Tumour infiltrating lymphocytes (TILs) are a key component of the tumour microenvironment. To establish a clinically relevant TILs cut-off for patients with oesophago-gastric (OG) cancer, it is essential to know whether TILs density varies by patient and/or disease characteristics. TILs were quantified as TILs/mm2 (TILs density) by a deep-learning algorithm applied to digitised Haematoxylin/Eosin (H&E)-stained biopsies and resection specimens from 4628 patients from nine phase III trials. 4533 patients with TILs density and matched clinicopathological data were included in the final analyses. Associations between TILs density, disease stage, geographical region (UK versus Asia), sex, age, and treatment were analysed. Median TILs density was higher in pre-treatment biopsies from patients with early-stage versus late-stage disease (962 vs 479 TILs/mm2, p < 0.001). Within the same geographical region and disease stage, TILs density was similar across different chemotherapy regimens. In UK-led trials of early-stage disease, post-chemotherapy resections showed higher TILs density than chemotherapy-naïve resections (618 vs 571 TILs/mm2, p = 0.003). TILs density was higher in Asian tumours compared to UK tumours (1419 vs 571 TILs/mm2, p < 0.001). No significant associations were observed with age or sex. This is the largest study to date evaluating TILs density in OG cancer. TILs density varied with stage and geographical region but not by age or sex. These findings may explain enhanced response to immunotherapy observed in published studies of patients with early-stage disease and highlight the need to account for baseline TILs heterogeneity when interpreting TILs as a possible biomarker in future studies.

Precision treatment of advanced non-small cell lung carcinoma (NSCLC) requires early biomarker availability, yet routine practice is constrained by small biopsies, sequential testing, and limited resources. We assessed whether PD-L1 and EGFR testing were completed from the initial biopsy using a tissue-efficient reflex workflow integrating histologic subtyping, PD-L1 scoring, EGFR analysis, and tumour-infiltrating lymphocyte (TIL) assessment. In this prospective single-arm feasibility study, 50 stage III or IV NSCLC cases were evaluated. Tumours were subtyped using morphology and minimal immunohistochemistry (TTF-1, Napsin A, p40). PD-L1 immunohistochemistry was performed using a locally validated CAL10-based laboratory-developed test and scored by tumour proportion score (TPS) and combined positive score (CPS). Reflex EGFR testing was attempted in adenocarcinomas with adequate tissue. TILs were graded morphologically. Tissue adequacy and biomarker associations were analysed. Squamous cell carcinoma comprised 56% and adenocarcinoma 44% of cases. Adequate tissue to complete PD-L1 and EGFR testing was available in 59% (13/22) of adenocarcinomas, and was reported within 30 days. EGFR mutations were detected in 31% of tested adenocarcinomas and were associated with TPS <50% and absent TILs. High PD-L1 expression (TPS ≥50%) was observed in 14% of tumours, exclusively in EGFR wild-type cases. TILs were present in 12% of cases and were significantly associated with higher CPS ( P = 0.002), exceeding TPS. A biopsy-level reflex workflow enables feasible, tissue-efficient concurrent molecular and immune profiling in advanced NSCLC. Integration of TPS, CPS, TIL assessment, and EGFR testing within a reflex workflow demonstrates the feasibility of coordinated immuno-molecular profiling from limited biopsy tissue in advanced NSCLC.

Targeting intratumoral heterogeneity in pancreatic cancer with sequential TIL infusions.

LONP1, a mitochondrial ATP-dependent protease, plays a crucial role in mitochondrial homeostasis by regulating protein turnover and mitophagy. Recent studies have highlighted its upregulation in various cancers, including colorectal cancer (CRC). This study investigates the expression of LONP1 in colon adenocarcinoma (COAD) and its correlation with mitophagy-related proteins and immune infiltration markers. Using publicly available databases and immunohistochemical analysis of 50 COAD patient samples, we confirmed that LONP1 expression is significantly elevated in COAD compared with normal tissue. High LONP1 levels were associated with tumor progression, TP53 mutation status, and poor prognosis. Correlation analyses revealed that LONP1 is closely linked to mitochondrial dynamics, mitophagy regulators (PINK1, AMBRA1, FUNDC1), and metabolic reprogramming. Additionally, LONP1 expression positively correlated with tumor-infiltrating lymphocytes, particularly CD8+ T cells, suggesting a potential role in immune evasion. Immunohistochemical analysis distinguished two patterns: high LONP1/low TOMM20 expression associated with aggressive tumors and low LONP1/high TOMM20 expression linked to better outcomes and stronger immune infiltration. These findings suggest that LONP1 contributes to tumor progression through mitochondrial regulation and immune modulation, highlighting its potential as both a prognostic biomarker and a therapeutic target in COAD.

