Density Of Immune Cells Research Articles

Low densities of immune cells indicate unfavourable overall survival in patients suffering from squamous cell carcinoma of the lung

Carcinogenesis and tumor proliferation are characterized by a complex interaction of cancer cells with the tumor microenvironment. In particular, a tumor-promoting effect can be assumed for the stroma and its fibroblasts. An influence of the immune system on non small cell lung cancer (NSCLC) is now also suspected. In our study, we examined 309 sections of squamous cell carcinoma (SCC), a subtype of NSCLC. We determined the cell densities and areas of the different tissues in SCC using the software QuPath. Spearman rank correlation showed a significant positive correlation between the different tumor cell densities and stromal cell densities, and between tumor cell densities and immune cell densities. Overall survival curves by the Kaplan–Meier method revealed a prominent negative curve in cases of low immune cell density. Based on our results, we can assume a positive influence of the tumor microenvironment, especially the stromal cells, on tumor proliferation in SCC. We have also revealed that low density of immune cells is prognostically unfavorable.

Association between Helicobacter pylori infection, mismatch repair, HER2 and tumor-infiltrating lymphocytes in gastric cancer.

The influence of Helicobacter-pylori (H. pylori) infection and the characteristics of gastric cancer (GC) on tumor-infiltrating lymphocyte (TIL) levels has not been extensively studied. Analysis of infiltrating-immune-cell subtypes as well as survival is necessary to obtain comprehensive information. To determine the rates of deficient mismatch-repair (dMMR), HER2-status and H. pylori infection and their association with TIL levels in GC. Samples from 503 resected GC tumors were included and TIL levels were evaluated following the international-TILs-working-group recommendations with assessment of the intratumoral (IT), stromal (ST) and invasive-border (IB) compartments. The density of CD3, CD8 and CD163 immune cells, and dMMR and HER2-status were determined by immunohistochemistry (IHC). H. pylori infection was evaluated by routine histology and quantitative PCR (qPCR) in a subset of samples. dMMR was found in 34.4%, HER2+ in 5% and H. pylori-positive in 55.7% of samples. High IT-TIL was associated with grade-3 (P = 0.038), while ST-TIL with grade-1 (P < 0.001), intestinal-histology (P < 0.001) and no-recurrence (P = 0.003). dMMR was associated with high TIL levels in the ST (P = 0.019) and IB (P = 0.01) compartments, and ST-CD3 (P = 0.049) and ST-CD8 (P = 0.05) densities. HER2- was associated with high IT-CD8 (P = 0.009). H. pylori-negative was associated with high IT-TIL levels (P = 0.009) when assessed by routine-histology, and with high TIL levels in the 3 compartments (P = 0.002-0.047) and CD8 density in the IT and ST compartments (P = 0.001) when assessed by qPCR. A longer overall survival was associated with low IT-CD163 (P = 0.003) and CD8/CD3 (P = 0.001 in IT and P = 0.002 in ST) and high IT-CD3 (P = 0.021), ST-CD3 (P = 0.003) and CD3/CD163 (P = 0.002). TIL levels were related to dMMR and H. pylori-negativity. Low CD8/CD3 and high CD163/CD3 were associated with lower recurrence and longer survival.

Role of tumor-infiltrating lymphocytes in melanoma prognosis and treatment strategies: A systematic review and meta-analysis

PurposeNumerous studies underscore the relevance of tumor-infiltrating-lymphocytes (TILs) as important prognostic factors for melanoma. This meta-analysis aims to provide a comprehensive literature overview elucidating their role in predicting patient outcomes, specifically investigating the association between TIL density and prognosis. MethodsFrom an initial pool of 6094 records, 16 met the eligibility criteria, encompassing a collective cohort of 16021 patients. Data on TIL counts, clinical characteristics, and survival metrics (5-year overall survival [5yOS], 10-year overall survival [10yOS], and 5-year melanoma-specific survival [5yMSS]) were extracted from each study and expressed as proportions. Results were graphically presented using forest plots, reporting the estimates from individual studies, summary estimates, and corresponding 95 % confidence intervals (CI). ResultsAnalysis revealed a statistically significant difference in 5yOS concerning subgroup differences However, 10yOS and 5yMSS did not exhibit statistical significance. Nonetheless, a consistent trend emerged indicating a higher survival rate corresponding to increased immune cell density, ranging from absent TILs to brisk levels. ConclusionsTILs present potential as a readily applicable prognostic factor. Yet, further investigations into their density and phenotypic subpopulation characteristics could enhance our understanding of their predictive value in tailoring optimal patient-specific therapies.

Tear film hTERT and corneal nerve characteristics in dry eye disease

ABSTRACT Clinical relevance The behaviour of human telomerase reverse transcriptase (hTERT) in tears reflects its role in maintaining the ocular surface homoeostasis, as it is increased after the initial fitting of contact lenses and post-overnight lid closure. Background hTERT has been shown to respond to cellular stress in neurodegenerative diseases and to enhance axonal regeneration after peripheral axotomy in an animal model. This work investigated whether the behaviour of hTERT in the tear film reflects ocular surface inflammation and neuronal changes in the presence of dry eye disease. Methods Flush tears were collected from 18 participants with dry eye disease (14 females, 4 males, mean age 34.7 ± 5.2 years) and from 18 healthy participants without dry eye disease (8 females, 10 males, mean age 31.9 ± 5.8 years). Dry eye disease status was defined using the TFOS DEWS II diagnostic criteria. hTERT levels in tears were measured using enzyme-linked immunosorbent assays. Confocal images were taken at the level of the subbasal nerve plexus at the central cornea and at the inferior whorl, and the densities of corneal immune cells were evaluated as well as corneal nerve morphology metrics using a fully automated technique (University of Manchester, United Kingdom). Results In participants with dry eye disease, hTERT levels were significantly higher compared to controls (median [interquartile range]: 434 [320–600] ng/ml, and 184 [42–390] ng/ml, respectively, p = 0.01). Increased nerve fibre width at the inferior whorl, was seen in those with dry eyes (0.0219 [0.0214–0.0236] mm/mm compared to controls 0.0217 [0.0207 0.0222] p < 0.001), but no significant differences were found in the density of corneal immune cells. Conclusions hTERT levels were elevated in participants with dry eye disease, and this was accompanied by increased nerve thickness in the inferior cornea. The hTERT response may reflect the stress induced to the ocular surface and corneal nerves due to having dry eye disease.

