Pancreatic Ductal Adenocarcinoma Samples Research Articles

Dysbiosis Signature of Fecal Microbiota in Patients with Pancreatic Adenocarcinoma and Pancreatic Intraductal Papillary Mucinous Neoplasms.

Pancreatic cancer (PC) ranks as the seventh leading cause of cancer-related deaths, with approximately 500,000 new cases reported in 2020. Existing strategies for early PC detection primarily target individuals at high risk of developing the disease. Nevertheless, there is a pressing need to identify innovative clinical approaches and personalized treatments for effective PC management. This study aimed to explore the dysbiosis signature of the fecal microbiota in PC and potential distinctions between its Intraductal papillary mucinous neoplasm (IPMN) and pancreatic ductal adenocarcinoma (PDAC) phenotypes, which could carry diagnostic significance. The study enrolled 33 participants, including 22 diagnosed with PDAC, 11 with IPMN, and 24 healthy controls. Fecal samples were collected and subjected to microbial diversity analysis across various taxonomic levels. The findings revealed elevated abundances of Firmicutes and Proteobacteria in PC patients, whereas healthy controls exhibited higher proportions of Bacteroidota. Both LEfSe and Random Forest analyses indicated the microbiome's potential to effectively distinguish between PC and healthy control samples but fell short of differentiating between IPMN and PDAC samples. These results contribute to the current understanding of this challenging cancer type and highlight the applications of microbiome research. In essence, the study provides clear evidence of the gut microbiome's capability to serve as a biomarker for PC detection, emphasizing the steps required for further differentiation among its diverse phenotypes.

Comprehensive Genomic Studies on the Cell Blocks of Pancreatic Cancer.

Pancreatic cancer is one of the deadliest malignancies, characterized by late-stage diagnosis and limited treatment options. Comprehensive genomic profiling plays an important role in understanding the molecular mechanisms underlying the disease and identifying potential therapeutic targets. Cell blocks (CBs), derived from EUS-FNA, have become valuable resources for diagnosis and genomic analysis. We examine the molecular profile of pancreatic ductal adenocarcinoma (PDAC) using specimens obtained from CB EUS-FNA, across a large gene panel, within the framework of next-generation sequencing (NGS). Our findings revealed that over half (55%) of PDAC CB cases provided adequate nucleic acid for next-generation sequencing, with tumor cell percentages averaging above 30%. Despite challenges such as low DNA quantification and degraded DNA, sequencing reads showed satisfactory quality control statistics, demonstrating the detection of genomic alterations. Most cases (84.6%) harbored at least one gene variant, including clinically significant gene mutation variants such as KRAS, TP53, and CDKN2A. Even at minimal concentrations, as long as the extracted DNA is of high quality, performing comprehensive molecular profiling on PDAC samples from cell blocks has remained feasible. This strategy has yielded valuable information about the diagnosis, genetic landscape, and potential therapeutic targets, aligning closely with a precision cytopathology approach.

Excavation of gene markers associated with pancreatic ductal adenocarcinoma based on interrelationships of gene expression.

Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% of all pancreatic cancer cases, posing grave challenges to its diagnosis and treatment. Timely diagnosis is pivotal for improving patient survival, necessitating the discovery of precise biomarkers. An innovative approach was introduced to identify gene markers for precision PDAC detection. The core idea of our method is to discover gene pairs that display consistent opposite relative expression and differential co-expression patterns between PDAC and normal samples. Reversal gene pair analysis and differential partial correlation analysis were performed to determine reversal differential partial correlation (RDC) gene pairs. Using incremental feature selection, the authors refined the selected gene set and constructed a machine-learning model for PDAC recognition. As a result, the approach identified 10 RDC gene pairs. And the model could achieve a remarkable accuracy of 96.1% during cross-validation, surpassing gene expression-based models. The experiment on independent validation data confirmed the model's performance. Enrichment analysis revealed the involvement of these genes in essential biological processes and shed light on their potential roles in PDAC pathogenesis. Overall, the findings highlight the potential of these 10 RDC gene pairs as effective diagnostic markers for early PDAC detection, bringing hope for improving patient prognosis and survival.

Abstract 7643: Prognostic significance and immune infiltration patterns related to Claudin heterogeneity in pancreatic ductal adenocarcinoma patients

