Tumor-infiltrating Lymphocytes Infiltration Research Articles

Stromal CD4 (+) T Cell Subsets Mediate Antitumor Cytotoxic Immune Responses in Human Colorectal Carcinoma.

The local immune response in colorectal cancer is closely related to prognosis and therapeutic efficacy. In this study, histological analyses were performed to determine the phenotype of tumor-infiltrating lymphocytes (TILs) and their infiltration in the stromal and intratumoral regions, aiming to elucidate their interactions and prognostic effects. Multiplex fluorescent labeling was performed using surgically resected colorectal cancer specimens to investigate the infiltration of CD45RO (+) TILs, which exhibit cytotoxicity, and subsets of CD4 (+) TILs, identified by their characteristic transcription factor expression. The degree of CD45RO (+) TIL infiltration in the entire observation field or stromal area was not associated with prognosis. However, a high degree of infiltration in the tumor nest (intratumoral area) was significantly associated with a favorable prognosis. CD4 (+) TILs and their subsets were not associated with prognosis. However, stratified analyses revealed that a high degree of infiltration of stromal CD4 (+) TILs and the subsets T helper (Th)1, Th2, Th17, and regulatory T cells is necessary for the association between high intratumoral CD45RO (+) TIL infiltration and favorable prognosis. A sufficient degree of infiltration of stromal CD4 (+) TIL subsets is required for intratumoral CD45RO (+) TILs to exert toxicity against cancer cells. This highlights the significance of stromal immune reactions in achieving effective cytotoxic immune responses in the intratumoral area and demonstrates the critical role of the spatial distribution pattern of TILs in exerting their functions.

Spatial heterogeneity and prognostic significance of TAMs and TILs infiltrates in different staging esophageal squamous carcinoma

BackgroundThe prognostic value and clinical relevance of tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) in esophageal squamous cell carcinoma (ESCC) remain unclear. AimsTo investigate the prognostic value and functional involvement of TILs in ESCC. MethodsWe included 40 patients across different stages of ESCC from Xinjiang. Multiplex fluorescent immunohistochemistry characterized TILs and TAMs. TILs in different tumor regions were quantified and correlated with overall survival (OS) using log-rank test and Cox regression analyses. ResultsInvasive ESCC exhibited increased CD4 T cells and Tregs compared to carcinoma in situ, with a higher Tregs/CD4 T cells ratio (p < 0.05). TAMs, primarily in stromal regions, were significantly associated with Foxp3+ cells (p < 0.05). Higher infiltration of stromal TAMs and a higher CD4/CD8 T cells ratio correlated with poorer OS, while a higher CD8 T/Foxp3+ cells ratio indicated better survival. Multivariate Cox analysis revealed TNM stage, tumor length, and stromal CD4/CD8 T cells ratio as independent prognostic factors (p < 0.05). An immune prognostic risk score-based nomogram was constructed to predict patient outcomes. ConclusionsThe spatial distribution and abundance of TILs significantly correlated with prognosis, providing a useful immune classification for ESCC.

The aminophospholipid transporter, ATP8B3, as a potential biomarker and target for enhancing the therapeutic effect of PD-L1 blockade in colon adenocarcinoma

BackgroundColon adenocarcinoma (COAD) is a prevalent malignant tumor globally, contributing significantly to cancer-related mortality. COAD guidelines label MSI (Microsatellite instability) and MSS (Microsatellite stability) subtypes as global classification criteria and treatment strategy selection criteria for COAD. Various combination therapies involving PD-L1 inhibitors and adjuvant therapy to enhance anti-tumor efficacy. MethodsDatasets from single-cell RNA sequencing and bulk RNA sequencing in the TCGA and GEO databases were utilized to identify differentially expressed genes (DEGs). Furthermore, the correlation between ATP8B3 and PD-L1 was validated using siRNA, shRNA, and western blot analysis. Additionally, the association between ATP8B3 and immune checkpoint blockade (ICB) therapy was investigated through immune infiltration analysis and flow cytometry in both in vivo and in vitro assays. ResultsIn the COAD patient group, ATP8B3 significantly contributed to the establishment of an immunosuppressive microenvironment. Inhibiting ATP8B3 led to a reduction in PD-L1 expression in colon cancer cell lines. Additionally, ATP8B3 expression levels could serve as a potential guide for PD-L1 treatment in MSI-H COAD patients, with higher ATP8B3 expression associated with increased sensitivity to PD-L1 therapy. However, due to the lack of immuno-killer cells in the microenvironment of MSS subtypes, elevated ATP8B3 expression couldn't increase the sensitivity of MSS COAD patients to PD-L1 inhibitors. ConclusionOur research results support that Inhibiting ATP8B3 could enhance TIL (tumor-infiltrating lymphocyte) infiltration by reducing PD-L1 expression in MSI-H COAD, thereby serving as an effective strategy to improve PD-L1 blocker efficacy. The treatment strategy of combining ATP8B3 inhibitors and immunotherapy for MSI/MSS COAD patients will be the best choice.

