Tumor-infiltrating Lymphocytes Research Articles

Deep learning–powered analysis of tumor-infiltrating lymphocytes (TILs) in colorectal cancer.

288 Background: TILs are a promising biomarker in various cancers, including colorectal cancer (CRC). Advances in deep learning enable objective and efficient TIL assessment. This study applies a novel deep learning model to quantify TIL density in a large cohort of CRC tumors, examining its relationship with clinicopathologic factors and survival. Methods: We applied a deep learning model (Deep-ICP) to H&E-stained images from CRC patients treated at Dana-Farber Cancer Center to quantify TIL density (immune cells/mm²). Kruskal-Wallis and Wilcoxon tests were used to compare categories. Overall survival (OS) was calculated from the date of diagnosis to the date of death or last follow-up. Cox proportional hazards models analyzed the association between TIL density tertiles (lower, middle, upper) and OS, adjusting for age, sex, stage, biopsy site, tumor mutational burden (TMB, mutations/megabase), and mismatch repair (MMR) status. Results: Of 1,477 tumor specimens tested, 1,039 (70%) were primary and 438 were metastatic. The most common sites of metastasis were liver (n=205), lung (n=86), and soft tissue (n=35). The median age at diagnosis was 56 (range 19–91) years and 706 were female (48%). Most tumors (1,214/1,300; 93%) were MMR proficient (MMRp); 86 were MMR deficient (MMRd). Median TIL density was 664 (interquartile range; 469–934) cells/mm² in primary tumors, 478 (358–685) cells/mm² in liver metastases, 415 (251–645) cells/mm2 in soft tissue (p<0.001). MMRd tumors had higher median TIL density than MMRp (881 vs. 620 cells/mm²; p<0.001). After adjusting for covariates, patients with highest TIL tertile had significantly longer OS than patients with lowest TIL tertile (median OS: 75.4 [95%CI 67.4-87] months; 100.5 [83.9-116] months and 99.1 [92.4-157] months for lower, middle and upper TIL tertiles respectively; Table). In the MMRp subset, higher TIL density was still predictive of improved OS (p adj=0.010). Conclusions: This study demonstrates the feasibility of deep learning powered TIL quantification in CRC, potentially informing differences in tumor microenvironment and prognostication. Future prospective studies are needed to validate the results and explore their therapeutic implications in personalized CRC management. Variable Hazard Ratio (95% CI) P-value TIL upper tertile (vs. lower) 0.70 (0.50-0.99) 0.04 TIL middle tertile (vs. lower) 1.04 (0.77-1.39) 0.80 Age (continuous) 1.02 (1.00-1.03) 0.008 Male (vs. female) 1.13 (0.86-1.47) 0.38 Stage II (vs. stage I) 4.8 (0.59-40) 0.14 Stage III (vs. stage I) 9.4 (1.20-73) 0.03 Stage IVA (vs. stage I) 23 (2.94-178) 0.003 Stage IVB (vs. stage I) 46 (5.6-369) 0.0003 Liver specimen (vs. primary) 1.39 (1.00-1.92) 0.050 Lung specimen (vs. primary) 0.78 (0.47-1.29) 0.33 Lymph node specimen (vs. primary) 0.77 (0.27-2.17) 0.62 Other metastasis (vs. primary) 1.23 (0.78-1.93) 0.37 TMB≥10 (vs. <10) 1.17 (0.82-1.68) 0.38 MMRd (vs. MMRp) 0.65 (0.29-1.46) 0.30

Tumor infiltrating lymphocytes (TILs) - Pathologia, quo vadis? - A global survey.

Tumor-infiltrating lymphocytes (TILs) and the tumor microenvironment have become increasingly important in cancer research, and immunotherapy has achieved major breakthroughs in improving patient outcomes. Despite the significant role of the pathologist in identifying, subtyping and reporting TILs, the implementation and assessment of TILs in pathology routine remains vague. To assess the actual use of TILs in routine clinical practice, a formal standardized questionnaire was disseminated on two social media platforms ("X" and LinkedIn) and by email in June 2024. Based on the results, we conducted a literature review on TILs via Medline/Pubmed in the two most scored and reported entities, namely malignant melanoma and colorectal cancer (CRC). 77 participants from 24 different countries around the world, mostly pathologists (n = 63, 82.0 %), completed the survey. More than half of the participants do not assess or report TILs in their daily (clinical) practice, a trend consistent across the countries included in the study. A variety of methods are used to report TILs, ranging from Artificial Intelligence (AI)-based scoring algorithms to quantification by eyeballing. Despite recognizing the importance of TIL assessment in clinical routine, many participants find it time-consuming and express a strong preference for AI-based quantification. Our survey reflects the perspective of mostly early career pathologists who recognize the importance of TILs in cancer but face challenges in implementation. The development of AI tools and consensus guidelines could alleviate these barriers. In addition, increasing the visibility and understanding of the role of pathologists within the medical community remains critical.