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) stands out for its aggressive nature, often manifesting through invasion, metastasis, and recurrence. These characteristics are partly attributable to the Epithelial-mesenchymal transition (EMT), a critical phenomenon that follows the disintegration of epithelial barriers, including tight junctions (TJs), where Claudin (CLDN) proteins are integral components. Objective: This study focused on elucidating the differential expression of claudins in PDAC and exploring their correlations with disease progression, invasiveness, and immune infiltration. Methodology: Utilizing UCSC TOIL RNA-seq, TCGA-PAAD genomic, and clinical data, we investigated the genomic alterations and differential expression of claudins in PDAC. Conducted survival analysis using log-rank tests. Assessed immune infiltration patterns using the EPIC deconvolution algorithm. Results: Of 177 PDAC samples examined, 23 (13%) exhibited at least one form of claudin gene alteration, including mutations, amplifications, or deep deletions. Remarkably, 10 claudins (CLDN1, 2, 4, 5, 7, 11, 12, 15, 18, and 23) were found to be significantly overexpressed in PDAC samples, meeting a log2FC threshold of ≥1 (p < 0.05). Further, we identified a direct correlation between mRNA expression and copy numbers, alongside an inverse relationship with methylation levels, particularly in CLDN1, CLDN12, CLDN15, CLDN23, and CLDN4. Intriguingly, claudin expression escalated from grades G1 to G3 but diminished in G4, with CLDN1, CLDN4, and CLDN7 showing significant variability (Kruskal-Wallis rank test p-values of 1.2E-5, 0.00064, and 0.023, respectively). Survival analysis indicated that high expression levels of CLDN1 and CLDN4 were linked to poorer patient outcomes, while higher levels of CLDN5 and CLDN15 correlated with improved survival (log-rank p-values of 0.007, 0.016, 0.0035, and 0.0012, respectively), findings consistent across both univariate and multivariate analyses. Furthermore, immune infiltration patterns, assessed via the EPIC deconvolution algorithm, varied significantly between high and low CLDN expression groups. Specifically, low CLDN1 expression was associated with increased presence of B-cells, CAF, and CD8+ T-cells but fewer endothelial cells, a pattern reversed in high CLDN1 expression cases. A similar trend was observed for CLDN4. Conclusion: Our study underscores the significant prognostic value of claudin proteins in PDAC, highlighting their potential as indicators of disease progression and survival and their influence on the tumor's immune environment. Citation Format: Ajaz A. Bhat, Shahabuddin Usmani, Hana Q. Sadida, Sheema Hashem, Tariq Masoodi, Ikhlak Ahmed, Rakesh Kumar, Mayank Singh, Punita Dhawan, Shahab Uddin, Muzafar A. Macha, Ammira S. Al-Shabeeb Akil. Prognostic significance and immune infiltration patterns related to Claudin heterogeneity in pancreatic ductal adenocarcinoma patients [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 7643.

Abstract 4414: Genome-wide DNA methylation classifier diagnoses pancreatic ductal adenocarcinoma in CUP setting

Abstract Introduction: The metastasis of a pancreatic ductal adenocarcinoma (PAAD) is a diagnosis of exclusion and one of the most common causes of cancer of unknown primary (CUP). We have recently developed a genome-wide DNA methylation-based neural network classifier that can accurately differentiate between liver metastasis of a PAAD and intrahepatic cholangiocarcinoma (iCCA) (PAAD-iCCA-Classifier). Therefore, the aim of our study was to test whether our PAAD-iCCA-Classifier can be extended to be used to correctly diagnose PAAD metastases from other sites in CUP setting. Methods: For this purpose, we enhanced the anomaly detection layer of the classifier by incorporating ten mimicker carcinomas to be excluded by this layer. We used a validation set 1 (n=3786) including primary PAAD (n=242), PAAD liver metastases (n=20), iCCA (n=151) and 10 other mimicker carcinomas (n=3373) and a validation set 2 (n=26) including primary PAAD from a real-life clinical cohort from an independent institution to validate the classifier. Next, we tested the classifier on 16 PAAD initially considered CUP samples (test set) from different sites: peritoneum, lung, liver, and lymph node. The clinical history and diagnostic imaging of these samples were used to confirm PAAD as the most probable origin. We further performed differentially methylated probes (DMP) and copy number alterations (CNA) analysis of primary PAAD and metastatic PAAD from different locations. Results: The improved version of the PAAD-iCCA-Classifier achieved an accuracy of 98.43% on the validation set 1 and was able to exclude most of the mimicker carcinomas. On validation set 2, the classifier achieved an accuracy of 88.46%. Medical history, imaging and immunohistochemical analysis of the test set samples confirmed the diagnosis of PAAD. The DNA methylation classifier correctly labeled 15/16 PAAD metastatic samples as PAAD (93.75% accuracy). We observed that the classifier performance was negatively affected by a high CD3+ immune infiltrate and positively affected by high tumor purity and high proliferation rate. CNA revealed that PAAD liver metastases have a distinct CNA profile characterized by chromosome 6, 9 (CDKN2A/B) and 18q (SMAD4) deletions. DMP analysis showed that PAAD liver metastases have global hypomethylation of both promoter- and enhancer-associated CpGs compared to primary PAAD and PAAD peritoneal carcinomatosis. Finally, gene ontology analysis revealed that different epithelial-mesenchymal transition pathways are activated in PAAD liver metastases compared to PAAD peritoneal carcinomatosis. Conclusion: Our tool performs well in classifying metastatic PAAD samples and could be of great clinical use when a PAAD origin is suspected in the case of a CUP. DMP and CNA profiles show that PAAD liver metastases may have a distinct DNA methylation and copy number profile compared to primary and peritoneal carcinomatosis PAAD. Citation Format: Teodor G. Calina, Eilís Perez, Simon Schallenberg, Horst David, Erik Knutsen, David Capper, Mihnea P. Dragomir. Genome-wide DNA methylation classifier diagnoses pancreatic ductal adenocarcinoma in CUP setting [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 4414.