Association of tumor budding and tumor infiltrating lymphocytes with clinicopathological parameters in gallbladder carcinoma.

Gallbladder carcinoma (GBC) poses significant challenges in oncology due to its aggressive nature and limited treatment options. The lack of effective biomarkers for early detection and prognosis exacerbates the prognosis for GBC patients. Tumor budding (TB) and tumor infiltrating lymphocytes (TILs) have emerged as potential prognostic indicators in various cancers, reflecting tumor-host immune interactions and tumor aggressiveness. The study of TB and TILs in GBC is particularly important due to the limited literature available. This retrospective observational study aimed to evaluate the association of TB and TILs with clinicopathological parameters in GBC patients. Clinicopathological data were collected from patients with histologically confirmed GBC who underwent surgical resection. The sections were evaluated for TB and TILs using standardized methods. Statistical analysis was performed to assess associations between these parameters and clinicopathological variables. Tumor stage and grade showed significant associations with TB and TILs, indicating their potential as prognostic markers. High TB correlated with advanced tumor stage and higher grade, while high TIL infiltration was associated with early tumor stage and lower grade. Additionally, TILs exhibited a significant association with lymphovascular invasion. Interestingly, an inverse association was observed between TB and TILs, highlighting the dynamic interplay between tumor aggressiveness and host immune response. TB and TILs hold prognostic significance in GBC, offering insights into its pathogenesis and potential therapeutic targets. Future research exploring the mechanistic underpinnings of tumor-host immune interactions in GBC is crucial for translating these findings into clinical applications and improving outcomes for patients.

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

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

Predictive and prognostic value of aurora kinase A combined with tumor-infiltrating lymphocytes in medullary thyroid carcinoma.

Aurora kinase A (AURKA) and tumor-infiltrating lymphocytes (TILs) are both known to play an essential role in tumorigenesis. However, the expression and prognostic value of the AURKA and TILs in medullary thyroid carcinoma (MTC) have not yet been investigated. Surgical specimens and clinical data of 137 patients diagnosed with MTC were collected. AURKA expression and TILs infiltration were quantified by immunohistochemistry and hematoxylin-eosin staining. Subsequently, the prognostic value of AURKA expression and TIL infiltration in MTC was evaluated. AURKA was highly expressed in patients with multifocal tumor, cervical lymph node metastasis, and an advanced TNM stage, indicating a high probability of recurrence. AURKA further exhibited a positive correlation with TILs (R = 0.44, P < 0.001). High expression of AURKA combined with a low numbers of TILs (AURKAhigh/TILslow) was identified as an independent prognostic factor for biochemical recurrence (odds ratio: 4.57, 95% confidence interval: 1.54-14.66, P < 0.01) and recurrence-free survival (hazard ratio: 3.64, 95% confidence interval: 1.52-8.71, P < 0.001). The combination of AURKA and TILs apparently improves the prognostic value for biochemical recurrence (area under the curve: 0.751) and structural recurrence (area under the curve: 0.836) of MTC. Notably, AURKAhigh/TILslow demonstrated a high value for prediction of distant or unresectable locoregional recurrence, with an overall accuracy of 86.9%. AURKAhigh is associated with the MTC malignancy. The combination of AURKAhigh/TILslow was identified as novel independent prognostic marker in MTC, predicting incurable disease recurrence with high accuracy.

The tumor immune microenvironment features and their prognostic value in rectal and sigmoid colon signet-ring cell carcinoma.