Association between muscle mass, visceral adiposity, and histologic tumor stromal features in colon cancer.

Sarcopenia and obesity are indicators for poor outcomes in colon cancer. Additionally, aggressive histopathologic tumor stromal features, such as a low tumor-stroma ratio (TSR) and low tumor-infiltrating lymphocytes (TILs) predict survival and treatment response. As their relationship remains underexplored, we studied the association between skeletal muscle mass, visceral adipose tissue (VAT), TSR, and TILs in patients with colon cancer. We studied 194 stage II/III colon carcinoma patients who underwent elective surgery. Preoperative computed tomography (CT) scans classified patients into four groups based on skeletal muscle index (normal/low) and visceral adipose tissue index (normal/high). Tumor tissues were assessed for TSR and TILs, and five-year disease recurrence and relative hazard were evaluated. Among the patients, 56 (28.9%) were classified as Normal Muscle, Normal VAT, 26 (13.4%) as Normal Muscle, High VAT, 75 (38.7%) as Low Muscle, Normal VAT, and 37 (19.1%) as Low Muscle, High VAT. Patients with low skeletal muscle mass were more often male (62.5% vs. 39%, P=0.005). Stroma-high tumors were less common in Low Muscle, Normal VAT patients (24%) compared to Normal Muscle, High VAT (50%), Low Muscle, High VAT (48.6%), and Normal Muscle, Normal VAT (41.1%) patients (P=0.020). Tumors with low TILs were similarly distributed across groups (P=0.679). Low Muscle, Normal VAT patients had a lower recurrence hazard compared to both Low Muscle, High VAT (hazard ratio [HR] 0.34, 95% CI 0.12-0.98, P=0.048) and Normal Muscle, Normal VAT (HR 0.31, 95% CI 0.11-0.87, P=0.027) patients. Low Muscle, Normal VAT colon cancer patients exhibited fewer aggressive tumor features and a lower recurrence risk compared to Low Muscle, High VAT patients. These findings highlight the importance of body composition in tumor biology and prognosis.

A combination treatment with a water extract from Euglena gracilis and anti-PD-1 antibody strongly inhibits growth of lung cancer in mice through stimulating tumor-infiltrating lymphocytes.

Here, we investigated the relationship between the attenuation of lung cancer growth due to oral administration of Euglena gracilis water extract (EWE) and T cell stimulation. Orally administered EWE was revealed to increase PD-1 and PD-L1 mRNA and proteins primarily in tumor-infiltrating lymphocytes (TILs), which was correlated with a significant decrease in the tumor weights in mice. A combination treatment with EWE and anti-PD-1 antibody significantly decreased the growth of murine lung tumors more than treatment with either alone by increasing the number of TILs and attenuating T cell exhaustion. Short-chain fatty acids, which were previously shown to be increased in intestines of mice treated with oral EWE, increased both PD-1 and PD-L1 expression in splenocytes, but not in lung cancer cells in cell culture. These results suggest there is a close relationship between the EWE-induced increase of short-chain fatty acids, the increase of PD-1 expression in TILs, and the attenuation of lung tumor growth. Furthermore, EWE enhances the efficacy of anti-PD-1 antibody-based immune checkpoint blockade therapy against non-small cell lung cancer.

Immunotherapy engagement in pancreatic adenocarcinoma: Provisional results of iLSTA study— Durvalumab, LSTA1 (certepetide), gemcitabine, and nab-paclitaxel for locally advanced pancreatic ductal adenocarcinoma.

