T cells using immunohistochemistry (IHC) by QuPath. These 7 immune metrics were used to perform unsupervised clustering. PD-L1 22C3 IHC expression was also evaluated. Clustering analysis demonstrated 3 distinct immune microenvironment groups: immune active, immune desert, and tumor dominant. The immune-active group was highly prevalent in POLEmut, and it was also seen in other molecular subtypes. Although the immune-desert group was more frequent in NSMP and p53mut, it was also detected in MMRd and POLEmut. POLEmut showed the highest levels of CD4+ and CD8+ T cells, total TIL, intratumoral TIL, and stromal TIL with the lowest levels of FOXP3+/CD8+ ratio. In contrast, p53abn in the immune-active group showed higher FOXP3+/CD4+ and FOXP3+/CD8+ ratios. The immune-active group was associated with favorable overall survival and recurrence-free survival. In the NSMP subtype, a significant association was observed between immune active and better recurrence-free survival. The PD-L1 22C3 combined positive score (CPS) showed significant differences among the 3 groups, with the immune-active group having the highest median CPS and frequency of CPS ≥ 1%. The immune microenvironment of EC was variable within molecular subtypes. Within the same immune microenvironment group, significant differences in immune metrics and T cell composition were observed according to molecular subtype. AI-based immune microenvironment groups served as prognostic markers in ECs, with the immune-active group associated with favorable outcomes.

Immune checkpoint inhibitors (ICIs) exert clinical efficacy against various types of cancers by reinvigorating exhausted CD8+ T cells that can expand and directly attack cancer cells (cancer-specific T cells) among tumor-infiltrating lymphocytes (TILs). Although some reports have identified somatic mutations in TILs, their effect on antitumor immunity remains unclear. In this study, we successfully established 18 cancer-specific T cell clones, which have an exhaustion phenotype, from the TILs of four patients with melanoma. We conducted whole-genome sequencing for these T cell clones and identified various somatic mutations in them with high clonality. Among the somatic mutations, an SH2D2A loss-of-function frameshift mutation and TNFAIP3 deletion could activate T cell effector functions in vitro. Furthermore, we generated CD8+ T cell-specific Tnfaip3 knockout mice and showed that Tnfaip3 function loss in CD8+ T cell increased antitumor immunity, leading to remarkable response to PD-1 blockade in vivo. In addition, we analyzed bulk CD3+ T cells from TILs in additional 12 patients and identified an SH2D2A mutation in one patient through amplicon sequencing. These findings suggest that somatic mutations in TILs can affect antitumor immunity and suggest unique biomarkers and therapeutic targets.

OBJECTIVE: Triple-negative breast cancer (TNBC) presents a clinical challenge as an aggressive tumor, correlated with unfavorable prognosis. Tumor-infiltrating lymphocytes (TILs) have garnered interest as a potential prognostic biomarker. However, the disparity in outcomes between varying TILs rates remains inadequately explored.
METHODS: PubMed, Scopus, Web of Science, and Cochrane databases were searched for studies about the prognostic value of TILs in patients with TNBC receiving neoadjuvant chemotherapy. The hazard ratios (HRs) or odds ratios (ORs) were computed for binary endpoints, with 95% confidence intervals (CIs).
RESULTS: Twenty-nine studies were included, involving a population of six thousand one hundred sixty-one (80.41%) with TNBC. The cut-off TILs value ranged from 10 to 60%, with 50% being the most related value. Compared with the low-TIL expression group, the disease-free survival (DFS) (HR 0.71; 95% CI 0.61-0.82; p < 0.00001) and overall survival (OS) (HR 0.76; 95% CI 0.63-0.90; p = 0.002) rates showed significant improvement with higher TIL infiltrations. In the subgroup analyses of the lymphocyte subtypes CD4 + and CD8 + , there was statistical significance favoring higher TILs rates in both subtypes, each associated with improved DFS (HR 0.48; 95% CI 0.33-0.71; p = 0.0002) and OS (HR 0.53; 95% CI 0.36-0.78; p = 0.001), regardless of which cell subtype was predominantly infiltrated. The complete pathological response analysis showed better rates for the higher TIL group than the control for both the TIL (OR 1.29; 95% CI 1.13-1.48; p = 0.0003) and Ki-67 (OR 2.74; 95% CI 2.01-3.73; p < 0.00001) analyses.
CONCLUSIONS: Higher expressions of TILs in patients with TNBC were associated with improved significantly DFS, OS, and pCR outcomes.

UNASSIGNED: Pancreatic cancer is the seventh leading cause of cancer-related deaths worldwide. Checkpoint immunotherapy has not yet shown encouraging results in pancreatic cancer possibly because of a poor immunogenicity and/or an immune suppressive microenvironment. The aim of this study was to develop patient-derived xenograft (PDX) models, compare their genetics to the original biopsies, and assess if autologous tumor-infiltrating lymphocytes (TILs) would have antitumoral activity in pancreatic cancer.
UNASSIGNED: We subcutaneously transplanted tumors from 29 patients into NOG mice to generate PDX models. We established TIL cultures and injected them into PDX mice. We analyzed histology and genetics of biopsies and PDX tumors.
UNASSIGNED: Tumor growths were confirmed in 11 of 29 transplantations. The PDX tumors histologically resembled their original biopsies, but because stromal cells in the PDX model tumors were from mouse, their gene expression differed from the original biopsies. Immune checkpoint ligands other than programmed death ligand-1 (PD-L1) were expressed in pancreatic cancers, but PD-L1 was rarely expressed. When it was expressed, it correlated with tumor take in PDX models. One of the 3 tumors that expressed PD-L1 was an adenosquamous cancer, and another had a mismatch repair deficiency. TILs were expanded from 6 tumors and were injected into NOG or human interleukin-2 transgenic-NOG mice carrying PDX tumors. Regression of tumors could be verified in human interleukin-2 transgenic-NOG mice in 3 of the 6 PDX models treated with autologous TILs, including the adenosquamous PDX model.
UNASSIGNED: PDX models of pancreatic cancer can be used to learn more about tumor characteristics and biomarkers and to evaluate responses to adoptive cell therapy and combination therapies. The major benefit of the model is that modifications of T cells can be tested in an autologous humanized mouse model to gain preclinical data to support the initiation of a clinical trial.