3544 Background: Signet-ring cell carcinoma (SRCC) of the rectum and sigmoid colon is a rare and highly malignant challenge in clinical practice. Although high PD-1+CD8+ tumor-infiltrating lymphocyte (TIL) infiltration has been reported as a positive prognostic factor in gastric cancer, pancreatic cancer, etc., its role in colorectal SRCC remains unclear. This study aims to explore the immune landscape of colorectal SRCC and its prognostic implications. Methods: The study included 34 patients with stage II-IV SRCC of the rectum and sigmoid colon. The immune contextures of tumor tissues were characterized using multiplex immunofluorescence assays and compared with a cohort of 57 cases of stage II-IV AC. The ratio density and spatial distribution of CD3+, CD8+, PD-1+, PD-L1+ cells, M1/M2 macrophages, and NK cells were quantitively analyzed in both parenchymal (p) and interstitial (i) regions. Characteristics of immune cell infiltration and their prognostic value for long-term survival were investigated in SRCC cases. Results: Compared to AC, SRCC exhibited significantly higher infiltration of CD8+ and CD3+ iTILs in the interstitial region (P=0.00024; P=0.00321, respectively) and elevated CD8+ pTILs infiltration in tumor parenchyma (P=0.041). Conversely, parenchymal infiltration of TILs co-expressing PD-1 was significantly lower in SRCC than AC (PD-1+CD8+ pTILs: P=0.00035; PD-1+CD3+ pTILs: P=5.0e-05). SRCC showed significantly lower infiltration of M1 macrophages in both parenchyma and interstitium (P=0.0035; P=5.4e-09, respectively), lower interstitial infiltration of M2 macrophages (P=1.5e-05), and a borderline lower parenchymal infiltration of M2 macrophages (P=0.0522), compared to AC. Additionally, patients with high parenchymal or interstitial infiltration of PD-1+CD8+ TILs exhibited significantly longer DFS (P=0.00024; P=0.0041, respectively) and OS (P=0.011; P=0.0044, respectively) than those with low infiltration. Multivariate analysis indicated that the level of PD-1+CD8+ pTILs infiltration is an independent prognostic factor for DFS, being independent of CEA levels and TNM stage (P = 0.018; P = 0.02, respectively). Among patients with normal CEA level or of stage II-III, high level of PD-1+CD8+ pTILs infiltration was still associated with significantly improved DFS (P&lt;0.0001; P=0.00018, respectively) and OS (P=0.00021; P&lt;0.0001, respectively). Conclusions: SRCC of rectum and sigmoid colon exhibits distinct immune profiles compared to conventional AC, characterized by higher infiltration of CD8+ and CD3+ TILs, but lower infiltration of PD-1+CD8+ TILs. The unique immune landscape in SRCC, particularly the level of PD-1+CD8+ TILs infiltration and its positive predictive value for DFS and OS, provides new perspectives for immune-based stratification and therapeutic strategies for this clinically significant cancer subtype.

PROGNOSTIC VALUE OF THE DENSITY OF TUMOR-INFILTRATING LYMPHOCYTES AND ITS ASSOCIATION WITH CLINICAL-MORPHOLOGICAL FEATURES OF COLON ADENOCARCINOMAS

Aim. To study the prognostic value of the density of tumor-infiltrating lymphocytes (TILs) and its association with other clinical-morphological parameters in colon adenocarcinomas (CAC). Materials and Methods. 236 CAC samples were examined. TILs density was estimated as the percentage of tumor stromal area occupied by TILs. By the index of TILs density, the patients were divided into 3 groups: TILs 0—9% (n = 88); TILs 10—39% (n = 106); TILs &gt; 40% (n = 42). Dependent on this index, their overall survival (OS) was analyzed. Results. Kaplan — Meier curves revealed a significant (p &lt; 0.001) difference in the OS for patients with different TILs infiltration intensities. Multivariate Cox’s proportional hazard regression model analysis has confirmed that patients with moderate TILs density (HR 0.57, 95% CI 0.34—0.96, p = 0.035) had better OS rates compared to low TILs density. TILs were associated with the stage (p &lt; 0.001), lymph node metastasis pN (p &lt; 0.001), distant metastasis M (p &lt; 0.001), and the patient’s outcome (p &lt; 0.001). Conclusion. TILs can be considered an additional prognostic tool during regular histological examination and are strongly associated with the most significant clinical-morphological features of CAC.

IMMUNOREACT 7: Regular aspirin use is associated with immune surveillance activation in colorectal cancer.