Correlation of gasdermin B staining patterns with prognosis, progression, and immune response in colorectal cancer

BackgroundPyroptosis is a type of programmed cell death mediated by the gasdermin family. Gasdermin B (GSDMB), as a member of gasdermin family, can promote the occurrence of cell pyroptosis. However, the correlations of the GSDMB expression in colorectal cancer with clinicopathological predictors, immune microenvironment, and prognosis are unclear.MethodsSpecimens from 267 colorectal cancer cases were analyzed by immunohistochemistry to determine GSDMB expression, CD3+, CD4+, and CD8+ T lymphocytes, CD20+ B lymphocytes, CD68+ macrophages, and S100A8+ immune cells. GSDMB expression in cancer cells was scored in the membrane, cytoplasm, and nucleus respectively. GSDMB+ immune cell density was calculated. Univariate and multivariate survival analyses were performed. The association of GSDMB expression with other clinicopathological variables and immune cells were also analyzed. Double immunofluorescence was used to identify the nature of GSDMB+ immune cells. Cytotoxicity assays and sensitivity assays were performed to detect the sensitivity of cells to 5-fluorouracil.ResultsMultivariate survival analysis showed that cytoplasmic GSDMB expression was an independent favorable prognostic indicator. Patients with positive cytoplasmic or nuclear GSDMB expression would benefit from 5-fluorouracil based chemotherapy. The assays in vitro showed that high GSDMB expression enhanced the sensitivity of colorectal cancer cells to 5-fluorouracil. Patients with positive membranous or nuclear GSDMB expression had more abundant S100A8+ immune cells in the tumor invasive front. Positive nuclear GSDMB expression indicated more CD68+ macrophages in the tumor microenvironment. Moreover, GSDMB+ immune cell density in the stroma was associated with a higher neutrophil percentage but a lower lymphocyte counts and monocyte percentage in peripheral blood. Furthermore, the results of double immunofluorescence showed that GSDMB co-expressed with CD68 or S100A8 in stroma cells.ConclusionThe GSDMB staining patterns are linked to its role in cancer progression, the immune microenvironment, systemic inflammatory response, chemotherapeutic efficacy, and prognosis. Colorectal cancer cells with high GSDMB expression are more sensitive to 5-fluorouracil. However, GSDMB expression in immune cells has different effects on cancer progression from that in cancer cells.

Abstract PO1-07-01: Artificial intelligence-based prediction of Oncotype DX Score from whole slide images using human-interpretable features and breast biomarkers

Abstract Background The Oncotype DX Breast Recurrence Score assay (ODX) is a commonly used genomic test for patients with estrogen receptor (ER)-positive, HER2-negative, early-stage invasive breast cancer. While ODX predicts patients’ recurrence risk and benefit from chemotherapy, it is tissue and time-consuming, and expensive. Previous deep-learning or non-linear ODX prediction models achieved promising performance using whole-slide images (WSI) or with other covariates (e.g., ER, progesterone receptor (PR), HER-2, Ki-67 scores and tumor stage) but systematic quantification of the contribution of individual histological features to the ODX score remained challenging. Here, we extracted a rich set of human-interpretable features (HIFs) quantifying nuclear morphology and the distribution of cells and tissues in the tumor microenvironment. We used these HIFs, along with manually assessed ER, PR, HER-2 scores, and tumor stage, to predict ODX scores. We also explored the role of Ki-67 features in augmenting our model predictions Methods We developed machine learning models to extract cell, tissue, and nuclear HIFs from WSI stained with hematoxylin and eosin (H&amp;E) and immunohistochemistry (IHC) against Ki-67. These models were deployed on 353 H&amp;E WSI to extract 276 HIFs quantifying cell densities, tissue areas, relative cell counts, and nuclei size, shape and color. Hierarchical agglomerative clustering was applied to cluster these features into 11 groups. Univariate regression was performed to identify correlations between ODX scores and feature clusters. To predict ODX score using HIFs, a multivariable regression model was fitted to the data where regressors are representative features from each cluster, in addition to ER, PR, HER-2 scores and stage as covariates. The model was fitted on the training set (N=266 slides) and evaluated on the held-out test set (N=87 slides). To examine the utility of Ki-67 IHC features in ODX prediction, a Ki-67 model was deployed on a subset of 194 matched Ki-67 IHC WSI to extract count proportions and densities of Ki-67 positive cells in cancer epithelium and stroma. Results Significant positive correlations were identified between ODX scores and feature clusters corresponding to cancer cell density (p &amp;lt; 10-7), macrophage density (p &amp;lt; 10-4), immune cell density (p &amp;lt; 10-16), and variations in cancer nuclear size (p &amp;lt; 10-5) and color (p &amp;lt; 10-3). Significant negative correlations were observed between ODX scores and clusters related to fibroblast density (p &amp;lt; 10-3), and variations of non-cancer cell nuclear color (p = 0.02). Evaluation of our model’s ability to predict ODX scores revealed an association between predicted and observed scores (Pearson r = 0.74). The AUROC for model predictions of high/low classifications (with reference to a cutoff ODX score of 20) was 0.80. Ki-67 features were clustered together with H&amp;E cancer cell density features and showed a stronger correlation with ODX scores compared to the H&amp;E features alone. Combining Ki-67 cell density features with H&amp;E features led to increased performance of the multivariable regression model on the test set compared to H&amp;E features only (r = 0.62 vs. r = 0.59). However, a model combining Ki-67, H&amp;E and covariates performed similarly to a model with H&amp;E and covariates (r = 0.73), suggesting that Ki-67 features may not be needed for ODX prediction when H&amp;E and covariate information is available. Conclusions Our model predicts ODX recurrence scores with comparable performance to other black-box approaches using WSI and breast biomarker status and without the need for, Ki-67 features. Using readily available information, our model has the potential to be a convenient screening tool for patient stratification, which may lead to better patient care, lower costs, and faster treatment. Citation Format: Nhat Le, John Van Arnam, Christian Kirkup, Ylaine Gerardin, Michael Drage, Neha Khaitan, Anurag Sharma, Georgi Galev, Nicholas Indorf, Emily Sansevere, Ilan Wapinski, Stephanie Hennek, Gloria Zhang. Artificial intelligence-based prediction of Oncotype DX Score from whole slide images using human-interpretable features and breast biomarkers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-07-01.