The increasing global burden of cancer necessitates innovative therapeutic strategies. Cell therapy represents a major breakthrough in oncology, evolving rapidly from the successful application of chimeric antigen receptor T (CAR-T) cells in hematologic malignancies to a multiplatform landscape characterized by the concurrent development of diverse strategies. Current research focuses on T cell receptor-engineered T (TCR-T) cells, tumor-infiltrating lymphocytes (TILs), gamma delta (γδ) T cells, CAR-natural killer (CAR-NK) cells, CAR-macrophages (CAR-Ms), and various strategies based on dendritic cells (DCs), B cells, and stem cells. The translational paradigm is expanding from the relatively mature field of hematologic malignancies to the more prevalent and mechanistically complex domain of solid tumors. In recent years, this field has exhibited a clear trend toward expansion from autologous therapies to allogeneic "off-the-shelf" platforms. Approaches such as CAR-NK and CAR-natural killer T (CAR-NKT) cell therapies exhibit significant clinical potential because of their low immunogenicity and reduced risk of graft-versus-host disease (GvHD). Concurrently, in vivo engineering technologies that directly deliver CAR genes in situ are emerging as promising approaches to lower costs and simplify manufacturing by bypassing complex ex vivo procedures. This review systematically outlines recent advances in these strategies, focusing on their mechanisms of action, target antigens, and clinical translation. Despite progress, formidable challenges remain, including tumor heterogeneity, the immunosuppressive tumor microenvironment (TME), and therapy-related toxicity. To address these challenges, future research will focus on novel target discovery, enhanced toxicity management, and scalable manufacturing processes. The integration of multidisciplinary technologies, such as multiomics analysis, artificial intelligence, and synthetic biology, will advance cell therapies toward safer, more effective, and widely accessible applications.

Autophagy meets tissue-resident memory: Emerging crosstalk and therapeutic opportunities in cutaneous melanoma.

Clinical relevance of the characterisation and quantification of CD8+ T-cell infiltration in high-grade serous ovarian carcinoma using machine learning-based image analysis of dual immunostaining.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway represents a central driver of innate immune activation, and manganese ion (Mn2+) has recently been identified as a potent modulator of this signaling axis. However, the application of Mn2+ is limited by its rapid clearance, nonspecific distribution and potential neurotoxicity. Inspired by the unique chemical structure and biological functions of adenosine triphosphate (ATP), we herein propose an ATP-Mn coordination nanoparticle (ATP-Mn CNP) to fuel cGAS-STING activation and antitumor immunity. We demonstrated that the phosphate groups of ATP could coordinate with Mn2+ and form stable, well-defined nanoparticles after lipid coating. ATP-Mn CNP significantly increased the expression of cGAS-STING-associated genes and activated the corresponding signaling cascades, and thus effectively polarized macrophages from tumor-supportive M2 to antitumor M1 phenotype. In vivo antitumor studies indicated that ATP-Mn CNP treatment significantly suppressed tumor growth, and reprogramed macrophages in tumors and draining lymph nodes, which thus facilitated the tumor infiltration of cytotoxic lymphocytes. Combination of ATP-Mn CNP with immune checkpoint inhibitors achieved 37.5% tumor eradication in MC38 murine models, and significantly prolonged mice survival. This study establishes an ATP-fueled coordination strategy that harnesses ATP as both an assembly ligand and an immune stimulator to enhance Mn-mediated STING activation.

The tumor microenvironment (TME) plays a pivotal role in immuno-oncology, influencing cancer progression and treatment outcomes. Among its components, stromal tumor-infiltrating lymphocytes (sTILs) represent important prognostic and predictive biomarkers in breast cancer. However, visual assessment of sTILs is complex, time-consuming, and subject to inter-observer variability. To address these challenges, we propose an AI-based framework for sTILs quantification in hematoxylin and eosin (H&E)-stained breast cancer histopathological images across different molecular subtypes, in accordance with the recommendations of the International Immuno-Oncology Biomarker Working Group (IIOBWG). The framework integrates two parallel and explainable segmentation models trained on multi-center data: an attention-based model for TILs detection and a stroma segmentation model designed to identify both tumoral and infiltrated stromal regions as a unified clinically relevant compartment. The resulting segmentation outputs are combined through a scanner resolution-aware quantification algorithm, enabling robust sTILs scoring across data acquired from different scanners and institutions. The proposed framework was externally validated on two independent private datasets comprising 60 and 112 whole-slide images with expert-annotated Region Of Interests (ROIs). Strong agreement with expert pathologist scoring was achieved (Pearson correlation up to 0.73; Concordance Correlation Coefficient (CCC) up to 0.72), and the framework consistently outperformed state-of-the-art approaches when evaluated on the same data. These results demonstrate that the proposed framework provides a reliable, efficient, and scalable solution for standardized sTILs quantification, supporting its potential utility in both clinical practice and translational research.