Long-term daily use of aspirin reduces incidence and mortality due to colorectal cancer (CRC). This study aimed to analyze the effect of aspirin on the tumor microenvironment, systemic immunity, and on the healthy mucosa surrounding cancer. Patients with a diagnosis of CRC operated on from 2015 to 2019 were retrospectively analyzed (METACCRE cohort). Expression of mRNA of immune surveillance-related genes (PD-L1, CD80, CD86, HLA I, and HLA II) in CRC primary cells treated with aspirin were extracted from Gene Expression Omnibus-deposited public database (GSE76583). The experiment was replicated in cell lines. The mucosal immune microenvironment of a subgroup of patients participating in the IMMUNOREACT1 (ClinicalTrials.gov NCT04915326) project was analyzed with immunohistochemistry and flow cytometry. In the METACCRE Cohort, 12% of 238 patients analyzed were aspirin users. Nodal metastasis was significantly less frequent (p=.008) and tumor-infiltrating lymphocyte infiltration was higher (p=.02) among aspirin users. In the CRC primary cells and selected cell lines, CD80 mRNA expression was increased following aspirin treatment (p=.001). In the healthy mucosa surrounding rectal cancer, the ratio of CD8/CD3 and epithelial cells expressing CD80 was higher in aspirin users (p=.027 and p=.034, respectively). These data suggested that regular aspirin use may have an active role in enhancing immunosurveillance against CRC.

Effect of Neoadjuvant Chemotherapy on Tumor-Infiltrating Lymphocytes in Resectable Gastric Cancer: Analysis from a Western Academic Center.

Tumor-infiltrating lymphocytes (TILs) are an emerging biomarker predictive of response to immunotherapy across a spectrum of solid organ malignancies. The characterization of TILs in gastric cancer (GC) treated with contemporary, multiagent neoadjuvant chemotherapy (NAC) is understudied. In this retrospective investigation, we analyzed the degree of infiltration, phenotype, and spatial distribution of TILs via immunohistochemistry within resected GC specimens treated with or without NAC at a Western center. We hypothesized that NAC executes immunostimulatory roles, as evidenced by an increased number of anti-tumor TILs in the tumor microenvironment. We found significantly elevated levels of conventional and memory CD8+ T cells, as well as total TILs (CD4+, CD8+, Treg, B cells), within chemotherapy-treated tumors compared with chemotherapy-naïve specimens. We also revealed important associations between survival and pathologic responses with enhanced TIL infiltration. Taken together, our findings advocate for an immunostimulatory role of chemotherapy and underscore the potential synergistic effect of combining chemotherapy with immunotherapy in resectable gastric cancer.

Evaluation of tumor response to immune checkpoint inhibitors by a 3D immunotumoroid model.

Only 20 percent of renal and bladder cancer patients will show a significant response to immune checkpoint inhibitor (ICI) therapy, and no test currently available accurately predicts ICI response. We developed an "immunotumoroid" cell model system that recapitulates the tumor, its microenvironment, and necessary immune system components in patient-derived spheroids to enable ex vivo assessment of tumor response to ICI therapy. Immunotumoroids were developed from surgically resected renal cell carcinomas and bladder carcinomas selected for high tumor-infiltrating lymphocytes (TILs) and survived more than a month without media exchange. Immunohistochemistry was used to detect immune and non-immune cells in cryopreserved source tumors and the resulting immunotumoroids. Immunotumoroid response to ICIs (nivolumab, pembrolizumab, and durvalumab) and chemotherapy (cisplatin, gemcitabine, and paclitaxel) was monitored in real-time with Cytotox Red staining in an Incucyte device, and the immunotumoroid response was compared to retrospective clinical drug responses. Six of the 13 cases tested grew viable immunotumoroid models, with failed cases attributed to extensive tumor tissue necrosis or excess lymphocytes preventing spheroid formation. One successfully cultured case was excluded from the study due to low TIL infiltration (<5%) in the primary tumor sample. The five remaining models contained immune cells (CD4+ and CD8+ T cells, and macrophages), non-immune cells (fibroblasts), and tumor cells. Chemotherapy and ICI drugs were tested in immunotumoroids from 5 cases and compared to clinical outcomes where data was available. Four/five models showed cell killing in response to chemotherapy and two/five showed sensitivity to ICI. In three cases, the immunotumoroid model accurately predicted the patient's clinical response or non-response to ICIs or chemotherapy. Our immunotumoroid model replicated the multicellular nature of the tumor microenvironment sufficiently for preclinical ICI screening. This model could enable valuable insights into the complex interactions between cancer cells, the immune system, and the microenvironment. This is a feasibility study on a small number of cases, and additional studies with larger case numbers are required including correlation with clinical response.