746 Background: Pancreatic ductal adenocarcinoma (PDAC) is characterised by a dense, extracellular matrix-rich stroma, which creates a physical barrier against drug penetration. Evidence suggests that the iRGD peptide, LSTA-1, can increase the penetration of anti-cancer drugs to tumours through selective targeting of tumour endothelial cell receptors. Additionally, LSTA-1 administration promotes CD8+ immune cell tumour infiltration, potentially enabling immunotherapy as a treatment modality. iLSTA examined safety, tolerability and effect of the combination. Methods: Participants diagnosed with locally advanced PDAC were divided into 3 cohorts in a 1:1:4 ratio. Cohort 1 (n=5) received gemcitabine (1000mg/m 2 ), nab-paclitaxel (125mg/m 2 ), placebo LSTA-1 (3.2mg/kg) and placebo durvalumab (750mg). Cohort 2 (n=5) received gemcitabine, nab-paclitaxel, active and placebo LSTA-1, and placebo durvalumab. Cohort 3 (n=20) received gemcitabine, nab-paclitaxel, LSTA-1 and durvalumab. Tissue biopsies via endoscopic ultrasound were obtained pre-treatment and between weeks 12 and 16 for analysis of tumour infiltrating lymphocytes (TILs). Patient tumour sizes were assessed every 8 weeks using radiological imaging according to RECIST v1.1, and serum CA19-9 levels were analysed monthly. Results: 6/17 patients showed significant RECIST partial response after 2 cycles of treatment (5 patients in cohort 3), with the remaining 11 patients exhibiting stable disease. After 4 cycles of treatment, 10/17 patients demonstrated partial response (9 patients in cohort 3). Of the remaining 7 patients, 6 demonstrated stable disease, and 1 patient (cohort 2) exhibited a RECIST complete response. 13/17 patients who completed 4 treatment cycles showed a decrease in CA19-9 levels. 6 patients demonstrated >90% reduction in CA19-9 (5 in cohort 3), with the remaining 7 patients showing a >50% reduction in CA19-9 levels (5 in cohort 3). 12 patients underwent repeat biopsies 12-16 weeks post-treatment to assess TILs. 10 patients showed immune cell infiltration (5% to 50% stroma infiltration), 2 had no tumour found (Cohort 3). The combination was safe with no unexpected toxicities. Conclusions: The preliminary results of this study suggest that the combination of gemcitabine and nab-paclitaxel with LSTA-1 and durvalumab is safe and potentially induces tumour infiltrating lymphocytes and RECIST response in locally advanced pancreatic adenocarcinomas. Clinical trial information: ACTRN12623000223639 .

Tumor-infiltrating lymphocytes in HER2-positive breast cancer: potential impact and challenges.

In this review, we evaluate the role of stromal tumor-infiltrating lymphocytes (sTILs) as a biomarker in human epidermal growth factor receptor 2 (HER2)-positive breast cancer, exploring the prognostic and predictive potential in various treatment settings. Data from multiple clinical trials in the early and metastatic settings, focusing on TILs' correlation with pathologic complete response (pCR), progression-free survival (PFS), and overall survival across early and metastatic HER2-positive breast cancer were summarized. This review also discusses TILs' assessment methods, interobserver variability, and emerging technologies to assess TILs. TILs have been identified as a highly reproducible biomarker that predicts pCR in patients receiving neoadjuvant therapy and serves as a prognostic indicator for long-term outcomes in several breast cancer subtypes, including HER2-positive. Studies indicate that higher TIL levels correlate with better recurrence-free survival rates. Despite these findings, there is no consensus on the optimal TIL threshold for clinical decision making, and further research is required on how to incorporate TILs into routine clinical practice. TILs represent a promising biomarker in HER2-positive breast cancer, particularly in early disease settings. This assessment could guide treatment de-escalation or intensification, tailoring therapies to individual patient profiles. Due to their prognostic importance, TILs can be added to pathology reports. However, further validation in clinical trials is essential for the widespread adoption of TILs in clinical practice.

The inhibitory effects of nimotuzumab on CD276 expression and immune escape in head and neck squamous cell carcinoma: Insights into anticancer mechanisms.

CD276 has been identified as a novel immune checkpoint, and its overexpression is associated with immune evasion and poor prognosis in various tumors, including head and neck squamous cell carcinoma (HNSCC). Nimotuzumab, a humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody, has been approved for various solid tumors. However, it remains unclear whether its anticancer efficacy involves a reduction in CD276 expression. The purpose of this study was to investigate the regulatory effects and potential mechanisms of nimotuzumab on CD276 expression both in vitro and in vivo. In a coculture system, nimotuzumab showed inhibitory effects on TGF-β-induced upregulation of CD276 at both the transcriptional and protein levels in HNSCC cell lines. Mechanistic studies revealed that nimotuzumab primarily suppressed TGF-β-induced CD276 upregulation by blocking EGFR/MEK/ERK, which was further validated by MEK and ERK inhibitors. In xenograft and mice HNSCC models, nimotuzumab exerted antitumor effects accompanied by significantly reduced CD276 expression during tumor progression. Analysis of tumor-infiltrating lymphocytes (TILs) profiles indicated that nimotuzumab orchestrated the tumor immune microenvironment (TIME) by notably increasing the frequency of T lymphocytes, including cytotoxic T lymphocytes and helper T lymphocytes, as well as macrophage cells. However, no significant changes were observed in the populations of NK cells, DC cells, and neutrophils. These findings offer new insights into the anticancer mechanisms of nimotuzumab and its underlying synergy in combined treatments with immunotherapy for HNSCC.

A multimodal deep learning approach for the prognostic stratification of patients with colorectal cancer (CRC).