Our research objective is to construct a prognostic model of tumor-infiltrating lymphocytes related genes (TILRGs) for predicting the survival of cases with diffuse large B-cell lymphoma (DLBCL). Based on the clinical data of DLBCL patients in GEO database, the TILRGs that were significantly associated with survival prognosis were screened. Cox regression analysis and LASSO analysis were used to construct prognostic risk models for TILRGs. Kaplan-Meier (K-M) curve and ROC curve were used to assess the predictive performance of the model. CIBERSORT algorithm and GDSC database were applied to quantify tumor immune infiltration and chemotherapy drugs. The levels of key genes were detected via qRT-PCR. Additionally, K-M curves were plotted to analyze the relationship between LCP2 expression and patient prognosis. A prognostic risk model consisting of 20 TILRGs was constructed. This model has high accuracy in predicting the survival of DLBCL patients, and reveals significant differences in tumor immune infiltration and chemotherapy drugs between two groups. Low expression of LCP2 in DLBCL patients is obviously related to serum LDH, IPI score, stage and poor prognosis. Further analysis revealed that LCP2 overexpression inhibited the growth of DLBCL cells. In the current study, an accurate and reliable prognostic risk model was constructed based on 20 TILRGs. Low expression of LCP2 indicates poor survival of DLBCL cases and is a potential prognostic indicator.

The addition of MEK inhibition to PD-L1 blockade improves progression-free survival (PFS) in patients with advanced biliary tract cancer (BTC). While MEK inhibitors may increase tumor cell immunogenicity, they can impair T-cell priming/effector function, limiting combination efficacy. We hypothesized that the addition of a CD27 agonist could restore T-cell function and enhance anti-tumor immunity in this combination. We conducted a randomized, phase 2 trial evaluating atezolizumab (840mg IV days 1,15) in combination with the CD27 co-stimulatory mAb (CDX-1127/varlilumab [3mg/kg IV days 1, 15]), with/without the addition of a MEK inhibitor (cobimetinib [60mg oral daily days 1-21, off 22-28]) in unresectable BTC following at least 1 metastatic therapy. Overall response rate (ORR) and PFS were co-primary endpoints. Treatment-related changs in CD8+ tumor infiltrating lymphocytes (TIL) was the primary correlative outcome. The trial was closed early following interim preplanned ORR analysis. At closure, 57 patients had been enrolled (n=29[CAV],n=28[AV]. A majority (67%) had intrahepatic cholangiocarcinoma and 32% were immunotherapy-experienced. Both regimens were well-tolerated without new safety signals. Objective responses were rare (0%[CAV],3.8%[AV]). Median PFS was 2.40(CAV) and 1.84(AV) months (HR 0.67,95%CI[0.38,1.18]). Among immunotherapy-experienced patients, mPFS was 3.62(CAV) and 1.84(AV) months (HR 0.54,95%CI:[0.18-1.62]). Treatment with CAV increased intratumoral CD8+ T-cell density compared with treatment with AV. The combinations of atezolizumab and varlilumab with/without cobimetinib were safe but neither meaningfully improved outcomes in BTC treated in the later lines. Correlative tissue studies validated preclinical work that MEK inhibition increases CD8+TILs.