Abstract 3918: Targeting MARCO in combination with anti-CTLA4 leads to enhanced melanoma regression and immune cell infiltration via macrophage reprogramming

Abstract Tumor-associated macrophages (TAM) are known to suppress responses to conventional therapeutics and the presence of TAM is generally related to poor prognosis in various cancers. Therefore, depletion of TAM has been one of the main strategies to break the ceiling of efficacy in cancer treatment. However, to date, TAM depletion strategies have not been successful in clinical trials1. Here, we introduce an approach to target TAM by employing Macrophage Receptor with Collagenous Structure (MARCO), a class A scavenger receptor, as a novel immune checkpoint. Previously, we showed MARCO to be the most upregulated mRNA in murine and human dendritic cells following their uptake of dead tumor cells (TP-DC)2. We also showed ex vivo treatment of TP-DC with anti-MARCO mAb (ED31) led to enhanced anti-tumor responses in vivo3,4. The aim of this current study was to explore whether targeting MARCO using ED31 could improve the efficacy of an immune checkpoint inhibitor in murine models of melanoma. In C57BL/6 mice with B16F10 tumors, adding ED31 to anti-CTLA4 mAb (αCTLA4) resulted in enhanced tumor regression and longer survival (p=0.001, p&amp;lt;0.05 vs αCTLA4 monotherapy; p&amp;lt;0.05, p&amp;lt;0.01 vs ED31 monotherapy, respectively). To explore the immune mechanism of ED31 with αCTLA4, we focused on the tumor microenvironment (TME) by flow cytometry. TME immune-profiling consistently revealed that one time treatment with this combination resulted in a rapid increase in the number of tumor infiltrating lymphocytes (TIL) including CD8+T (2.8-fold) and CD4+T cells (2.9-fold), NK cells (2.5-fold), activated CD11c+DC (2.2-fold) and F4/80+Mφ (2.5-fold), compared to αCTLA4 monotherapy (all with p&amp;lt;0.0001; vs αCTLA4 monotherapy, vs ED31 monotherapy, vs control, respectively). As measured by chemokine production using murine bone marrow-derived, in vitro M-CSF induced Mφ phenotypes (M1: polarized by IFNγ and LPS, M2: polarized by IL4, TAM-like Mφ: polarized by a supernatant from B16F10 cell line culture), ED31 exposure switched the pattern of chemokine productions from M2 and TAM-like Mφ into an M1 pattern. In vivo depletion of each immune cell type had a diminishing effect on the treatment efficacy (p&amp;lt;0.001 for CD8 depletion, p=0.01 for CD4 depletion, p&amp;lt;0.01 for NK1.1 depletion). However, Mφ depletion had a more profound impact by completely abrogating the anti-tumor efficacy and the associated survival benefit, in addition to completely reversing the increase in TIL infiltration and canceling the increase of M1 chemokines in vivo (all with p&amp;lt;0.05 vs Mφ depletion). These preclinical data support the proof-of-concept that a combination strategy of targeting MARCO along with αCTLA4 can lead to enhanced anti-tumor responses through utilizing TAMs rather than their depletion. 1 Immunity 2023;56:2188. 2 J Immunol 2003;171:2879. 3 Cancer Immunol Immunother 2010;59:875. 4 PLoS One 2013;8:e67795. Citation Format: Hidenori Takahashi, Patricio Perez-Villarroel, Rana Falahat, James J. Mulé. Targeting MARCO in combination with anti-CTLA4 leads to enhanced melanoma regression and immune cell infiltration via macrophage reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3918.