287 Background: Traditional prognostic systems like the AJCC TNM staging for colorectal cancer (CRC) often fall short in predicting long-term patient outcomes. These systems often rely on limited pathologic features, leading to generalized approaches to treatment despite diverse tumor heterogeneity, such as the complex interaction between the tumor-host microenvironment. There is therefore a pressing need for more accurate, scalable tools to enhance decision-making and predict outcomes for patients with CRC. Methods: Tumor sections from 191 CRC patients were comprehensively stained for CD8 to examine CD8-positive tumor-infiltrating lymphocytes (TILs). To account for tumor heterogeneity, multiple areas of the tumor were evaluated to include intertumoral and peritumoral areas. Slides were digitized and quantitatively analyzed using a cloud-based platform (SpatialX Diagnostics, Inc. USA). Additionally, clinicopathologic features including patient demographics, 8th edition AJCC staging, lymphovascular invasion (LVI), perineural invasion (PNI), mismatch repair (MMR) protein status, tumor location, presence/development of distant metastases, and patient follow-up were collected. Merging CD8 AI findings with clinicopathologic features, a complete data set was available for 175 patients. Cases were randomly assigned into 70% training and 30% validation. In the training set, unsupervised artificial intelligence (AI) models integrated clinicopathologic data and CD8-positive TIL counts using vision transformer models. The final model combined spatially resolved pathologic features with clinical variables to statistically predict overall survival (OS) and distant metastasis risk (DMR). Results: In the validation cohort, the model stratified patients into two groups based on a deep learning-derived risk score, with statistically significant differences between low-risk and high-risk groups for OS (p<0.02) and DMR prediction (p<0.0001), For OS prediction, the model selected tumor site, PNI, MMR status, epithelial and stromal CD8-positive TILs, and 5 deep vision features as the top 10 clinically significant features. For DMR prediction, the age, T- and N-stage, PNI, and 6 deep vision-derived features as key predictors. Conclusions: As captured by vision transformers, the distribution and spatial arrangement of CD8-positive TILs are critical factors in patient stratification for OS and DMR. AI-driven models like the one herein offer the potential for more personalized management strategies, advancing CRC management and improving patient outcomes beyond traditional prognostic staging systems and parameters.

GOBLET platform study: Preliminary safety and tumor response results for the relapsed anal carcinoma cohort in patients treated with pelareorep and atezolizumab.

6 Background: Patients with relapsed unresectable squamous cell carcinoma of the anal canal (SCCA) have limited treatment options after failure of standard first-line therapy. While checkpoint inhibitor monotherapy is possible, response rates are low (10-24%). Therapies that induce an inflammatory tumor microenvironment may improve the susceptibility of SCCA to immunotherapy. Pelareorep (pela) is a non-genetically modified, intravenously administered reovirus that stimulates the expansion of tumor-infiltrating lymphocyte (TIL) clones and simultaneously makes tumors visible to the immune system by upregulating interferon-induced gene expression. Pela-based combination therapies have demonstrated activity in metastatic breast and pancreatic cancer. In the GOBLET platform study, the efficacy of pela plus atezolizumab (atezo) in SCCA is being evaluated. Methods: GOBLET is a phase 1/2, open-label, non-randomized, Simon two-stage platform study in patients with advanced or metastatic GI cancers. SCCA patients in the trial are treated with pela plus atezo. Ten evaluable patients will be enrolled in Stage 1 and an additional 18 evaluable patients will be enrolled in Stage 2 if the prespecified Stage 1 success criterion is met (≥2 responses). The coprimary endpoints are tolerability of the treatment regimen and objective response rate (based on RECIST v1.1). Safety data are reviewed in an ongoing manner by an independent Data Safety Monitoring Board (DSMB). In addition, tumor and blood samples are being collected to assess TIL clonal expansion and other biomarkers. Results: Twelve patients are currently evaluable for tumor response in the SCCA cohort. Four out of 12 patients have achieved an objective response to therapy including 1 prolonged complete response (>15 months) and 3 partial responses- resulting in an ORR of 33.3%. Final ORR, progression-free survival, and overall survival results are pending. The DSMB has identified no safety signals and has recommended that enrollment continues without modification. TIL clonal expansion in the blood has been observed at treatment cycle 4 in all 3 responding patients for whom data are available. Conclusions: Twelve patients are currently evaluable for tumor response in the SCCA cohort. Four out of 12 patients have achieved an objective response to therapy including 1 prolonged complete response (>15 months) and 3 partial responses resulting in an ORR of 33.3%. Final ORR, progression-free survival, and overall survival results are pending. The DSMB has identified no safety signals and has recommended that enrollment continues without modification. TIL clonal expansion in the blood has been observed at treatment cycle 4 in all 3 responding patients for whom data are available. Clinical trial information: 2020-003996-16.

GOBLET study: Results of the safety run-in for first-line metastatic pancreatic ductal adenocarcinoma (PDAC) patients treated with pelareorep + modified FOLFIRINOX +/- atezolizumab.