Hepatocellular carcinoma (HCC) shows heterogeneous responses to treatment with immune checkpoint inhibitors, and predictive biomarkers for dual immune checkpoint blockade remain insufficiently defined. The STRIDE regimen, combining tremelimumab and durvalumab, improves the survival of patients with unresectable HCC, but a substantial proportion of patients experience early progression. We investigated whether pretreatment intratumoral CD8+ tumor-infiltrating lymphocyte (TIL) density was associated with treatment response and progression-free survival (PFS) in patients treated with STRIDE. This retrospective, single-center study included 26 consecutive patients with unresectable HCC who received STRIDE and underwent pretreatment liver tumor biopsy. CD8+ TIL density was assessed using immunohistochemistry. Tumors were classified as CD8-high or CD8-low using a prespecified cutoff (15.9 cells/high-power field). Fifteen patients were classified as CD8-high and 11 as CD8-low. The objective response rate was significantly higher in the CD8-high group than in the CD8-low group (60.0% vs. 9.1%, p=0.005). The median PFS was significantly longer in the CD8-high group [8.8 months, 95% confidence interval (CI)=3.0-24.5] than in the CD8-low group (2.0 months, 95%CI=0.8-7.2; p=0.019). The frequency of grade ≥2 immune-related adverse events did not differ significantly between groups. Pretreatment intratumoral CD8+ TIL density was associated with an improved response and a longer PFS in STRIDE-treated HCC. Baseline tumor immune contexture may serve as a practical predictive biomarker for STRIDE. These findings are exploratory and hypothesis-generating, and require validation in larger prospective studies.

VG161 is an oncolytic HSV-1 with an ICP34.5 deletion and armed with multiple immunomodulatory factors. The absence of ICP34.5 restricts viral replication in neurons while conferring tumor specificity. However, this modification attenuates viral replication in tumor cells compared to wild-type HSV-1. Radiotherapy (RT) has been reported to promote viral replication and exert an immune-priming effect. Based on this, we hypothesize that RT could potentiate both the VG161 replication and its antitumor efficacy in breast cancer (BC). Our findings suggest that the optimal regimen for combining VG161 with RT involves administering 5Gy irradiation 6h after VG161 infection, which ensures that RT maximally promotes VG161 replication in BC. This enhancement in VG161 replication is mediated by the upregulation of GADD34 and HVEM induced by RT. Moreover, RT augments the expression of immunostimulatory transgenes carried by VG161 and immunogenic cell death of BC cells. In vivo, VG161 combined with RT demonstrates superior antitumor efficacy compared with either monotherapy in BC. Mechanistic investigations reveal that this combination therapy increases the abundances of tumor-infiltrating lymphocytes and elicits potent systemic antitumor immunity that inhibits local tumors and regresses abscopal metastases. The observed synergistic effect of VG161 and RT encourages further clinical translation.

Tailored HER2 ECD mRNA-LNP vaccines boost CD8+ T cell-mediated antitumor immunity for HER2-positive tumor suppression.

Treatment strategies for patients with advanced melanoma resistant to adoptive cell therapy with tumor-infiltrating lymphocytes (TILs) remain undefined. The efficacy of immune checkpoint inhibitors (ICIs) after TIL has not been previously described. Patients with unresectable melanoma who received ICI therapy both before and after TIL treatment were retrospectively identified at 14 international centers. Safety was evaluated among all patients that received one dose of ICI therapy after TIL. Objective response rates (ORR) were determined based on investigator-assessed Response Evaluation Criteria in Solid Tumors (RECIST) V.1.1 responses based on radiology reports. Kaplan-Meier methodology was used to estimate overall survival (OS) and melanoma-specific survival. Among 133 patients assessed for response to ICI therapy after progression on TIL therapy, the ORR was 11% (n=14; 95% CI 5.9% to 17%), including 3 complete responses (2.3%) and 11 partial responses (8.3%). Among patients treated with a programmed death-1 inhibitor prior to TIL (n=121), the ORR was 8.3% (95% CI 4.0% to 15%). At a median follow-up among survivors of 21 months, median OS from post-TIL ICI was 8.8 months (95% CI 6.5 to 12 months). Among 14 patients with response to post-TIL ICI, 13 received an ICI they had not previously received. In the safety cohort (n=138), post-TIL ICI therapy-related toxicity occurred in 31 patients(22%), among which 7 patients (5.1%) experienced recurrence of an adverse event experienced with prior ICI treatment. Efficacy with ICI after TIL was limited, with no evidence of synergistic toxicity. As TIL therapy becomes more widely available, these data highlight the need for novel therapies in this setting and support the inclusion of this population in future clinical trials.