Abstract PS09-08: Genomic characterization of endocrine resistance in ER+HER2+ breast cancers in the POETIC Trial

Abstract Background: Mechanisms of resistance to endocrine therapy are not well understood within ER+HER2+ breast cancer (BC). Our prior work suggested that intrinsic HER2-Enriched (HER2E) molecular subtype predicts early resistance to aromatase inhibitors (AI) (Bergamino eBioMedicine 2022) and high on-treatment (on-Txt) Ki67 levels predict poor survival (Smith Lancet Oncol 2020). Improved early detection of persistent proliferating tumor cells with endocrine resistance pathways could be targeted by pre-emptive personalized therapy and reduction in recurrence. In this study, we proposed to further identify additional alterations/features from genomic and spatial data to provide unprecedented new insight into intrinsic and adaptive resistant pathways in tumor cells that may assist to identify molecular targets for treatment. Materials: POETIC was a phase III trial of post-menopausal patients with ER/PR+ invasive BC (n = 4480) randomized 2:1 to 2 weeks of peri-operative AI (POAI) vs control, followed by standard-of-care treatment. Ki67 was assessed by IHC and intra-tumor heterogeneity was evaluated (5-15 regions) for all the POETIC POAI samples (N = 2487). ER+HER2+ samples were classified as good responders (GR) or poor responders (PR) based on a reduction in Ki67 between pre-treatment (pre-Txt) and 2-week on-Txt samples. Tumor-infiltrating lymphocytes were assessed; multiplex Immunofluorescence (mIF) was performed to measure immune cell densities in tumor and stroma compartments (CD3, CD20, CD68, FOXP3, and CD3 FOXP3 co-expression). Gene expression profiles by BC360™ (Nanostring) on all 210 pairs of POAI treated ER+/HER2+; whole exome sequencing (WES, 100X) were performed on pre-Txt tumor and blood samples from 13 GR, 17 PR, and 9 HER2E GR. We performed GeoMx Whole Transcriptome on 4 pairs (pre-Txt and on-Txt) of GR and GeoMx Proteins (77 including IO proteins) on 6 pairs of GRs and 6 pairs of PRs. Results: The most frequently mutated genes were TP53, PIK3CA, GATA3, and CHD4. Only TP53 was associated with PR (Fisher’s exact p=0.01). TP53 mutated cases had higher expression of TP53 mutant-like gene expression signature compared to wild-type cases (Wilcoxon test p=0.001), mIF FOXP3 (Wilcoxon test p = 0.0005), and CD68 (Wilcoxon test p = 0.019) density score. However, within the HER2-E subset, we found that TP53 mutations were associated with GR (Fisher’s exact p=0.02). We found spatial heterogeneity of Ki67 IHC levels across POAI samples. Examining IHC, while there was higher heterogeneity of Ki67 in the ER+HER2- samples (n = 2264) with 3% of pre-Txt and 9% on-Txt, 6% of ER+HER2+ samples (13/223, 6 LumA, 5 LumB, and 2 HER2E) showed heterogeneity of Ki67 exclusively on-Txt. Even in GR tumors with Ki67 &amp;lt; 10% on-Txt, we identified hotspots with retained proliferating Ki67+ cells after 2 weeks of POAI. The lobular tumors were GR and had characteristic CDH1 mutations. Importantly, cases with persistent areas of Ki67+ cells, regardless of Her2 status, were associated with late relapse. To further explore intratumoral heterogeneity, we performed spatial whole transcriptomics profiling on 95 regions from 4 pairs of GR samples (Ki67 &amp;gt; 10% at baseline and Ki67 &amp;lt; 10% on-Txt) and found low intratumoral heterogeneity in the pre-Txt samples that increased at 2 weeks on-Txt. In a larger set of samples including both GR and PR with the GeoMx protein method, we found increased intratumoral heterogeneity in the PR vs GR. Conclusion: While TP53 mutation was generally a predictor of poor response; in HER2-E it paradoxically was associated with a good early response to aromatase inhibitor which warrants further investigation. Ki67 levels in ER+HER2+ showed higher intratumoral heterogeneity in a subset of patients on treatment suggesting the potential of persistent, proliferating cells leading to later recurrence. Our spatial RNA and protein data further observe the intratumoral heterogeneity that identifies pathways for use as potential spatial biomarkers. Citation Format: Maggie Chon U Cheang, Xixuan Zhu, Orsolya Sipos, Anastasia Alataki, Mikayla Feldbauer, Elena López-Knowles, Holly Tovey, Lucy Kilburn, Milana Bergamino Sirvén, Dhrusti Patel, Hui Xiao, Perry Maxwell, Anthony Skene, Chris Holcombe, Manuel Salto-Tellez, Nicholas Turner, Andrew Dodson, Ian Smith, John Robertson, Judith Bliss, Gene Schuster, Roberto Salgado, Mitch Dowsett, Katherine A Hoadley. Genomic characterization of endocrine resistance in ER+HER2+ breast cancers in the POETIC Trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS09-08.

Contribution of tumour and immune cells to PD-L1 expression as a predictive biomarker in metastatic triple-negative breast cancer: exploratory analysis from KEYNOTE-119.

The efficacy of pembrolizumab monotherapy versus chemotherapy increased with increasing programmed death ligand 1 (PD-L1) expression, as quantified by combined positive score (CPS; PD-L1 expression on both tumour cells and immune cells) in patients with previously treated metastatic triple-negative breast cancer (mTNBC) in the phase 3 KEYNOTE-119 study. This exploratory analysis was conducted to determine whether the expression of PD-L1 on tumour cells contributes to the predictive value of PD-L1 CPS in mTNBC. PD-L1 expression in tumour samples was assessed using PD-L1 IHC 22C3 pharmDx and quantified using both CPS and tumour proportion score (TPS; PD-L1 expression on tumour cells alone). Calculated immune cell density (CID) was defined as CPS minus TPS. The ability of each scoring method (CPS, TPS, and CID) to predict clinical outcomes with pembrolizumab was evaluated. With pembrolizumab, the area under the receiver operating characteristic curve was 0.69 (95% CI = 0.58-0.80) for CPS, 0.55 (95% CI = 0.46-0.64) for TPS, and 0.67 (95% CI = 0.56-0.77) for CID. After correction for cutoff prevalence, CPS performed as well as, if not better than, CID with respect to predicting objective response rate, progression-free survival, and overall survival. Data from this exploratory analysis suggest that, although PD-L1 expression on immune cells alone is predictive of response to programmed death 1 blockade in mTNBC, adding tumour PD-L1 expression assessment (i.e. CPS, which combines immune cell and tumour cell PD-L1 expression) may improve prediction. PD-L1 CPS thus remains an effective and broadly applicable uniform scoring system for enriching response to programmed death 1 blockade with pembrolizumab in mTNBC as well as other tumour types.

Lectin-like Transcript-1 (LLT1) Expression in Oral Squamous Cell Carcinomas: Prognostic Significance and Relationship with the Tumor Immune Microenvironment.