730 Background: Immunotherapies are effective in only the small subset of metastatic PDAC patients with MSI-H or dMMR tumors. Pelareorep (pela) is a non-genetically modified, intravenously administered reovirus that selectively infects cancer cells. It stimulates the expansion of pre-existing tumor-infiltrating lymphocyte (TIL) clones, and it makes tumors visible to the immune system by upregulating interferon-induced gene expression including PD-L1, CXCL9, CXCL10, and CXCL11. PD-L1 upregulation also provides a basis for potential synergy between pela and immune checkpoint inhibitors. Pela combined with gemcitabine/nab-paclitaxel/atezolizumab showed promising tumor responses in mPDAC. Here, we are assessing the safety and preliminary efficacy of pela + mFOLFIRINOX +/- atezolizumab in a new cohort in the ongoing GOBLET study. Methods: GOBLET is a phase 1/2, open-label, Simon two-stage study in patients with advanced or metastatic GI cancers. In this new GOBLET cohort (Cohort 5), patients with newly diagnosed mPDAC are randomized to receive either pela + mFOLFIRINOX or pela + mFOLFIRINOX + atezolizumab. The primary endpoints are safety and objective response rate (ORR). In Stage 1, 15 evaluable patients will be enrolled in each arm. Both arms include a 3-6 patient safety run-in that must be successfully completed prior to opening the study to full enrollment. If pre-specified ORR success criteria are met, one or both arms may be advanced to Stage 2 during which 17 additional evaluable patients/per arm will be enrolled. Results: The 3-patient safety run-in for both arms of Cohorts 5 (6 patients total) have been enrolled, and all patients have completed the required 1-month evaluation period. Cohort 5 safety data have been reviewed by the independent Data Safety Monitoring Board (DSMB). The DSMB identified no safety signals attributable to the combination of pela and mFOLFIRINOX +/- atezolizumab and recommended that enrollment into these arms of Cohort 5 continue without modification. Reported adverse events are consistent with the known toxicities of the components of the treatment regimen. Tumor response results are pending. Conclusions: The results of the GOBLET Cohort 5 safety run-in indicate that pela can be given safely in combination with mFOLFIRINOX +/- atezolizumab to newly diagnosed mPDAC patients. Safety and efficacy of these combination therapies will continue to be monitored (Eudra-CT: 2020-003996-16). Clinical trial information: 2020-003996-16 .

The characterization of tumor immune microenvironment after neoadjuvant immunotherapy in head and neck squamous cell cancer using multiplex immunohistochemistry.

Optimizing clinical decision-making in head and neck squamous cell carcinoma (HNSCC) is challenging due to the ambiguous understanding of the immune cell dynamics and immune checkpoints regulation in the disease after the administration of neoadjuvant immunotherapy (NIT). HNSCC biopsy samples collected before and after the neoadjuvant treatment are classified into the pathologic response (PR) and the non-pathologic response (NPR) groups according to treatment responses and the expression of immune cells and checkpoints was labeled using multiplex immunohistochemistry (m-IHC). The populations of CD4+ T cells, CD8+ T cells, regulatory T cells (Treg), PD-1, and PD-L1 were particularly higher in the PR group than the NPR group in pre-treatment tissues, with the p-values of log-transformed positive cell density <0.05. Almost all markers showed a lower expression in the PR patients after treatment, resulting lower post/pre-treatment ratios of positive cell densities in the PR patients relative to the NPR patients. Following treatment, TIM3+ T cells and LAG3+ T cells exhibited significantly diminished levels in the PR cohort relative to the NPR cohort, with post/pre-treatment expression ratios showing significant differences (P<0.05). Tumor infiltration lymphocyte analysis revealed that the PR group exhibited a considerably higher average density of CD8+ T cells infiltrating in the tumor marginal zone. The presence of T cells demonstrated significant predictive capability for responses to neoadjuvant immunotherapy in HNSCC patients. Furthermore, TIM3+ T cells and LAG3+ T cells were found to be remarkably lower in the partial response (PR) cohort than in the non-partial response (NPR) cohort post-treatment. This research contributes critical understanding of the physiological changes occurring in immune cell responses.

Sex differences in the colorectal tumor immune microenvironment.