Abstract 3878: Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis

Abstract The aggressive nature of pancreatic ductal adenocarcinoma (PDAC) is driven by cell-intrinsic features and cell-extrinsic interactions between tumor cells and the desmoplastic stroma, which is infiltrated with heterogeneous populations of cancer-associated-fibroblasts (CAFs) and immune cells. These features are able to drive emergent properties such as chemoresistance through diverse and not yet fully elucidated mechanisms. Through single-nucleus RNA-seq and whole-transcriptome digital spatial profiling of PDAC patient tumors, we previously reported marked heterogeneity in the malignant cells and CAF composition of PDAC. In this project, we performed high-plex single-cell spatial molecular imaging (990-plex SMI, Nanostring) to dissect the therapy-remodeled multicellular neighborhoods and malignant-stroma cell interactions in primary resected human pancreatic cancer with (n = 6) or without (n = 7) neoadjuvant chemotherapy and radiotherapy. SMI of the PDAC samples yielded ~753,000 high-confidence single cells and captured the heterogeneity of malignant cells and CAFs. We identified three malignant subtypes: classical (CLS), basal-like (BSL), and the recently characterized neural-like progenitor (NRP) malignant subtype that exhibits stem-like and neurodevelopmental features. To leverage the single-cell spatial resolution of SMI to further dissect treatment-associated changes in cell-cell interactions, we developed Spatially Constrained Optimal Transport Interaction Analysis (SCOTIA), an optimal transport model with a cost function that includes both spatial distance and ligand-receptor gene expression. Our results uncovered a marked enrichment in ligand-receptor interactions between CAFs and malignant cells in response to treatment. In combination with several complementary methods, including examination of downstream target gene expression and ex vivo 3D co-culture of CAF and PDAC cells, we found that IL-6 family signaling was the most consistently enriched interaction between CAFs and malignant cells after treatment. This led us to hypothesize that IL-6 signaling between CAFs and malignant cells may induce chemoresistance in cancer cells. To test this hypothesis, we treated three human cancer cell lines (AsPC1, Panc1, Panc0203) with 5FU with or without IL-6 and discovered that IL-6 induced chemoresistance for two of the lines (Panc1, Panc0203). Taken together, we have integrated novel experimental and computational approaches to enable high-resolution, spatially-guided discovery of treatment-associated remodeling in the pancreatic cancer microenvironment. Further studies will be conducted to dissect additional mechanisms by which candidate cell-cell interactions promote disease progression and/or resistance to cytotoxic therapy, with the ultimate goal of guiding novel therapeutic development. Citation Format: JINGYI CAO, Carina Shiau, Dennis Gong, Mark T. Gregory, Xunqin Yin, Jae-Won Cho, Peter L. Wang, Jennifer Su, Jason W. Reeves, Jimmy A. Guo, Nicole A. Lester, Jung Woo Bae, Ryan Zhao, Martin Hemberg, William L. Hwang. Therapy-associated remodeling of pancreatic cancer revealed by single-cell spatial transcriptomics and optimal transport analysis [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 3878.

Abstract 1395: Unraveling the interplay between tumor heterogeneity and microenvironment in pancreatic ductal adenocarcinoma evolution and its clinical implications

Abstract Intratumoral heterogeneity (ITH) and tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) play important roles in tumor evolution and patient outcomes. However, the precise characterization of diverse cell populations and their crosstalk associated with PDAC progression and metastasis is still challenging. We performed single-cell RNA sequencing of primary PDAC samples with and without paired liver metastasis samples to understand the interplay between ITH and TME in the PDAC evolution and its clinical associations. We discovered that even a small proportion (~30%) of basal-like ductal cells in primary PDAC was associated with poor chemotherapy response and patient survival and that epithelial-mesenchymal transition programs were largely subtype specific. The clonal homogeneity significantly increased with more prevalent and pronounced copy number gains of oncogenes, such as KRAS and ETV1, and losses of tumor suppressor genes, such as SMAD2 and MAP2K4, along PDAC progression. Diverse immune cell populations, including naive SELLhi regulatory T cells (Tregs) and activated TIGIThi Tregs, contributed to shaping immunosuppressive TMEs of PDAC through cellular interactions with malignant ductal cells. The proportion of basal-like ductal cells negatively correlated with that of immunoreactive cell populations, such as cytotoxic T cells, but positively correlated with that of immunosuppressive cell populations, such as Tregs. Overall, our study provides a valuable resource for better understanding the roles of ITH and TME in PDAC evolution and their clinical relevance in the treatment of PDAC. Citation Format: Hyemin Kim, Hyong-oh Jeong, Se-Hoon Lee, Kwang Hyuck Lee, Kyu Taek Lee, Jong Kyun Lee, Min Woo Lee, Semin Lee, Joo Kyung Park. Unraveling the interplay between tumor heterogeneity and microenvironment in pancreatic ductal adenocarcinoma evolution and its clinical implications [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 1395.