Lectin-like transcript-1 (LLT1) expression is detected in different cancer types and is involved in immune evasion. The present study investigates the clinical relevance of tumoral and stromal LLT1 expression in oral squamous cell carcinoma (OSCC), and relationships with the immune infiltrate into the tumor immune microenvironment (TIME). Immunohistochemical analysis of LLT1 expression was performed in 124 OSCC specimens, together with PD-L1 expression and the infiltration of CD20+, CD4+, and CD8+ lymphocytes and CD68+ and CD163+-macrophages. Associations with clinicopathological variables, prognosis, and immune cell densities were further assessed. A total of 41 (33%) OSCC samples showed positive LLT1 staining in tumor cells and 55 (44%) positive LLT1 in tumor-infiltrating lymphocytes (TILs). Patients harboring tumor-intrinsic LLT1 expression exhibited poorer survival, suggesting an immunosuppressive role. Conversely, positive LLT1 expression in TILs was significantly associated with better disease-specific survival, and also an immune-active tumor microenvironment highly infiltrated by CD8+ T cells and M1/M2 macrophages. Furthermore, the combination of tumoral and stromal LLT1 was found to distinguish three prognostic categories (favorable, intermediate, and adverse; p = 0.029, Log-rank test). Together, these data demonstrate the prognostic relevance of tumoral and stromal LLT1 expression in OSCC, and its potential application to improve prognosis prediction and patient stratification.

Induction of Immunological Antitumor Effects by the Combination of Adenovirus-Mediated Gene Transfer of B7-1 and Anti-Programmed Cell Death-1 Antibody in a Murine Squamous Cell Carcinoma Model.

The goal of this study was to evaluate the antitumor immune effects of B7-1 gene expression in addition to immune checkpoint inhibitor against squamous cell carcinoma. A murine SCC cell line, KLN205, was infected with adenoviral vector carrying B7-1 (AdB7). Infected cells were injected subcutaneously in the flanks of DBA/2 mice. Three weeks after implantation, anti-mouse PD-1 antibody (antiPD1) was intraperitonially administrated twice a week for a total of six times. CD80 was significantly overexpressed in the AdB7-infected tumors. IFN-gamma in the T cells in the spleen was significantly increased and tumor size was significantly reduced in the mice treated with both AdB7 and antiPD1. Targeted tumors treated with both AdB7 and antiPD1 exhibited significantly increased cell densities of total immune cells as well as Ki-67+ CD8+ T cells and decreased regulatory T cells. These results suggest that the B7-1 gene transfer may enhance the antitumor effect of anti-PD1 antibody against SCC.

Extracellular vesicles derived from dendritic cells loaded with VEGF-A siRNA and doxorubicin reduce glioma angiogenesis in vitro

BackgroundNumerous attempts have been devoted to designing anti-angiogenic agents as a strategy to slow tumor growth and progression. Clinical applications of conventional anti-angiogenic agents face some challenges, e.g., off-target effects for TKIs and also low solid tumor penetration for mAbs. Furthermore, although anti-angiogenic therapy provides a normalization window for better chemo-RT response, in long-term treatments, tumor hypoxia as a result of total removal of VEGF-A by mAbs from the TME or complete blockade of TK receptors induces over-activation of compensatory angiogenic pathways, causing escape. Herein, we investigate the efficacy of si-DOX-DC-EVs to reduce glioma angiogenesis and invasiveness. MethodsMature DCs were generated from PBMC and EVs were isolated from the DCs culture media. siRNA and Doxorubicin were loaded into EVs by EP and incubation. Afterward, the uptake of DC-EVs was assessed by flow cytometry, and the subcellular localization of EVs was tested by confocal imaging. Tube formation assay was performed to assess the efficacy of si-DOX-DC-EVs to reduce tumor angiogenesis which was analyzed by DHM. Morphometric analysis of apoptotic cells was performed by DHM and confocal imaging and further, ELISA was performed for hypoxia-related and angiogenic cytokines. The impact of our theranostic system “si-DOX-DC-MVs” on the formation of vascular mimics, colonies, and invasion of C6 cells was checked in vitro. Afterward, orthotropic rat models of glioma were generated and the optimal administration route was selected by in vivo fluorescent analysis. Then, the microvessel density, vimentin expression, and accumulation of immune cells in tumoral tissues were assessed by IHC. Finally, necropsy and autopsy analyses were performed to check the safety of our theranostic agent. ResultsDC-EVs loaded with si-DOX-DC-EVs were successfully uptaken by cells with different subcellular trafficking for MVs and exosomes, reduced tumor angiogenesis in DHM analysis, and induced apoptosis in tumoral cells. Moreover, using DHM, we performed a detailed label-free analysis of tip cells which suggested that the tip cells in si-DC-MV treatments lost their geometrical migration capacity to form tube-like structures. Furthermore, the ELISAs performed highlighted that there is a mild overactivation of compensatory Tie2/Ang2 pathway after VEGF-A blockade which confers with severe hypoxia and sustains normal angiogenesis which is the optimal goal of anti-angiogenesis therapy for cancer to avoid resistance.The results of our VM analyses indicated that si-DOX-DC-MVs completely inhibited VM process. Moreover, the invasion, migration, and colony formation of the C6 cells treated with si-DOX-MVs were the least among all treatments. IN was the optimal route of administration. The MVD analyses indicated that si-DOX-DC-MVs reduced the number of tumoral microvessels and normalized vessel morphology. Intense CD8+ T cells were observed near the tumoral vessels in the si-DOX-DC-MVs group and with minimal activation of MT (low Vimentin expression). Necropsy and toxicology results proved that the theranostic system proposed is safe. ConclusionsDC-EVs loaded with VEGF-A siRNA and Doxorubicin were more potent than BV alone as a multi-disciplinary strategy that combats glioma growth by cytotoxic impacts of DOX and inhibits angiogenesis by VEGF-A siRNAs with excess immunologic benefits from DC-EVs. This next-generation anti-angiogenic agent normalizes tumor vessel density rather than extensively eliminating tumor vessels causing hypoxia and mesenchymal transition.

Abstract 1206: Three-dimensional immune atlas of pancreatic cancer precursor lesions reveals large inter- and intra-lesion heterogeneity