283 Background: Males have increased risk and mortality rates of colorectal cancer (CRC) compared to females, but the reasons for these disparities are not well understood. Immune responses differ by sex, and a robust T cell response is an important prognostic indicator in CRC. We hypothesized that females exhibit a stronger CRC-associated T cell response, contributing to reduced mortality in females compared to males. Methods: The study cohort included 2,751 incident CRC patients (1,337 female; 1,414 male) from a population-based case-control study. T cell receptor (TCR) abundance and clonality were quantified using immunoSEQ, and a gastrointestinal pathologist scored tumor-infiltrating lymphocyte counts per high-powered field (TILs/hpf). We used logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between T cell metrics and sex, adjusting for age, anatomic site, and microsatellite instability (MSI) status. Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% confidence intervals for the association between sex and 5-year all-cause and CRC-specific survival, adjusting for known prognostic indicators. We also conducted a formal test for interaction between sex and T cell features in relation to survival outcomes. Results: There were no associations between sex and TCR abundance (OR=1.05, 95% CI=0.98-1.13, p=0.14), TCR clonality (OR=0.99, 95% CI=0.91-1.06, p=0.72), or TILs/hpf (OR=1.05, 95% CI=0.88-1.25, p=0.60). We observed no differences in overall or disease-specific five-year survival between males and females. Further, the associations between T cell responses and survival did not significantly differ by sex. Conclusions: In a well-powered analysis, no robust sex differences were observed in the CRC immune microenvironment or in the relationship between T cell infiltration and CRC outcomes. The high burden of CRC and the emergence of immunotherapy as an important therapeutic approach suggest a need for further research investigating immunological sex differences in the setting of treatment response. Summary of associations between the colorectal tumor T cell response and sex. T cell Features Odds Ratio(Female vs. Male) Lower 95% CI Upper 95% CI P Value TCR Abundance 1.05 0.98 1.13 0.14 TCR Clonality 0.99 0.91 1.06 0.72 TILs/hpf* &lt;2 1.05 0.88 1.25 0.60 *A total of 99 patients (45 female; 54 male) with missing TILs/hpf data were excluded from the regression analysis.

Biological correlates associated with high-risk breast cancer patients identified using a computational method

Using a novel unsupervised method to integrate multi-omic data, we previously identified a breast cancer group with a poor prognosis. In the current study, we characterize the biological features of this subgroup, defined as the high-risk group, using various data sources. Assessment of three published hypoxia signatures showed that the high-risk group exhibited higher hypoxia scores (p < 0.0001 in all three signatures), compared to the low-risk group. Our analysis of the immune cell composition using CIBERSORT and leukocyte fraction showed significant differences between the high and low-risk groups across the entire cohort, as well as within PAM50 subtypes. Within the basal subtype, the low-risk group had a statistically significantly higher spatial fraction of tumor-infiltrating lymphocytes (TILs) compared to the high-risk group (p = 0.0362). Our findings indicate that this subgroup with poor prognosis is driven by a distinct biological signature with high activation of hypoxia-related genes as well as a low number of TILs.

Radiomic signatures derived from baseline 18F FDG PET/CT imaging can predict tumor-infiltrating lymphocyte values in patients with primary breast cancer.

To determine the value of radiomics data extraction from baseline 18F FDG PET/CT in the prediction of tumor-infiltrating lymphocytes (TILs) among patients with primary breast cancer (BC).We retrospectively evaluated 74 patients who underwent baseline 18F FDG PET/CT scans for BC evaluation between October 2020 and April 2022. Radiomics data extraction resulted in a total of 131 radiomic features from primary tumors. TILs status was defined based on histological analyses of surgical specimens and patients were categorized as having low TILs or moderate & high TILs. The relationships between TILs groups and tumor features, patient characteristics and molecular subtypes were examined. Features with a correlation coefficient of less than 0.6 were analyzed by logistic regression to create a predictive model. The diagnostic performance of the model was calculated via receiver operating characteristics (ROC) analysis.Menopausal status, histological grade, nuclear grade, and four radiomics features demonstrated significant differences between the two TILs groups. Multivariable logistic regression revealed that nuclear grade and three radiomics features (Morphological COMShift, GLCM Correlation, and GLSZM Small Zone Emphasis) were independently associated with TIL grouping. The diagnostic performance analysis of the model showed an AUC of 0.864 (95% CI: 0.776-0.953; p < 0.001). The sensitivity, specificity, PPV, NPV and accuracy values of the model were 69.6%, 82.4%, 64%, 85.7% and 78.4%, respectivelyThe pathological TIL scores of BC patients can be predicted by using radiomics feature extraction from baseline 18F FDG PET/CT scans.

Tumor-Infiltrating Lymphocytes Assessed Using the International TILs Working Group System Are Not Prognostic in Medullary Thyroid Cancer.