Abstract 6181: Digital pathology workflow: Building a bridge for translational biomarker development in humanized mice

Abstract The introduction of immune checkpoint inhibitors (ICIs) directed against programmed death protein 1 (PD-1), as well as cytotoxic T lymphocyte antigen 4 (CTLA-4), has revolutionized the treatment of non-small cell lung cancer (NSCLC) in the last decade. These therapies have shown significant success in a subset of NSCLC patients, leading to durable response and improved overall survival. However, clear predictive biomarkers for response are still yet to be defined. Tumor-infiltrating lymphocytes (TILs) as one important part of the tumor microenvironment (TME) have proven to be predictive biomarkers in other tumor types such as melanoma, whereas not fully understood in NSCLC. The assessment of TILs typically involves techniques such as flow cytometry (FC) or immunohistochemistry (IHC). While the former has the advantage of being quantitative and relatively easy to standardize it lacks translational value as most clinical samples will be examined using IHC. The current study aims to compare both methods at terminal end point and for treatment outcome, using a panel of 14 NSCLC PDX models, implanted in humanized NSG mice and treated with anti-CTLA4, anti-PD1 and the combination thereof. Based on FC analyses of the untreated tumor tissue the PDX models were stratified into seven hot (&amp;gt;5% TILs) and seven cold (&amp;lt;5% TILs) tumors. The IHC samples were analyzed by digital pathology for TIL infiltration by making use of automated segmentation of the three main tissue classes: tumor, stroma and necrosis. Small number of IHC slides were manually annotated to form a training set, which were then used to train a deep learning model using Visiopharm, a commercial AI platform for image analysis. By quantification of TILs in a spatial context of the tumor tissue, across all models, tumor was the most prominent tissue class. The tissue class distribution as well as the TIL rate (FC or IHC measure) is a model immanent feature. Under treatment, the ratio was stable in case of the cold tumors. In the hot tumors the stroma compartment increased while tumor tissue decreased. Treatment sensitive models displayed an increase of TILs confirmed by FC and IHC. The latter revealed that the TILs specifically increased in the tumor tissue whereas decreased or was stable in the stroma. This observation was significantly more pronounced in cold tumors compared to hot tumors (Oneway ANOVA, p &amp;lt; 0.001). Furthermore, a positive correlation between FC and IHC results on CD45 positive cells in the tumors (p&amp;lt; 0.002) could be confirmed. In this study, we proofed the feasibility of software-based image analysis as translational relevant read-out in preclinical animal experiments. The possibility to reproducibly quantify tissue classes, immune cell markers and including spatial information paves the way towards discovery of novel features predicting response in translational immuno-oncology research. Citation Format: Matti Hakkarainen, Timmo Bragge, Jussi Rytkönen, Eva Oswald, Julia B. Schueler. Digital pathology workflow: Building a bridge for translational biomarker development in humanized mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6181.

Intraepithelial tumor-infiltrating lymphocytes shape loco-regional PET/CT spread of locally advanced cervical cancer

BackgroundData suggest an association between positron emission tomography/CT (PET/CT) metabolic metrics and tumor microenvironment in several malignancies, and a potential role of PET/CT to monitor response to immunotherapy.ObjectiveTo evaluate the...

Platinum-based neoadjuvant chemotherapy upregulates STING/IFN pathway expression and promotes TILs infiltration in NSCLC.

To evaluate the effects of platinum-based neoadjuvant chemotherapy (NACT) on the STING/IFN pathway and tumor-infiltrating lymphocytes (TILs) in non-small cell lung cancer (NSCLC), as well as clinicopathological factors affecting patient survival. A total of 68 patients aged 34-77 years with NSCLC who received neoadjuvant chemotherapy and surgical treatment from March 2012 to February 2019 were reviewed, and the clinical pathological data and paired tissue specimens before and after NACT were collected. Immunohistochemistry and immunofluorescence were used to detect the protein levels of STING, PD-L1 and IFN-β, and the infiltration density of CD3+ TILs and CD8+TILs. The correlation between the expression of STING, PD-L1, IFN-β and the infiltration density of CD3+ TILs and CD8+ TILs as well as the clinicopathological characteristics before and after NACT was analyzed. The relationship between the related indexes, clinicopathological features and prognosis was also discussed. NACT increased the expression of STING, IFN-β and PD-L1 in tumor cells, and the infiltration of CD3+ and CD8+ TILs. In addition, ypTNM stage, ypN stage, changes in CD3+ TILs and in PD-L1 were associated with DFS (disease-free survival). CD3+ TILs changes and ypN stage were associated with OS (overall survival). Notably, ypN stage and CD3+ TILs changes were independent prognostic factors for DFS and OS. NACT stimulates STING/IFN-β pathway, promotes infiltration of CD3+ and CD8+ TILs, triggers innate and adaptive immunity, and also upregulates PD-L1, which complemented the rationale for neoadjuvant chemotherapy in combination with immunotherapy. In addition, DFS was longer in patients with ypTNM I, ypN0-1, and elevated CD3+TILs after NACT. Patients with ypN0 and elevated CD3+ TILs after NACT had better OS benefits.