Abstract 1603: Intra-pancreatic fat promotes the progression of PDAC by activating thermogenesis

Abstract Background: The presence of minimal intra-pancreatic fat deposition (IPFD) in the healthy human pancreas has been demonstrated in numerous studies. But excess IPFD, or fatty pancreas disease, leads to a wide array of diseases, including but not limited to diabetes, pancreatitis, and pancreatic ductal adenocarcinoma (PDAC). Remarkably, PDAC patients with excessive intra-pancreatic fat accumulation are commonly associated with a poor prognosis compared to those with minimal deposition. However, the interaction between tumor and intra-pancreatic adipose tissue remains elusive. Here, we evaluated the pancreatic fat infiltration in normal people and patients with PDAC respectively, and explored the mechanism of how intra-pancreatic fat affects the progression of PDAC. Methods: Magnetic resonance imaging (MRI) and clinical biopsy specimens were used to assess the degree of pancreatic fat infiltration in PDAC patients and healthy control individuals. The C57BL/6J mice injected orthotopically with KPC cells and adipocytes, high-fat diet (HFD)-induced obesity KC (Pdx1-Cre; KrasLSL-G12D/WT) mice, and KC; ob/ob mice were established to dynamically observe how adipocytes affect tumor progression in vivo. The snRNA-seq of tumor samples and proteomics of conditional medium of PDAC cell lines were performed to identify the mechanism of interaction between cancer cells and adipocytes. Results: We found that patients with PDAC presented excess intra-pancreatic fat deposition more frequently than healthy individuals, and the degree of fat infiltration was positively correlated with the tumor burden of PDAC patients. HFD-induced obesity KC mice and KC; ob/ob mice showed a higher degree of IPFD, which contributed to tumor growth and low survival rates of mice. Additionally, the expression of uncoupling protein 1 was upregulated in PDAC patients and KPC (Pdx1-Cre; KrasLSL-G12D/WT; p53LSL-R172H/WT) mice compared to normal controls. Constantly, the snRNA-seq of PDAC samples demonstrated the increase in thermogenic signature. In vitro, C3H10T1/2 cells treated with conditional medium from pancreatic cancer cell lines or co-cultured with pancreatic cancer cell lines showed increased expression of browning markers and elevated oxygen consumption rate. Specifically, combined analysis of the proteomic of T3M4 and CFPAC-1 conditional medium and the snRNA-seq revealed that pancreatic cancer cells secreted integrin β4 (ITGB4) to improve the thermogenesis of intra-pancreatic adipocytes (IPA), which in turn promoted cancer cell progression. Conclusion: Parts of PDAC patients present excess intra-pancreatic fat accumulation, a process associated with tumor growth and poor prognosis. Mechanistically, pancreatic cancer cells activate the thermogenesis of IPA by secreting ITGB4, and then the thermogenetic adipocytes fuel the progression of the tumor in response. Citation Format: Xinpeng Yin, Yuan Chen, Ruiyuan Xu, Chenglin Hu, Chengcheng Wang, Yupei Zhao. Intra-pancreatic fat promotes the progression of PDAC by activating thermogenesis [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 1603.

Abstract 4277: CREB-ROCK driven extracellular matrix remodeling exasperates pancreatic cancer progression

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by a heightened oncogenic mutational burden, an immunosuppressive tumor microenvironment, and a dense desmoplastic stroma. During the tumor progression from in situ carcinoma to intraductal neoplasia, cancer cells manage to evade the stromal compartment and infiltrate into the ducts. This invasive tumor progression is often associated with the overexpression of extracellular matrix (ECM) modulating genes. Despite this correlation, there are currently no effective and specific inhibitors of ECM-altering pathways. By understanding the signaling mechanism, the ECM remodeling pathways may be intercepted to prevent invasive tumor progression. This study aims to elucidate the role of cAMP-response element binding protein 1 (CREB1) and its downstream effectors in regulating PDAC ECM remodeling. Methods: The Cancer Genome Atlas (TCGA) was quired for CREB1 expression and other ECM modulators in PDAC patients. In vitro, CRISPR/CAS9-based genomic editing was utilized to knockout CREB1 in the murine KPC cell line, on which RNA-seq analysis was performed. To investigate the effects of CREB in PDAC progression, a novel conditional CREB1 knockout (CREBfl/fl) was created in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice (KPCC-/-). Tumors were extracted and histologically evaluated. Immunoblotting and staining were conducted on both in vitro and in vivo samples. Chromatin immunoprecipitation sequencing (ChIP-seq) and qPCR were performed on the KPC CREBKO. Eukaryotic Promoter Database (EPD) and ENCODE databases were explored to evaluate the transcriptional role of CREB in regulating ECM genes. Results: Patient PDAC samples data from TCGA revealed elevated levels of CREB1, ROCK1, and ROCK2 compared to normal pancreatic tissues. When comparing high CREB expression to low CREB expression, upregulation of several ECM genes was noted, including ROCK1 and ROCK2 in patients with high CREB expression. In CREBKO, RNA-seq analysis revealed the downregulation of key ECM genes, including Mmp3, Mmp10, Lama3, and Fn1 compared to CREB wildtype. In our novel murine conditional knockout model, a substantial reduction in fibrosis was observed. In both in vitro and in vivo, immunoblot and staining demonstrated a marked reduction in the expression of ECM remodeling proteins upon CREB deletion. Through ChIP-seq, the direct involvement of CREB1 in the transcriptional regulation of Rock1 and Rock2 was established. EPD and ENCODE were utilized to confirm and validate our CREB regulation experimental findings. Conclusion: These findings demonstrate the role of CREB in PDAC progression, which may provide a potential target to intercept ECM remodeling via inhibition of the CREB-ROCK axis to suppress PDAC tumor progression and invasion. Citation Format: Sudhakar Jinka, Siddharth Mehra, Varunkumar Krishnamoorthy, Anna Bianchi, Karthik Rajkumar, Andrew Adams, Haleh Amirian, Samara Singh, Edmond W. Box, Erin Dickey, Nivelo L. Alberto, Ban Yuguang, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. CREB-ROCK driven extracellular matrix remodeling exasperates pancreatic cancer progression [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 4277.