Background: Tumor-infiltrating lymphocytes (TILs) are a protective prognostic factor in several solid tumors and predict response to immune checkpoint inhibitor therapy. The prognostic impact of TILs in medullary thyroid cancer (MTC) is poorly understood. Materials and Methods: In this retrospective cohort study, we assessed the TILs profile of primary MTC tumors using the International TILs Working Group system and correlated this with clinicopathological prognostic variables, including the International Medullary Thyroid Cancer Grading System (IMTCGS) grade and survival outcomes. Results: We identified 71 patients with primary MTC tumors who were treated surgically between 1995 and 2016 at the Royal North Shore Hospital in Sydney, Australia. The median (interquartile range) duration of follow-up was 69 (90) months. Using the ITWG system, all patients with MTC had low TILs, with a median (range) of 3% (0-10%). This group was further subdivided into "very low" (0-4%) and "low" (5-10%), and on Cox regression analysis, increasing TILs were associated with increased local recurrence (log-rank p = 0.022, odds ratio [OR] 1.94 [confidence interval or CI 0.61-6.16], p = 0.26), reduced disease-specific survival (log-rank p = 0.015, OR 5.11 [CI 1.01-26.0], p = 0.049), and a trend to decreased distant metastasis-free survival (log-rank p = 0.14). When examining the association between TILs and other prognostic factors, only "high IMTCGS grade" was significantly associated with increased TILs (OR 7.29 [CI 1.21-43.90], p = 0.015). In the multivariable logistic regression analysis, there was no significant association between TILs and local recurrence or disease-specific survival. Conclusions: In our study, the prognostic value of TILs in MTC was limited. Even high-grade MTC can be considered an immune quiescent tumor, and the adverse prognostic factors associated with higher grade tumors outweigh the marginal increase in immune recognition associated with a slight increase in TILs. The low level of TILs in MTC and their lack of correlation with survival suggest that immune checkpoint inhibitor therapy may not be effective.

Current Landscape of Adoptive Cell Therapy and Challenge to Develop "Off-The-Shelf" Therapy for Hepatocellular Carcinoma.

Adoptive cell therapy (ACT) is a type of immunotherapy in which autologous or allogeneic immune cells, such as tumor-infiltrating lymphocytes or engineered lymphocytes, are infused into patients with cancer to eliminate malignant cells. Recently, autologous T cells modified to express a chimeric antigen receptor (CAR) targeting CD19 showed a positive response in clinical studies for hematologic malignancies and have begun to be used in clinical practice. This article discusses the current status and promise of ACT research in hepatocellular carcinoma (HCC), focusing on challenges in off-the-shelf ACT using primary cells or induced pluripotent stem cells (iPSCs) with or without genetic engineering. Early clinical trials of autologous GPC-3-, MUC1-, or CEA-targeted CAR-T cell therapies are underway for HCC. There is a growing demand for the development of off-the-shelf therapies due to the high cost and manufacturing issues associated with autologous CAR-T. The development of ACT from various cell sources, such as NK cells, NKT cells, macrophages, and γδ T cells without MHC restriction other than T cells has been proposed. Advances in genome editing, including HLA gene knockout to avoid GvHD, and strategies to enhance efficacy in overcoming the suppressive tumor microenvironment, are used to create universal 'off-the-shelf' CAR-T cells which can be used immediately as therapeutic products from healthy donors or iPSC-derived immune cells. Despite several limitations, cell-based immunotherapy is expected to become a key cancer treatment modality for both hematologic malignancies and solid tumors including HCC, thanks to technological advancements overcoming these challenges.

A panel of cancer testis antigens in squamous cell carcinoma of the lung, head and neck, and esophagus: implication for biomarkers and therapeutic targets

This study aims to investigate the expression of seven cancer testis antigens (MAGE-A1, MAGE-A4, MAGE-A10, MAGE-A11, PRAME, NY-ESO-1 and KK-LC-1) in pan squamous cell carcinoma and their prognostic value, thus assessing the potential of these CTAs as immunotherapeutic targets. The protein expression of these CTAs was evaluated by immunohistochemistry in 60 lung squamous cell carcinoma (LUSC), 62 esophageal squamous cell carcinoma (ESCA) and 62 head and neck squamous cell carcinoma (HNSC). The relationship between CTAs expression and progression-free survival (PFS) was assessed. PD-L1 expression and tumor-infiltrating lymphocytes were also collected and correlated with CTAs expression. The prognostic impact of CTAs gene expression was evaluated using the Kaplan–Meier Plotter website. CTAs expression was 0–48% in ESCA, 3%-77% in LUSC, and 3%-71% in HNSC. Analysis of PFS showed that MAGE-A1 expression in HNSC (**p < 0.01), PRAME in LUSC (p = 0.008, **p < 0.01), MAGE-A10 (p = 0.012, *p < 0.05) and PRAME (p = 0.021, *p < 0.05) in ESCA were significantly correlated with PFS. In all three cancers, coexpression of three CTAs was used as a cutoff value for grouping, and the results showed a significant difference in PFS between these two groups. Moreover, CTAs expression was significantly correlated with PD-L1 expression and T cell infiltration. These findings indicate a high incidence of CTA expression in HNSC, LUSC and ESCA, which was correlated with PD-L1 expression, T cell infiltration, and tumor progression. The results suggest that cancer testis antigens could be feasible vaccine targets in squamous cell carcinoma.