Abstract Immunotherapies are generally ineffective in pancreatic ductal adenocarcinoma (PDAC). Paradoxically, inflammation is believed to play a key role in PDAC development and invasion: patients suffering from chronic pancreatitis have a 13-fold increase in risk of developing PDAC. PDAC cells are surrounded by a dense network of fibrotic tissue containing immunosuppressive cells such as regulatory T cells, tumor-associated macrophages, and cancer-associated fibroblasts. Better understanding of the role of inflammation in PDAC could lead to the design of effective immunotherapies. Pancreatic intraepithelial neoplasia (PanIN) is a precursor to PDAC. While most of us will develop PanINs, few of these lesions will progress to invasive cancer. Efforts to determine which PanINs are likely to progress have shown that the size, incidence, and genetic variation of these lesions is high. Here, we add to these efforts through in-depth quantitative analysis of the pancreatic immune microenvironment. CODA is a novel 3D imaging technique used extensively to study the structure, transcriptomic signatures, and genetic heterogeneity of PanINs. In serial histological samples, CODA integrates image registration, cell detection, and segmentation of nine pancreatic structures: PanIN, normal ducts, islets, acini, nerves, vasculature, fat, stroma, and immune aggregates. Through integration with CD45, CD3, and FoxP3 labelled images, we explored immune infiltration in 48 large (cm3) samples of human pancreas tissues containing &amp;gt;1,000 PanIN lesions to describe spatial correlations between local immune hotspots, 3D tumor morphology, and the surrounding pancreas structures. We quantified inter- and intra-sample heterogeneity in immune response to pancreatic cancer precursor lesions. We determined that this cohort varied greatly in both the number of PanIN (range 3 - 92 per cm3) and overall immune cell density (range 3,000 - 40,000 immune cells/mm3). We determined that, while PanIN size is not related to local immune cell density, overall PanIN burden is highly correlated to global immune cell density. This suggests that PanIN content does not have a short-range relationship to inflammation, but does have a long-range relationship with pancreatic structures and immune infiltration. As chronic inflammation in the pancreas shows correlation with development of pancreatic cancer, increased knowledge of the heterogeneous, local immune cell environment in precancerous samples will help us determine the role of inflammation in reacting to and controlling malignant progression. Citation Format: Ashley L. Kiemen, Cristina Almagro-Pérez, Valentina Matos-Romero, Alicia M. Braxton, Jaanvi Mahesh-Babu, Elizabeth D. Thompson, Toby C. Cornish, PeiHsun Wu, Laura D. Wood, Arrate Muñoz-Barrutia, Ralph H. Hruban, Denis Wirtz. Three-dimensional immune atlas of pancreatic cancer precursor lesions reveals large inter- and intra-lesion heterogeneity [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 1206.

Abstract 3467: Tumor immune and microbial characteristics of colorectal cancer according to age at diagnosis and detailed anatomical location

Abstract Background: Colorectal cancer is a heterogeneous disease, exhibiting spectra of tumor characteristics that vary across age at diagnosis and anatomical location. We hypothesized that tumor immune and microbial characteristics of colorectal cancer might change with age at diagnosis and detailed anatomical location. Methods: We utilized a pooled dataset of colorectal cancer cases (N= 958 to1413 depending on variables) with available immune cell density or microbial data in the Nurses' Health Study, Health Professionals Follow-up Study, and Ontario Familial Colon Cancer Registry. We examined whether there were statistically significant trends in tumor immune and microbial according to age at diagnosis and detailed primary tumor location (cecum, ascending, hepatic flexure, transverse, splenic flexure, descending, sigmoid, rectosigmoid, rectum) using Spearman’s correlation test. The densities of immune cells were determined by using multiplex immunofluorescence, including T cells (CD3, CD4, CD8, CD45RA, CD45RO, FOXP3) and macrophages (CD68, M1-like, and M2-like) in tumor intraepithelial and stromal regions. We conducted stratified analyses by microsatellite instability (MSI) status. Results: The density of memory (both CD4+ and CD8+) T cells in tumor epithelial areas continuously increased as age at diagnosis increased (P for trend &amp;lt;0.005). Compared to later-onset colorectal cancer, early-onset colorectal cancer had a lower density of memory T-cells. With respect to tumor site, CD3+CD4+ and CD3+CD8+ T densities in tumor epithelial areas were highest in the cecum compared to other subsites, and there was a statistically significant decreasing trend from the cecum to rectum (P for trend &amp;lt;0.005). We also observed significant decreasing trends for CD68+, M1-like, and M2-like macrophage densities in combined tumor (intraepithelial and stromal) regions from the cecum to rectum (P for trend &amp;lt;0.005). The proportion of F. nucleatum-positive colorectal cancers gradually decreased from the cecum to rectum (P for trend &amp;lt;0.005). Similar trends were observed in separate analyses in non-MSI-high cases. Conclusions: This study suggests that certain tumor immune and microbial characteristics in the tumor microenvironment, specifically T cell and macrophage densities and Fusobacterium nucleatum positivity, change gradually according to age at diagnosis and/or detailed anatomical location. Age and anatomical location spectra in tumor immune and microbial characteristics will shed light on the etiology of colorectal cancer (including early-onset colorectal cancer), leading to better-personalized cancer prevention and treatment. Citation Format: Tomotaka Ugai, Yasutoshi Takashima, Yuxue Zhong, Juha P. Väyrynen, Daniel Buchanan, Jeroen Huyghe, Li Hsu, Conghui Qu, Sushma Thomas, Claire Thomas, Steve Gallinger, Robert Grant, Ulrike Peters, Amanda I. Phipps, Jonathan Nowak, Shuji Ogino. Tumor immune and microbial characteristics of colorectal cancer according to age at diagnosis and detailed anatomical location [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 3467.

Abstract 5532: Differences in immune contexture across metastatic sites and tissue compartments in ovarian cancer

Abstract Introduction: Characterizing the immune contexture within the tumor microenvironment (TME) can provide valuable biological insights that may impact treatment regimens and improve clinical outcomes. The advent of neoadjuvant chemotherapy has provided a unique opportunity to study matched, pre- and post- treatment sample pairs, but these samples are often obtained from different metastatic sites. Innate differences in the immune profiles across metastatic sites could therefore be a confounding factor when attempting to determine treatment-related effects. Furthermore, immune contexture can also vary by region within the TME, so results could vary depending on the regions of interest (ROIs) selected for analysis. Methods: 36 matched, advanced primary ovarian cancer samples were obtained pre- and post- treatment with neoadjuvant chemotherapy aimed at reducing tumor burden. The first biopsy was taken at the time of initial diagnosis, whereas the second biopsy was collected after 3-4 rounds of carboplatin paclitaxel chemotherapy. Each slide was processed using a 26-plex multiplex immunohistochemistry (mIHC) assay, which interrogates immune cell densities and functional states for a broad range of immune cell populations, including CD8+ T cells, Tregs, TH1s, B cells, dendritic cells (DCs), granulocytes, macrophages, and monocytes. Tumor and stroma ROIs were selected by a board-certified pathologist using H&amp;E slides immediately adjacent to the mIHC samples, and cell densities were calculated separately for each of the two tissue compartments. Results: When comparing pre-treatment samples from the omentum (n = 8) with post-treatment samples from the omentum (n = 14), all of the statistically significant changes were found within the tumor compartment, with higher densities in the post-treatment samples for CD8+ T cells (p = 0.009), Tregs (p = 0.012), DCs (p = 0.04), and monocytes (p = 0.027). In contrast, when comparing post-treatment samples from the omentum (n = 14) and the ovary (n = 14), all of the significant differences were found within the stroma compartment, with higher densities in the omentum for CD8+ T cells (p = 0.002), Tregs (p = 0.043), TH1s (p = 0.003), and macrophages (p = 0.048). Conclusions: These results demonstrate that the site of metastasis can be a significant confounding factor when making statistical comparisons between variables such as timepoint and that differences in immune cell density before and after treatment may be more apparent in the tumor, while differences between tissue types may be more apparent in the stroma. Further research into the immune profiles of different metastatic sites is warranted, but our findings demonstrate the importance of considering both tissue type and ROI selection strategy when designing a study with the intent to detect differences between longitudinal patient samples. Citation Format: Benjamin Tate, Elias Pavlatos, Teresa Kim, Kiaya Wilson, Erik Cid, Jian Rao, Gottfried Konecny, Joanna Pucilowska. Differences in immune contexture across metastatic sites and tissue compartments in ovarian cancer [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 5532.