Combining bulk and scRNA-seq to explore the molecular mechanisms governing the distinct efferocytosis activities of a macrophage subpopulation in PDAC.

Pancreatic ductal adenocarcinoma (PDAC), a very aggressive tumour, is currently the third leading cause of cancer-related deaths. Unfortunately, many patients face the issue of inoperability at the diagnostic phase leading to a quite dismal prognosis. The onset of metastatic processes has a crucial role in the elevated mortality rates linked to PDAC. Individuals with metastatic advances receive only palliative therapy and have a grim prognosis. It is essential to carefully analyse the intricacies of the metastatic process to enhance the prognosis for individuals with PDAC. Malignancy development is greatly impacted by the process of macrophage efferocytosis. Our current knowledge about the complete range of macrophage efferocytosis activities in PDAC and their intricate interactions with tumour cells is still restricted. This work aims to resolve communication gaps and pinpoint the essential transcription factor that is vital in the immunological response of macrophage populations. We analysed eight PDAC tissue samples sourced from the gene expression omnibus. We utilized several software packages such as Seurat, DoubletFinder, Harmony, Pi, GSVA, CellChat and Monocle from R software together with pySCENIC from Python, to analyse the single-cell RNA sequencing (scRNA-seq) data collected from the PDAC samples. This study involved the analysis of a comprehensive sample of 22,124 cells, which were classified into distinct cell types. These cell types encompassed endothelial and epithelial cells, PDAC cells, as well as various immune cells, including CD4+ T cells, CD8+ T cells, NK cells, B cells, plasma cells, mast cells, monocytes, DC cells and different subtypes of macrophages, namely C0 macrophage TGM2+, C1 macrophage PFN1+, C2 macrophage GAS6+ and C3 macrophage APOC3+. The differentiation between tumour cells and epithelial cells was achieved by the implementation of CopyKat analysis, resulting in the detection and categorization of 1941 PDAC cells. The amplification/deletion patterns observed in PDAC cells on many chromosomes differ significantly from those observed in epithelial cells. The study of Pseudotime Trajectories demonstrated that the C0 macrophage subtype expressing TGM2+ had the lowest level of differentiation. Additionally, the examination of gene set scores related to efferocytosis suggested that this subtype displayed higher activity during the efferocytosis process compared to other subtypes. The most active transcription factors for each macrophage subtype were identified as BACH1, NFE2, TEAD4 and ARID3A. In conclusion, the examination of human PDAC tissue samples using immunofluorescence analysis demonstrated the co-localization of CD68 and CD11b within regions exhibiting the presence of keratin (KRT) and alpha-smooth muscle actin (α-SMA). This observation implies a spatial association between macrophages, fibroblasts, and epithelial cells. There is variation in the expression of efferocytosis-associated genes between C0 macrophage TGM2+ and other macrophage cell types. This observation implies that the diversity of macrophage cells might potentially influence the metastatic advancement of PDAC. Moreover, the central transcription factor of different macrophage subtypes offers a promising opportunity for targeted immunotherapy in the treatment of PDAC.

Integrating Tertiary Lymphoid Structure-Associated Genes into Computational Models to Evaluate Prognostication and Immune Infiltration in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor response to all therapeutic modalities and dismal prognosis. The presence of tertiary lymphoid structures (TLSs) in various solid cancers is of crucial prognostic significance, highlighting the intricate interplay between the tumor microenvironment and immune cells aggregation. However, the extent to which TLS and immune status affect PDAC prognosis remains incompletely understood. Here, we sought to unveil the unique properties of TLS in PDAC by leveraging both single-cell and bulk transcriptomics, and culminating in a risk model that predicts clinical outcomes. We used TLS score based on 12 genes (CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21, CXCL9, CXCL10, CXCL11 and CXCL13) and 9 genes (PTGDS, RBP5, EIF1AY, CETP, SKAP1, LAT, CCR6, CD1D and CD79B) signature, respectively, and examined their distribution in cell clusters of single-cell data from PDAC samples. The markers involved in these clusters were selected to develop a prognostic model using The Cancer Genome Atlas Program (TCGA) database as the training cohort and Gene Expression Omnibus (GEO) database as the validation cohort. Further we compared the immune infiltration, drug sensitivity, enriched and differentially expressed genes between the high-risk and low-risk groups in our model. Therefore, we established a risk model that has significant implications for the prognostic assessment of PADC patients with remarkable differences in immune infiltration and chemo-sensitivity between the low-risk and high-risk groups. And this paradigm established by TLS-related cell marker genes provides a prognostic prediction and a panel of novel therapeutic targets for exploring potential immunotherapy.