Reverse-engineering the FLT3-PI3K/AKT axis to enhance TILs function and improve prognosis in ovarian and cervical cancers

BackgroundOvarian cancers (OC) and cervical cancers (CC) have poor survival rates. Tumor-infiltrating lymphocytes (TILs) play a pivotal role in prognosis, but shared immune mechanisms remain elusive.MethodsWe integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) to explore immune regulation in OC and CC, focusing on the PI3K/AKT pathway and FLT3 as key modulators. Seurat and Harmony were employed for batch correction and dimensionality reduction. FLT3 expression was mapped with spatial data from 10 × Genomics.ResultsFLT3, identified as a regulator through the PI3K/AKT pathway, showed positive correlations with T cells, NK cells, and B cells. FLT3-high regions exhibited increased immune infiltration, particularly in CC, enhancing survival outcomes.ConclusionThis study provides the first spatially resolved evidence of FLT3's immune-modulatory role in OC and CC, positioning it as a promising immunotherapeutic target. FLT3-targeted strategies may offer new options for patients resistant to conventional therapies.

Timing Anti-PD-L1 Checkpoint Blockade Immunotherapy to Enhance Tumor Irradiation

Background: The ability of radiotherapy (RT) to drive anti-tumor immunity is limited by adaptive resistance. While RT induces inflammation and recruits activated tumor-infiltrating lymphocytes (TILs), including cytotoxic T lymphocytes (CTLs), the resulting radiation- and IFNγ-dependent PD-L1 expression restores an immunosuppressed tumor microenvironment. Unleashing an effective anti-tumor response may require the precise sequencing of RT and checkpoint blockade immunotherapy (CBI) to block PD-L1 signaling before it can mediate its suppressive effects. Methods: Flank tumors formed in BALB/c mice with syngeneic CT26 colon or 4T1 mammary carcinoma cells were treated with otherwise ineffective doses of ionizing radiation (10 Gy) followed by CBI (0.2 mg anti-PD-L1, i.v.) after 0, 1, 3, 5, or 7 days, comparing tumor response. Anti-PD-L1 delivery was measured by fluorescence, TILs by flow cytometry and immunofluorescence, PD-L1 expression by immunohistochemistry, and tumor size by calipers. Results: In both CT26 and 4T1 tumors, 10 Gy alone resulted in a transient growth delay associated with infiltrating CTLs peaking at 3 days and PD-L1 at 5 days. CTLs returned to baseline after 7 days, consistent with adaptive resistance. Anti-PD-L1 failed to potentiate radiation except when injected 5 days after 10 Gy, which prevented CTL depletion and led to tumor elimination. Potentially contributing to compound effects, anti-PD-L1 penetrated tumors and bound PD-L1 more efficiently after irradiation. Conclusions: Optimal timing to exploit radiation-induced permeability to enhance CBI delivery and interrupt adaptive resistance by blocking PD-L1 as it peaks may offer a general strategy to enhance external beam radiotherapy by protecting activated TILs and potentiating anti-tumor immune response.

Co-blocking TIGIT and PVRIG using a novel bispecific antibody enhances anti-tumor immunity.

TIGIT and PVRIG are immune checkpoints co-expressed on activated T and NK cells, contributing to tumor immune evasion. Simultaneous blockade of these pathways may enhance therapeutic efficacy, positioning them as promising dual targets for cancer immunotherapy. This study aimed to develop a bispecific antibody (BsAb) to co-target TIGIT and PVRIG. Expression of TIGIT and PVRIG was assessed on tumor-infiltrating lymphocytes (TILs) from patients with various cancers, including non-small cell lung cancer (n=63) and colorectal cancer (n=26). The BsAb was engineered by fusing anti-PVRIG nanobodies to the N terminus of anti-TIGIT antibodies. Functional characterization of the BsAb was performed in vitro and in vivo, including assessments of T and NK cell activation and cytotoxicity. Pharmacokinetics and safety profiles were evaluated in cynomolgus monkeys. Statistical analyses were conducted using the Student's t-test. The results showed that the BsAb effectively blocked TIGIT and PVRIG from binding their respective ligands, CD155 and CD112, leading to significant increases in T cell activation (2.8-fold, p<0.05) and NK cell cytotoxicity (1.8-fold, p<0.05). In vivo, the BsAb demonstrated potent anti-tumor activity, both as a monotherapy and in combination with anti-PD-1 or anti-PD-L1, in humanized PBMC and transgenic mouse models. Pharmacokinetic studies in cynomolgus monkeys revealed a favorable profile, with no dose-limiting toxicities observed after four repeated doses of 200 mg/kg. These findings provide compelling preclinical evidence for the therapeutic potential of targeting the TIGIT-PVRIG axis with a bispecific antibody. This approach shows promise for enhancing anti-tumor immunity and warrants further investigation in clinical trials.