Abstract 4912: Unsupervised detection of stromal phenotypes with distinct fibrogenic and inflamed properties in NSCLC

Abstract Background: Understanding the composition of cancer-associated stroma (CAS) is vital, as the number and location of immune cells and fibroblasts, as well as the degree of extracellular matrix deposition, have implications for cancer progression and response to treatment, including in non-small cell lung cancer (NSCLC). Manual analysis of CAS does not fully describe the stromal milieu, especially from a spatial perspective, and is highly subjective. To this end, we have developed an unsupervised machine learning (ML) model to characterize the CAS in NSCLC from hematoxylin and eosin (H&amp;E) stained whole slide images (WSI) at scale. Methods: PathExploreTM models were deployed to predict stromal tissue and cell types, while another ML model was used to detect collagen fibers from H&amp;E stained WSIs from the TCGA LUAD (N=536) and LUSC (N=464) datasets. Stroma was divided into small regions (median = 0.02 mm2), and 88 features characterizing cell distribution, tissue composition and fiber density were extracted from each region. Graphs were generated connecting neighboring regions (nodes), and an unsupervised variational graph auto-encoder (VGAE) model was trained to learn 8 latent features through dimensionality reduction. Stromal phenotypes were then derived from the latent features using k-means clustering. The fraction of each phenotype in the stroma was correlated against immune- and stroma-related gene expression signatures (GES) and overall survival (OS). Results: Deployment of VGAE on LUAD and LUSC WSIs revealed three distinct stromal phenotypes - P0, P1 and P2. Fibroblast density was elevated in P0 and P1 regions (p&amp;lt;0.001), immune cell density was elevated in P2 regions (p&amp;lt;0.001), and collagen fiber intensity was highest in P1 regions (p&amp;lt;0.001). P2 enrichment was correlated with elevated expression of the T cell-inflamed gene expression profile (TGEP; Spearman ρ = 0.43 in LUAD; ρ = 0.27 in LUSC) and with improved OS (HR = 0.696; 95% CIs: 0.571-0.847 in LUSC). Conversely, P1 enrichment was positively associated with a transforming growth factor-β-induced cancer associated fibroblast GES (TGFβ-CAF: ρ = 0.19 in LUAD and ρ = 0.12 in LUSC) and poor OS (HR = 1.358; 95% CIs: 1.149-1.603 in LUSC). These phenotypes are consistent with fibroblast-enriched, collagen-depleted stroma (P0), collagen-rich, fibroblast-enriched tumor-promoting stroma (P1), and immune cell-enriched, tumor-suppressive stroma (P2). Conclusions: We describe an unsupervised, data-driven method of predicting stromal regions with discrete patterns of cell composition and collagen deposition in NSCLC. This approach identified three phenotypes of NSCLC stroma. These results highlight the ability of ML models to characterize and find meaningful patterns within the cell, tissue, and matrix components of a tumor. This work provides further evidence of the potential of ML to discover novel precision medicine biomarkers in NSCLC. Citation Format: Neel Patel, Nhat Le, Tan Nguyen, Fedaa Najdawi, Sandhya Srinivasan, Adam Stanford-Moore, Deeksha Kartik, Jun Zhang, Jacqueline Brosnan-Cashman, Robert Egger, Justin Lee, Matthew Bronnimann. Unsupervised detection of stromal phenotypes with distinct fibrogenic and inflamed properties in NSCLC [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 4912.

Abstract 7630: Therapeutic relevance of systemic immune status to treatment effects of abemaciclib

Abstract Background: The addition of a CDK4/6 inhibitor (CDK4/6i) on endocrine therapies (ET) has been currently a standard treatment for hormone receptor (HR)-positive HER2-negative metastatic breast cancers (MBCs). Especially abemaciclib has been implicated in multiple mechanisms of action including cell cycle arrest, senescence. In addition, immunomodulatory effects have been suggested but are not yet clear. The aim of the study was to investigate the clinical relevance of abemaciclib to systemic immune status by examining the status of peripheral immune cells in patients with MBC who received abemaciclib plus ET. Methods: Patients with MBC who were treated with abemaciclib plus ET in our hospital were included in this study. Response was evaluated by RECIST 1.1. Peripheral blood at the initiation of abemaciclib treatment, 4 weeks, 12 weeks and 24weeks after abemaciclib treatment initiation were analyzed using mass cytometry by time of flight (CyTOF). Changes of cellular densities of each immune cell and survival by immune cell density were analyzed without calculating p values because of small sample number. Results: Twenty patients were enrolled, among whom 14 and 6 received an aromatase inhibitor and fulvestrant, respectively, as concomitant ET. 7 premenopausal patients received an LH-RH analogue. The analysis of serial blood collection showed an increase in CD8-positive T cell density by 12.5% and conversely a decrease in monocyte density by 52.3% at 4 weeks after treatment initiation. In responders (N = 10), an increase of the ratio between CD8-positive T cell density and Treg density (CD8/Treg) at 4 weeks after the treatment initiation were observed (65.2%), while not in non-responders (7.3%). In the analysis of the blood samples at the treatment initiation, lower Treg density and lower NK density at treatment initiation indicated longer PFS than the counterparts (median PFS for Treg: low, not reached; high, 8.8 months; for NK: low, not reached; high, 8.8 months). Conclusion: An increase of CD8/Treg at 4 weeks after the treatment initiation was associated with a good response in patients with HR-positive HER2-negative MBC treated with abemaciclib plus ET. Our results suggest an association between systemic immune status and clinical outcome in MBC patients who received abemaciclib plus ET treatment. A larger study is needed to validate the results. Citation Format: Takayuki Ueno, Shigehisa Kitano, Takayuki Kobayashi, Makiko Yamashita, Fumikata Hara, Yukinori Ozaki, Meiko Nishimura, Toshimi Takano, Naoko Toriguchi, Tsutomu Kawaguchi. Therapeutic relevance of systemic immune status to treatment effects of abemaciclib [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 7630.