The association of PD-L1 expression and CD8-positive T cell infiltration rate with the pathological complete response after neoadjuvant treatment in HER2-positive breast cancer.

Achieving a pathological complete response (pCR) after neoadjuvant therapy in HER2-positive breast cancer patients is the most significant prognostic indicator, suggesting a low risk of recurrence and a survival advantage. This study aims to investigate clinicopathological parameters that can predict the response to neoadjuvant treatment in HER2 + breast cancers and to explore the roles of tumour-infiltrating lymphocytes (TILs), CD8 + T lymphocytes and PD-L1 expression. This single-centre retrospective study was conducted with 85 HER2-positive breast cancer patients who underwent surgery after receiving neoadjuvant therapy between January 2017 and January 2020. Paraffin blocks from these patients were selected for immunohistochemical studies. A complete pathological response to neoadjuvant treatment was determined in 39 (45.9%) patients. High Ki-67 index (> 30%), moderate to high TIL infiltration, PD-L1 positivity and high CD8 cell count (≥ 25) were significantly associated with pCR in univariate analyses (p: 0.023, 0.025, 0.017 and 0.003, respectively). Multivariate regression analysis identified high Ki-67 index (> 30%) and CD8 cell infiltration as independent predictors for pCR in HER2-positive breast cancer. High Ki-67 index, and high CD8 cell count are strong predictors for pCR in HER2-positive breast cancer. Tumours with high Ki-67 index, high TILs and CD8 infiltration may represent a subgroup where standard therapies are adequate. Conversely, those with low TILs and CD8 infiltration may identify a subgroup where use of novel strategies, including those that increase CD8 infiltration could be applied.

A Dysfunctional T-cell Gene Signature for Predicting Nonresponse to PD-1 Blockade in Non-small Cell Lung Cancer That Is Suitable for Routine Clinical Diagnostics.

Because PD-1 blockade is only effective in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), biomarkers are needed to guide treatment decisions. Tumor infiltration by PD-1T tumor-infiltrating lymphocytes (TIL), a dysfunctional TIL pool with tumor-reactive capacity, can be detected by digital quantitative IHC and has been established as a novel predictive biomarker in NSCLC. To facilitate translation of this biomarker to the clinic, we aimed to develop a robust RNA signature reflecting a tumor's PD-1T TIL status. mRNA expression analysis using the NanoString nCounter platform was performed in baseline tumor samples from 41 patients with advanced-stage NSCLC treated with nivolumab that were selected on the basis of PD-1T TIL infiltration by IHC. Samples were included as a training cohort (n = 41) to develop a predictive gene signature. This signature was independently validated in a second cohort (n = 42). Primary outcome was disease control at 12 months (DC 12 m), and secondary outcome was progression-free and overall survival. Regularized regression analysis yielded a signature using 12 out of 56 differentially expressed genes between PD-1T IHC-high tumors from patients with DC 12 m and PD-1T IHC-low tumors from patients with progressive disease (PD). In the validation cohort, 6/6 (100%) patients with DC 12 m and 23/36 (64%) with PD were correctly classified with a negative predictive value (NPV) of 100% and a positive predictive value of 32%. The PD-1T mRNA signature showed a similar high sensitivity and high NPV as the digital IHC quantification of PD-1T TIL. This finding provides a straightforward approach allowing for easy implementation in a routine diagnostic clinical setting.

Presence, Subtypes, and Prognostic Significance of Tertiary Lymphoid Structures in Urothelial Carcinoma of the Bladder.