Pathologic complete response after neoadjuvant treatment is considered a surrogate of cure in triple-negative breast cancer, yet around 10% of patients still relapse. Whether baseline stromal tumor-infiltrating lymphocytes can stratify this residual risk is unknown. Here, we report on GAMBIT, a multicentric real-world retrospective study of 2457 patients with triple-negative breast cancer or estrogen receptor-low disease, HER2-negative, of whom 1192 obtained a pathological complete response and 690 have evaluable tumor infiltrating lymphocytes. Among patients with pathological complete response,clinical nodal status and tumor infiltrating lymphocytes are independently prognosticand patients withclinical node-positive/low-tumor infiltrating lymphocytestumors experience substantially worse outcomes, with five-year distant relapse-free survival of 83.4% and overall survival of 85.8%. In this high-risk subgroup, five-year cumulative incidence of central nervous system reaches 7.5%, including 6.9%presenting as isolated central nervous system relapse. In this work, we identify a high-risk subgroup despite pathologic complete response and provide a framework supporting risk-adapted trial design incorporating central nervous system-directed strategies.

PROGNOSTIC and PREDICTIVE VALUE of HRD in EARLY TRIPLE NEGATIVE BREAST CANCER (TNBC).

Current advances in the treatment of melanoma brain metastases through immune checkpoint blockade and targeted therapy has significantly improved outcomes and patient survival. Yet, half of all patients continue to die from brain metastases despite use of these therapies and the addition of localized therapies like surgery and stereotactic radiosurgery. Novel treatments are needed to improve the morbidity and mortality associated with melanoma brain metastases. We have identified the inflammatory myddosomal pathway as a novel potential target of therapy in melanoma brain metastases, with gross upregulation in both tumor tissue and tumor-infiltrating immune cells. These cells display upregulation in MYD88, IRAK-1, IRAK-4, and downstream mediators of inflammatory activation at both the gene and protein level. We have previously shown the ability of the oral IRAK-4 inhibitor emavusertib (CA-4948) to penetrate the blood brain barrier and inhibit the myddosomal pathway in metastatic melanoma and primary CNS lymphoma. In this study we further show the capacity for IRAK-4 inhibition with emavusertib to improve the therapeutic efficacy of anti-PD-1 immune checkpoint blockade in melanoma brain metastases. Emavusertib in combination with anti-PD-1 therapy results in improved tumor-infiltrating lymphocyte recruitment, decreased myeloid derived suppressor cell function, and upregulation of interferon γ signaling resulting in improved survival in aggressive mouse models of melanoma brain metastases. This work supports the development of combination strategies of emavusertib with immune checkpoint blockade in melanoma brain metastases.

Pathologic tumor response and changes in the tumor microenvironment (TME) predict outcomes to immune checkpoint inhibitors, but are understudied in rare tumors. We investigated whether artificial intelligence (AI)-powered analyses of pretreatment and on-treatment biopsies may inform treatment outcomes to pembrolizumab. We evaluated 256 baseline and 248 on-treatment biopsies from 84 patients with rare tumors (10 cohorts) in a phase II pembrolizumab trial. Intratumoral tumor-infiltrating lymphocyte (iTIL) density and tumor content (TC) were assessed on H&E-stained slides using a deep learning-based analyzer (Lunit SCOPE IO). Baseline iTIL and changes in iTIL and TC were correlated with progression-free survival (PFS) and overall survival (OS). Multiplex immunofluorescence was performed in 27 paired samples to assess TME changes. In the high-iTIL tumor group, a baseline iTIL of ≥60 cells/mm2 was associated with favorable PFS (HR 0.49, 95% CI 0.25 to 0.99, p=0.046) and higher CD8+ and CD8+PD-1+ and lower FoxP3+CD8+PD-1+ T-cell density. However, this association with PFS was not seen in the overall cohort (HR 0.62, 95% CI 0.37 to 1.06, p=0.082). In paired biopsies, on-treatment increase in iTIL showed a trend toward improved PFS (HR 0.64, 95% CI 0.40 to 1.06, p=0.084) and was significantly associated with improved OS (HR 0.55, 95% CI 0.35 to 1.01, p=0.037). This increase was also associated with reduced spatial distance between CD8+ immune and tumor cells. Decreased TC during treatment was significantly associated with prolonged PFS and OS (PFS: HR 0.51, p=0.019; OS: HR 0.54, p=0.042). The combination of increased iTIL and decreased TC was significantly associated with better PFS (HR 0.36, p=0.009) and OS (HR 0.36, p=0.029). AI-powered assessment of the TME before and during treatment may help inform treatment outcomes to pembrolizumab in patients with rare tumors. NCT02721732.

Peptide-MHC-targeted engineered virus-like particles enable selective priming and gene editing of tumor-specific T cells.