Abstract 169: Prevalence, immune checkpoint expression and spatial interplay of intraepithelial lymphocytes, macrophages, and dendritic cells in 4740 human carcinomas

Abstract Background: Although there is raising evidence that the density and the level of immune checkpoint expression of immune cell subpopulations that are in direct contact to the tumor cells (intraepithelial) can predict response to immune checkpoint therapy and patient’s outcome, a comprehensive assessment of intraepithelial immune cell subpopulations and their spatial interplay is lacking. Material and methods: To comprehensively assess the density, level of immune checkpoint expression, and spatial interplay of 48 immune checkpoint expressing intraepithelial leukocyte subpopulations in 46 carcinoma entities, 4740 tumor samples in a tissue microarray format were stained with 21 antibodies using our BLEACH&amp;STAIN mfIHC approach. A deep learning-based framework comprising 18 different convolutional neuronal networks (U-Net and DeepLabv3+) were used for image analysis. Results: The mean intraepithelial immune cell density of CD8+ cytotoxic T-cells, CD4+ T-helper cells, FOXP3+ Tregs, CD20+ B-cells, M1/M2 macrophages and CD11c+ dendritic cells varied markedly between tumor entities and individual tumors. For example, 88 (±90) cells/mm2 were found in tubular breast cancer, 661 (±729) cells/mm2 in colorectal cancer, and 1280 (±1334) to 2325 (±2131) cells/mm2 in squamous cell cancers from various origins. The abundance of T-cell subpopulations, M2 macrophages, and dendritic cells was highly concordant (range r=0.49 to 0.92) across all tumor entities, while the density of M1 macrophages was unrelated (r=0.1, p&amp;lt;0.001). Unsupervised hierarchical clustering across all tumor entities revealed a cluster A of 634 patients from almost all different tumor entities with an exceptionally high density of intraepithelial immune cells, thus a highly inflamed tumor microenvironment that was characterized by the strongest interaction between intraepithelial CD8+ T-cell and CD4+ T-helper cells, M2 macrophages, as well as dendritic cells (DC, p&amp;lt;0.002 each) along with the highest expression level of TIM3, PD-1, CLTA-4 and PD-L1. In contrast, a cluster C of 2245 patients with the lowest intraepithelial immune cell infiltration was characterized by a significantly increased M1 proportion (p&amp;lt;0.001) and an enriched proportion of regulatory T-cells (p&amp;lt;0.001). Across all analyzed tumor entities, the intraepithelial highly inflamed cluster A was significantly linked to low pT (p&amp;lt;0.001). Conclusion: The data from this study provide a comprehensive characterization of intraepithelial immune cells across 46 different human carcinomas and identify an inflamed phenotype defined by strong interactions of intraepithelial CD8+ cytotoxic T-cells, CD4+ T-cells, dendritic cells, and M2 macrophages along with highest levels of TIM3, PD-1, PD-L1, and CTLA-4 expression that is linked to a favorable tumor phenotype. Citation Format: Zhihao Huang, Tim Mandelkow, Jan H. Müller, Andreas H. Marx, Till Krech, Guido Sauter, Julia Ebner, Maximilian Lennartz, Ronald Simon, Martina Kluth, Claudia Hube-Magg, Sarah Minner, Elena Bady, Niclas C. Blessin. Prevalence, immune checkpoint expression and spatial interplay of intraepithelial lymphocytes, macrophages, and dendritic cells in 4740 human carcinomas [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 169.

Abstract 5528: Adaptive immune cells in colorectal cancer between normal mucosa, primary tumor and liver metastasis

Abstract Introduction: We aimed to assess the role of local adaptive immunity in the metastatic process by comparing densities of adaptive immune cells between normal colorectal mucosa (NM), primary colorectal cancer (pCRC) and liver metastases (LM) in patients with synchronous and metachronous disease. Patients and Methods: We enrolled patients, who underwent resection of pCRC and LM in Pilsen University Hospital between 1999 and 2022. 56 patients presented with LM (stage IV, synchronous) and 43 patients (stage II/III) developed LM later (metachronous). After immunohistochemical staining and whole slide scanning densities of CD3+ and CD8+ T cells and CD20+ B cells were quantified in NM, pCRC and LM using QuPath software. In pCRC and LM cell densities were measured in tumor center (TC), inner margin (IM), outer margin (OM) and peritumor (PT) region. IM and OM were defined as 500 µm on each side of the invasive tumor border. Results: For all examined cells in both groups we found smaller densities in TC of pCRC and LM compared to NM (Table). In pCRC of synchronous group compared to LM densities of all cells were smaller in IM and OM and densities of CD3+ and CD8+ cells were smaller in PT region. In pCRC of metachronous group compared to LM densities of all cells were greater in TC, densities of CD3+ were smaller in OM and PT region, whereas densities of CD20+ cells were smaller only in OM. Compared to metachronous group patients with synchronous metastases had smaller densities of CD3+ T cells in IM and CD8+ T cells in TC and IM of pCRC. Conclusion: Development of pCRC is accompanied by decreased densities of T and B cells in TC compared to NM, which further decrease in metachronous LM. Lower densities of T and B cells in IM and OM and T cells in PT region of pCRC compared to LM is a hallmark of synchronous group. TC of pCRC harbors larger numbers of T and B cells compared to LM in metachronous group. Low numbers of CD3+ and CD8+ T cells in IM and CD8+ T cells in TC of primary CRC may contribute to development of synchronous metastases. Densities of CD3+, CD8+ and CD20+ cells per mm2 of the tissue (median) Patients witn liver metastases Synchronous Metachronous NM pCRC LM NM pCRC LM CD3 NM 1014 1042 CD3 TC 313p&amp;lt;0.05 to NM 249p&amp;lt;0.05 to NM 338p&amp;lt;0.05 to NM 199p&amp;lt;0.05 to NM and pCRC CD3 IM 252 431p&amp;lt;0.05 to pCRC 393p&amp;lt;0.05 to pCRC in synchronous group 483 CD3 OM 521 1437p&amp;lt;0.05 to pCRC 649 1953p&amp;lt;0.05 to pCRC CD3 PT 479 873p&amp;lt;0.05 to pCRC 447 987p&amp;lt;0.05 to pCRC CD8 NM 319 390 CD8 TC 58p&amp;lt;0.05 to NM 52p&amp;lt;0.05 to NM 102p&amp;lt;0.05 to NM and to pCRC in synchronous group 52p&amp;lt;0.05 to NM and pCRC CD8 IM 65 96p&amp;lt;0.05 to pCRC 102p&amp;lt;0.05 to pCRC in synchronous group 120 CD8 OM 206 559p&amp;lt;0.05 to pCRC 310 363 CD8 PT 177 369p&amp;lt;0.05 to pCRC 218 291 CD20 NM 331 215 CD20 TC 32p&amp;lt;0.05 to NM 11p&amp;lt;0.05 to NM 27p&amp;lt;0.05 to NM 12p&amp;lt;0.05 to NM and pCRC CD20 IM 20 39p&amp;lt;0.05 to pCRC 20 33 CD20 OM 137 327p&amp;lt;0.05 to pCRC 134 303p&amp;lt;0.05 to pCRC CD20 PT 153 224 196 213 Citation Format: Andriy Trailin, Wenjing Ye, Esraa Ali, Sergii Pavlov, Lenka Cervenkova, Filip Ambrozkiewicz, Ondrej Vycital, Ondrej Daum, Vaclav Liska, Kari Hemminki. Adaptive immune cells in colorectal cancer between normal mucosa, primary tumor and liver metastasis [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 5528.