Alternative Splicing in Pancreatic Ductal Adenocarcinoma Leads to Dysregulated Immune System

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy that poses a significant global health threat, marked by a substantial increase in prevalence and mortality rates. Accounting for 90 % of pancreatic cancer cases, PDAC carries a dismal prognosis, and current therapeutic approaches, including immunotherapy, face challenges due to poor immunogenicity. This study aimed to discover differentially expressed immune genes shared between PDAC and normal samples from two datasets obtained from the NCBI GEO Dataset. The RNA-seq pipeline was employed for gene expression analysis, and enrichR facilitated functional enrichment analysis of biologically and statistically significant genes. Predictions of immune infiltration cells and corresponding genes, along with their immune responses, were made using the ScType database and the immunedeconv package, respectively. Verification of gene expression levels was conducted through GEPIA2, Expression Atlas, and literature review. Additionally, isoform-switching analysis of dysregulated genes aimed to uncover alternatively spliced pathogenic isoforms in PDAC. Notably, four immune genes (EPHA2 upregulated, GNG11, CRHBP, and FCER1A downregulated) were found to be common in both datasets and were highly implicated in PDAC. The dysregulated immune genes influenced molecular functions, including protein binding, transmembrane receptor protein tyrosine kinase activity, protein tyrosine kinase activity, and cadherin binding for upregulated genes. Downregulated genes were associated with GTPase activity and ribonucleoside triphosphate phosphatase activity. This study suggests these immune genes as potential prognostic biomarkers for effective PDAC treatment. However, further investigations are essential to unravel the functional perspectives of potential isoforms.

Serum Carbohydrate Antigen 19-9 and Metabolite Hypotaurine Are Predictive Markers for Early Recurrence of Pancreatic Ductal Adenocarcinoma.

A significant number of patients experience early recurrence after surgical resection for pancreatic ductal adenocarcinoma (PDAC), negating the benefit of surgery. The present study conducted clinicopathologic and metabolomic analyses to explore the factors associated with the early recurrence of PDAC. Patients who underwent pancreatectomy for PDAC at Kagawa University Hospital between 2011 and 2020 were enrolled. Tissue samples of PDAC and nonneoplastic pancreas were collected and frozen immediately after resection. Charged metabolites were quantified by capillary electrophoresis-mass spectrometry. Patients who relapsed within 1 year were defined as the early recurrence group. Frozen tumor tissue and nonneoplastic pancreas were collected from 79 patients. The clinicopathologic analysis identified 11 predictive factors, including preoperative carbohydrate antigen 19-9 levels. The metabolomic analysis revealed that only hypotaurine was a significant risk factor for early recurrence. A multivariate analysis, including clinical and metabolic factors, showed that carbohydrate antigen 19-9 and hypotaurine were independent risk factors for early recurrence ( P = 0.045 and P = 0.049, respectively). The recurrence-free survival rate 1 year after surgery with both risk factors was only 25%. Our results suggested that tumor hypotaurine is a potential metabolite associated with early recurrence. Carbohydrate antigen 19-9 and hypotaurine showed a vital utility for predicting early recurrence.

Interplay between B7–H3 and HLA class I in the clinical course of pancreatic ductal adenocarcinoma

Human leukocyte antigen (HLA) class I defects are associated with cancer progression. However, their prognostic significance is controversial and may be modulated by immune checkpoints. Here, we investigated whether the checkpoint B7–H3 modulates the relationship between HLA class I and pancreatic ductal adenocarcinoma (PDAC) prognosis.PDAC tumors were analyzed for the expression of B7–H3, HLA class I, HLA class II molecules, and for the presence of tumor-infiltrating immune cells. We observed defective HLA class I and HLA class II expressions in 75% and 59% of PDAC samples, respectively. HLA class I and B7–H3 expression were positively related at mRNA and protein level, potentially because of shared regulation by RELA, a sub-unit of NF-kB. High B7–H3 expression and low CD8+ T cell density were indicators of poor survival, while HLA class I was not. Defective HLA class I expression was associated with unfavorable survival only in patients with low B7–H3 expression.Favorable survival was observed only when HLA class I expression was high and B7–H3 expression low.Our results provide the rationale for targeting B7–H3 in patients with PDAC tumors displaying high HLA class I levels.

Unraveling the potential of CD8, CD68, and VISTA as diagnostic and prognostic markers in patients with pancreatic ductal adenocarcinoma.

Pancreatic cancer is a truculent disease with limited treatment options and a grim prognosis. Immunotherapy has shown promise in treating various types of cancer, but its effectiveness in pancreatic cancer has been lacking. As a result, it is crucial to identify markers associated with immunological pathways in order to improve the treatment outcomes for this deadly cancer. The purpose of this study was to investigate the diagnostic and prognostic significance of three markers, CD8, CD68, and VISTA, in pancreatic ductal adenocarcinoma (PDAC), the most common subtype of pancreatic cancer. We analyzed gene expression data from Gene Expression Omnibus (GEO) database using bioinformatics tools. We also utilized the STRING online tool and Funrich software to study the protein-protein interactions and transcription factors associated with CD8, CD68, and VISTA. In addition, tissue microarray (TMA) and immunohistochemistry (IHC) staining were performed on 228 samples of PDAC tissue and 10 samples of normal pancreatic tissue to assess the expression levels of the markers. We then correlated these expression levels with the clinicopathological characteristics of the patients and evaluated their survival rates. The analysis of the GEO data revealed slightly elevated levels of VISTA in PDAC samples compared to normal tissues. However, there was a significant increase in CD68 expression and a notable reduction in CD8A expression in pancreatic cancer. Further investigation identified potential protein-protein interactions and transcription factors associated with these markers. The IHC staining of PDAC tissue samples showed an increased expression of VISTA, CD68, and CD8A in pancreatic cancer tissues. Moreover, we found correlations between the expression levels of these markers and certain clinicopathological features of the patients. Additionally, the survival analysis revealed that high expression of CD8 was associated with better disease-specific survival and progression-free survival in PDAC patients. These findings highlight the potential of CD8, CD68, and VISTA as diagnostic and prognostic indicators in PDAC.