To evaluate the presence and subtypes of tertiary lymphatic structures (TLSs) in urothelial carcinoma of the bladder (UCB) and to analyze their associated clinicopathological characteristics and prognostic significance. The study enrolled 580 patients with surgically treated UCB, including 313 non-muscle invasive bladder cancer (NMIBC) and 267 muscle-invasive bladder cancer (MIBC). The presence and subtypes of TLSs were identified by immunohistochemistry (CD20, CD3, Bcl-6, and CD21). TLSs were classified into non-GC (nGC) TLS and GC TLS subtypes based on germinal center (GC) formation. Disease-free survival (DFS) was used as an endpoint outcome to evaluate the prognostic significance of TLS and its subtypes in UCB. TLSs were more common in MIBC than in NMIBC (67.8% vs 48.2%, P < .001), and the tumor-infiltrating lymphocyte (TIL) mean density was significantly higher in MIBC than in NMIBC (24.0% vs 17.5%, P < .001). Moreover, a positive correlation was found between TLS presence and GC structure formation and TIL infiltration in UCB. Endpoint events occurred in 191 patients. Compared to patients with endpoint events, patients without disease progression exhibited higher TIL density and more TLSs (P < .05). Kaplan-Meier curves showed that TLS was associated with better DFS in NMIBC (P = .041) and MIBC (P = .049). However, the Cox multivariate analysis did not demonstrate the prognostic significance of TLS. TLS is heterogeneous in UCB, and that TLS and GC structures are related to TIL density and prognostic events. However, TLS as a prognostic indicator remains unclear, warranting further investigation.

Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes as a predictive biomarker for axitinib in adenoid cystic carcinoma.

This study analyzed the predictive value of artificial intelligence (AI)-powered tumor-infiltrating lymphocyte (TIL) analysis in recurrent or metastatic (R/M) adenoid cystic carcinoma (ACC) treated with axitinib. Patients from a multicenter, prospective phase II trial evaluating axitinib efficacy in R/M ACC were included in this study. H&E whole-side images of archival tumor tissues were analyzed by Lunit SCOPE IO, an AI-powered spatial TIL analyzer. Twenty-seven patients were included in the analysis. The best response was stable disease, and the median progression-free survival (PFS) was 11.1 months (95% CI, 9.2-13.7 months). Median TIL densities in the cancer and surrounding stroma were 25.8/mm2 (IQR, 8.3-73.0) and 180.4/mm2 (IQR, 69.6-342.8), respectively. Patients with stromal TIL density >342.5/mm2 exhibited longer PFS (p = 0.012). Cancer and stromal area TIL infiltration were generally low in R/M ACC. Higher stromal TIL infiltration was associated with a longer PFS with axitinib treatment.

The levels of immunosuppressive checkpoint protein PD-L1 and tumor-infiltrating lymphocytes were integrated to reveal the glioma tumor microenvironment.

In spite of significant strides in the realm of cancer biology and therapeutic interventions, the clinical prognosis for patients afflicted with glioblastoma (GBM) remains distressingly dismal. The tumor immune microenvironment (TIME), a crucial player in the progression, treatment response, and prognostic trajectory of glioma, warrants thorough exploration. Within this intricate microcosm, the immunosuppressive checkpoint protein PD-L1 and tumor-infiltrating lymphocytes (TILs) emerge as pivotal constituents, underscoring their potential role in deciphering glioma biology and informing treatment strategies. However, prognostic models based on the association between PD-L1 expression and TIL infiltration in the tumor immune microenvironment have not been established. The aim of this study was to explore TIME genes associated with PD-L1 expression and TIL invasion and to construct a risk score for predicting the overall survival (OS) of GBM patients based on these genes. The samples were separately classified according to the PD-L1 expression level and TIL score and TIME-related genes were identified using differential expression and weighted gene co-expression network analysis. The DEGs were subjected to least absolute contraction and selection operator (LASSO) -Cox regression to construct TIME associated risk score (TIMErisk). A TIMErisk was developed based on STEAP3 and CXCL13 genes. The STLEAP3 was demonstrated to be involved in glioma progression. The results showed that the patients in the high TIMErisk group had poor OS compared with subjects in the low TIMErisk group. The biological phenotypes associated with TIMErisk were analyzed in terms of functional enrichment, tumor immune profile, and tumor mutation profile. The results on tumor immune dysfunction and exclusion dysfunction (TIDE) score and immune surface score (IPS) showed that GBM patients with different TIME risks had different responses to immunotherapy. Tumor purity analysis indicated that PD-L1 and TIL scores were positively correlated with TIMErisk score and negatively correlated with tumor purity. These results show that the TIMErisk-based prognostic model had high predictive value for the prognosis and immune characteristics of GBM patients. Immunohistochemical staining images of patients in the high and low TIMErisk groups were analyzed, showing that the degree of immune cell infiltration was higher in the high TIMErisk group relative to the low TIMErisk group. The present study provides a basis for understanding glioma tumor microenvironment and a foundation for conducting comprehensive immunogenomic analysis.