Abstract 2312: Integrating spatial multi-omics data with spatial quantitative pharmacology (spQSP) model to simulate human neoadjuvant immunotherapy clinical trial of hepatocellular carcinoma

Abstract Human clinical trials provided tremendous insights to advance novel systemic therapies and to improve treatment outcomes for cancer patients. The few durable treatment options have led to a critical need to advance new therapeutics in hepatocellular carcinoma (HCC). Recent human clinical trials have demonstrated that new combination of immunotherapeutic regimens provide augmented clinical response in a subset of patients. Computational methods that can simulate tumors from mathematical equations describing cellular and molecular interactions are emerging as promising tools to simulate the impact of therapies entirely in silico. To facilitate designing dosing regimens and selecting potential predictive biomarkers, we developed a new computational model to track tumor progression at organ scale while reflecting the spatial heterogeneity in the tumor at cellular and molecular scale in HCC. This computational model is called a spatial quantitative systems pharmacology (spQSP) platform and it is also designed to simulate the effects of combination immunotherapy. The model is first calibrated using clinical outcomes and measured immune cell densities based on recent neoadjuvant HCC clinical trial combining anti-PD-1 immunotherapy and a multitargeted tyrosine kinase inhibitor (TKI) cabozantinib (NCT03299946). Then, we validate the results from the spQSP model by leveraging real-world spatial multi-omics data from the same clinical trial. The model output is compared with spatial data from Imaging Mass Cytometry (IMC). Both IMC data and simulation results suggest closer proximity between CD8 T cell and Arginase 1 (Arg1) positive macrophages among non-responders while the reverse trend was observed for responders. We also compared the model output with Visium spatial transcriptomics analyses of samples from post-treatment tumor resections in the original clinical trial. Both spatial transcriptomic data and simulation results identify the role of spatial patterns of tumor vasculature and TGFβ in tumor and immune cell interactions. To our knowledge, this is the first spatial tumor model for virtual clinical trials at a molecular and cellular scale that is grounded in high-throughput spatial multi-omics data from a human clinical trial. Citation Format: Shuming Zhang, Atul Deshpande, Babita K. Verma, Hanwen Wang, Haoyang Mi, Long Yuan, Won Jin Ho, Elizabeth M. Jaffee, Qingfeng Zhu, Robert A. Anders, Mark Yarchoan, Luciane T. Kagohara, Elana J. Fertig, Aleksander S. Popel. Integrating spatial multi-omics data with spatial quantitative pharmacology (spQSP) model to simulate human neoadjuvant immunotherapy clinical trial of hepatocellular carcinoma [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 2312.

Abstract 2916: Discoidin domain receptor 1 (DDR1) expression is associated with degree of immune exclusion across epithelial tumors

Abstract Background: Discoidin domain receptor 1 (DDR1) is highly expressed in epithelial cancers and has been implicated in tumor growth, invasion, and lack of response to therapy. DDR1 contributes to immune exclusion by promoting tumor collagen alignment in in vivo models. However, it is unclear how DDR1 expression impacts immune cell infiltration in human tumors. A first-in-human trial of PRTH-101, a DDR1-targeted therapeutic antibody, is underway. Establishing a correlation between DDR1 expression and immune infiltration in the tumor microenvironment (TME) will shed light on the role of DDR1 in the TME and inform indication and patient selection strategies for DDR1-targeted therapies. Methods: Adjacent formalin fixed paraffin embedded slides from colorectal (CRC), non-small cell lung (NSCLC), ovarian (OC), pancreatic (PDAC), and triple-negative breast cancers (TNBC) were stained by H&amp;E and a multiplex immunofluorescence (mIF) panel containing DDR1 and immune cell markers CD8 and CD45. Tumor-stromal segmentation and cell identification was done from H&amp;E using AI-powered models developed by PathAI (PathExplore™) or from mIF by image analysis using QuPath. DDR1 mRNA expression was measured by bulk RNA-sequencing, while DDR1 protein expression was measured by mIF. Immune Exclusion Scores (IESs) were calculated for each tumor based on lymphocyte density (from H&amp;E), CD8+ T cell density (from mIF), or CD45+ cell density (from mIF). Each IES is the orthogonal distance between the coordinate of immune cell density in tumor epithelium and stroma, and the regression line representing equal density in epithelium and stroma. Correlations between lymphocyte, CD8, and CD45 IESs and DDR1 mRNA or protein expression levels were evaluated. Results: Both DDR1 mRNA and protein expression levels were significantly correlated with all Immune Exclusion Scores (IESs) measured by the distribution of lymphocytes (H&amp;E), CD8+, or CD45+ cells (mIF) in tumor epithelium and stroma (R: 0.31-0.55, adjusted p: &amp;lt;0.001-0.01) at a pan-cancer level. While the degree of the correlation varied between tumor indications by IES immune cell type, pancreatic cancer exhibited the strongest correlation between the lymphocyte-based IES and DDR1 mRNA and protein expression (R: 0.48-0.54, adjusted p: 0.04-0.07). DDR1 mRNA was also moderately correlated with lymphocyte IES in NSCLC and TNBC (R: 0.37-0.48, adjusted p: 0.12-0.14). Conclusions: We developed a continuous scoring method to quantify the degree of immune exclusion in tumors based on the spatial distributions of lymphocytes, CD8+ T cells, and CD45+ immune cells from H&amp;E and mIF images. DDR1 mRNA and protein expression are correlated with immune exclusion at both the pan-cancer and specific indication level. This adds additional insight into the role of DDR1 in human cancers and may be useful in selecting indications and stratifying patients for DDR1-targeted therapies. Citation Format: Xinwei Sher, Fredrick D. Gootkind, Thomas Schürpf, Florent Peyraud, Jean-Philippe Guégan, Antoine Italiano, G. Travis Clifton, Laura A. Dillon. Discoidin domain receptor 1 (DDR1) expression is associated with degree of immune exclusion across epithelial tumors [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 2916.