Reconstitution of human PDAC using primary cells reveals oncogenic transcriptomic features at tumor onset

Animal studies have demonstrated the ability of pancreatic acinar cells to transform into pancreatic ductal adenocarcinoma (PDAC). However, the tumorigenic potential of human pancreatic acinar cells remains under debate. To address this gap in knowledge, we expand sorted human acinar cells as 3D organoids and genetically modify them through introduction of common PDAC mutations. The acinar organoids undergo dramatic transcriptional alterations but maintain a recognizable DNA methylation signature. The transcriptomes of acinar organoids are similar to those of disease-specific cell populations. Oncogenic KRAS alone do not transform acinar organoids. However, acinar organoids can form PDAC in vivo after acquiring the four most common driver mutations of this disease. Similarly, sorted ductal cells carrying these genetic mutations can also form PDAC, thus experimentally proving that PDACs can originate from both human acinar and ductal cells. RNA-seq analysis reveal the transcriptional shift from normal acinar cells towards PDACs with enhanced proliferation, metabolic rewiring, down-regulation of MHC molecules, and alterations in the coagulation and complement cascade. By comparing PDAC-like cells with normal pancreas and PDAC samples, we identify a group of genes with elevated expression during early transformation which represent potential early diagnostic biomarkers.

Characterization of the cachexia pathway in pancreatic ductal adenocarcinoma.

699 Background: Cancer cachexia is characterized by progressive weight loss and skeletal muscle degradation, contributing to 33% of pancreatic ductal adenocarcinoma (PDAC) deaths. Targeting myostatin in the myostatin-activin pathway has been shown to reverse cachexia. Here, we present comprehensive clinical and molecular characterization of the myostatin-activin pathway in PDAC. Methods: 9,607 samples of PDAC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Cachexia gene scores (GS) were calculated by averaging the positive z scores of activators and negative z scores of repressors in the myostatin-activin pathway. Activators were ACVR1B, ACVR1C, ACVR2A, ACVR2B, SMAD2, SMAD3, SMAD4, and TGFBR1, while repressors were SMAD1, SMAD5, SMAD6, SMAD7, SMURF1, and SMURF2. The top quartile (Q4) and bottom quartile (Q1) of GS were compared using chi-squared and Fisher-Exact tests. RNA deconvolution analysis with QuantiSEQ estimated cell infiltration in the tumor microenvironment (TME). Differences in overall survival (OS) were analyzed from insurance claims data and calculated from time of tissue collection using Kaplan-Meier estimates. Statistical significance was determined as a P-value adjusted for multiple comparisons ( q<0.05). Results: GS were higher in primary tumors compared to metastases (median: -0.71 vs -0.86, q<0.05). GS was associated with increased PD-L1 IHC expression (Q1 21.2% vs Q4 10.3%, q<0.001), but not TMB-high (1.9% vs 2.1%, q=1) or MSI-H status (1.1% vs 1.3%, q=1). GS correlated with increased expression of immune related genes ( CD274, CD80, IDO1, CD86, PDCD1, LAG3, CTLA4, HAVCR2, and IFNG, q<0.05). However, TME immune cell infiltration did not vary based on cachexia GS. Mutation rates of TP53 (Q1 79.4% vs Q4 73.7%), ARID1A (Q1 11.8% vs Q4 6.9%) and KRAS (Q1 92.7% vs Q4 86.4%) were associated with Q1-GS (all q<0.01), while STK11 mutations (1.1% vs 3.0%, q=0.001) were associated with Q4-GS. Spearman correlation linked cachexia GS with the lipid metabolizing genes UCP2 (rho=0.49) and UCP3 (rho=0.30), as well as the inflammatory markers CCL2/MCP-1(rho=0.34) and IL1B (rho=0.33). Decreased OS was seen with higher tumor expression of the myostatin-activin pathway activator, SMAD3 (6.6 vs 8.5 mo, HR=1.22, CI 1.12-1.33, P<0.001), and lower expression of the repressor SMAD7 (6.9 vs 8.9 mo, HR=0.91, CI 0.79-0.90, P=0.034) in metastatic PDAC. Conclusions: This is the largest molecular and clinical characterization of the myostatin-activin cachexia pathway in PDAC. Our data show that increased activation of the myostatin-activin pathway is associated with immune mediators, lipid metabolism, and inflammatory gene activation. Activators and repressors are significant predictors of survival in PDAC, suggesting possible novel therapeutic targets.

Abstract B044: Targeting integrin alpha V beta 3 remodels the tumor microenvironment in pancreatic cancer

Abstract In pancreatic ductal adenocarcinoma (PDAC) the tumor microenvironment (TME) plays a crucial role in therapy resistance, metastasis, and recurrence. ProAgio is a novel therapeutic cytotoxin rationally designed to bind outside the RGD ligand pocket of the alphaV-beta3 integrin pair and then kill cells expressing this target. Pre-clinical studies demonstrated the safety and anti-tumor efficacy of ProAgio in animal models, and ProAgio is currently being tested in a Phase I clinical trial. The effect of ProAgio on TME cellular components has not been closely examined. We hypothesized that ProAgio remodels the PDAC TME to decrease stromal density and local immunosuppression by killing stromal cells thought to express the integrin pair, such as cancer associated fibroblasts (CAFs), proliferating endothelial cells, and tumor associated macrophages (TAMs). To identify potential cellular targets of ProAgio in the PDAC TME, we re-analyzed publicly available single cell RNA-seq datasets of human and mouse PDAC samples. We assessed integrin co-expression using flow cytometry, performed immunophenotyping and histopathological analysis to identify TME cellular and structural alterations induced by ProAgio. Integrins αV and β3 were both expressed at the RNA level in TAMs, myeloid cells, lymphocytes (T, B and NK), endothelial cells and CAFs. In the autochthonous KPC mouse model, we confirmed that granulocytes, macrophages, monocytes, CD4+ and CD8+T cells co-expressed integrin αVβ3 at the protein level. Amongst CAFs, inflammatory CAFs (iCAFs) had the highest percentage of cells co-expressing αV and β3 integrins. No anti-tumor effect was observed after 14 days of ProAgio treatment in a syngeneic KPC-derived PDAC orthotopic model, however, in the KPC autochthonous model the same treatment retarded tumor growth within 1 week. ProAgio treated autochthonous tumors displayed no changes in immune components or CAF subsets, but increased collagen area was observed. In addition, ProAgio treatment resulted in higher blood vessel area while integrin β3 protein levels remained stable. In conclusion, short-course ProAgio remodels the TME and delays tumor growth in an autochthonous PDAC model with mature stromal components, but does not deplete CAFs, TAMs or collagen area. Future studies will seek to refine our understanding of the cell subtypes affected by more prolonged ProAgio treatment and to determine the cellular and inflammatory mediators responsible for these changes. Citation Format: Mayrel Palestino Dominguez, Philip Homan, Xianyu Zhang, Sandra Navas Reyes, Theresa Guerin, Laura Bassel, Liu Zhi-ren, Serguei Kozlov, Christine Alewine. Targeting integrin alpha V beta 3 remodels the tumor microenvironment in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B044.

IQ Motif Containing GTPase-Activating Protein 3 Is Associated with Cancer Stemness and Survival in Pancreatic Ductal Adenocarcinoma

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of malignancy, with poor prognosis and rising incidence. IQ motif containing GTPase-activating protein 3 (IQGAP3) is a member of the IQGAPs family of scaffolding proteins that govern multiple cellular activities like cytoskeletal remodeling and cellular signal transduction. This study aimed to analyze the expression and biological function of IQGAP3 in PDAC. Methods: We analyzed IQGAP3 expression in 81 PDAC samples by immunohistochemistry. RNA interference was used to inhibit IQGAP3 expression in PDAC cell lines. Results: Immunohistochemical analysis of IQGAP3 showed that 54.3% of PDACs were positive for cytoplasmic expression of IQGAP3, with no expression found in non-neoplastic tissue. Furthermore, IQGAP3 expression was an independent poor prognostic factor in our immunostaining-based studies and analyses of public databases. Our cohort and the Cancer Genome Atlas database indicated that IQGAP3 is co-localized with kinesin family member C1 (KIFC1), which we previously reported as a cancer stem cell-associated protein. IQGAP3 small interfering RNA treatment decreased PDAC cell proliferation and spheroid colony formation via ERK and AKT pathways. Discussion/Conclusion: These results suggest that IQGAP3, a transmembrane protein, is involved in survival and stemness and may be a promising new therapeutic target for PDAC.

Caprin-1 influences autophagy-induced tumor growth and immune modulation in pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is characterized by rapid progression and poor prognosis. Understanding the genetic mechanisms that affect cancer properties and reprogram tumor immune microenvironment will develop new strategies to maximize the benefits for cancer therapies.MethodsGene signatures and biological processes associated with advanced cancer and unfavorable outcome were profiled using bulk RNA sequencing and spatial transcriptome sequencing, Caprin-1 was identified as an oncogenesis to expedite pancreatic cancer growth by activating autophagy. The mechanism of Caprin-1 inducing autophagy activation was further explored in vitro and in vivo. In addition, higher level of Caprin-1 was found to manipulate immune responses and inflammatory-related pathways. The immune profiles associated with increased levels of Caprin-1 were identified in human PDAC samples. The roles of CD4+T cells, CD8+T cells and tumor associated macrophages (TAMs) on clinical outcomes prediction were investigated.ResultsCaprin-1 was significantly upregulated in advanced PDAC and correlated with poor prognosis. Caprin-1 interacted with both ULK1 and STK38, and manipulated ULK1 phosphorylation which activated autophagy and exerted pro-tumorigenic phenotypes. Additionally, the infiltrated CD4+T cells and tumor associated macrophages (TAMs) were increased in Caprin-1High tissues. The extensive CD4+T cells determined poor clinical outcome in Caprin-1high patients, arguing that highly expressed Caprin-1 may assist cancer cells to escape from immune surveillance.ConclusionsOur findings establish causal links between the upregulated expression of Caprin-1 and autophagy activation, which may manipulate immune responses in PDAC development. Our study provides insights into considering Caprin-1 as potential therapeutic target for PDAC treatment.