Pancreatic Ductal Adenocarcinoma Specimens Research Articles

Analysis of Pancreatic Cancer Genetic Risk Factors in a Multi-Ethnic Population Sample.

Pancreatic cancer (PC) has one of the highest mortality to incidence ratio of all cancers. Early identification of at-risk individuals should permit early diagnosis. Genome-wide association studies showed the association of several genetic variants with PC risk in multi-ethnic populations. Our objective was to examine the association of these genetic variants with PC in a population sample from Kuwait. DNA samples from 103 pancreatic ductal adenocarcinoma (PDAC) specimens and 132 healthy controls were used for genotyping ABO rs505922, BCAR1 rs7190458, LINC-PINT rs6971499, HNF1B rs4795218, VDR rs2228570 rs731236, and PRSS1 rs111033565 rs111033568 rs387906698 and rs267606982 using TaqMan genotyping assays, and VDR expression was performed by immunocytochemistry. ABO rs505922C and VDR rs2228570A were associated with PDAC risk (odds ratio (OR): 1.55, 95% confidence interval (CI): 1.07 - 2.24, P = 0.027; OR: 1.64, 95% CI: 1.09 - 2.48, P = 0.024; respectively). An unweighted polygenic risk score (ABO rs505922, BCAR1 rs7190458, LINC-PINT rs6971499, and HNF1B rs4795218) was significantly associated with PDAC risk (β: -0.11, 95% CI: -0.15 to -0.05, P < 0.001). VDR expression was downregulated or absent in most PDAC specimens regardless of VDR haplotype. ABO rs505922C and VDR rs2228570A are PDAC genetic risk factors in our population. Ethnicity influences the association of reported genetic PDAC risk factors and should be adjusted for when performing PDAC genetic risk estimations. Investigation of these genetic risk factors in other ethnic populations is a necessity to evaluate their PDAC risk prediction potential.

Abstract C044: PI5P4Kα regulates cell fitness through iron homeostasis in pancreatic cancer

Abstract PI5P4Ks (Phosphatidylinositol 5-phosphate 4-kinases) are a family of lipid kinases that phosphorylate phosphatidylinositol 5-monophosphate (PI-5-P) at the 4-position to generate distinct pools of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) largely on intracellular membranes. These lipid species are involved in cellular and metabolic stress responses, signaling and organelle communication. PI5P4Ks have been implicated in multiple cancers, with compelling evidence showing synthetic lethality when PI5P4Ks are inhibited in TP53-mutant cancer cells. Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive metabolic rewiring but despite the critical roles of PI5P4Ks in cellular metabolism, their role in PDAC remains completely unexplored. We have found that depletion of PI5P4Kα selectively induces apoptotic cell death in PDAC cells. To gain insights into the metabolic mechanisms underlying this apoptotic cell death, we quantified polar metabolites using gas chromatography- mass spectrometry (GC/MS) and found significant reductions in the ratio of monounsaturated to saturated fatty acids, lactate and TCA metabolites. Orthogonal rescue assays demonstrated that exogenous supplementation with fatty acids, pyruvate or lactate fail to rescue the observed apoptosis in PI5P4Kα-knockdown cells; however, TCA cycle metabolites did reverse the extent of apoptosis. Interestingly, we identify that these altered metabolic read-outs and the effects on cell fitness are dependent on iron. Consistent with these findings, intracellular iron levels are reduced upon PI5P4Kα inhibition, and this is directly linked to PI5P4Kα depletion disturbing the intracellular labile iron pools via iron import. Using a heterotopic xenograft mouse model, we demonstrate that PI5P4Kα knockdown leads to the abrogation of PDAC tumor growth due to increased intratumoral cell death. Validating the physiological relevance of PI5P4Kα in human PDAC, we find that PIP4K2A, the gene that encodes PI5P4Kα, is upregulated in human PDAC specimens relative to normal pancreas, and that PIP4K2A gene expression is strongly correlated with iron uptake related gene signatures. This study highlights the critical role of PI5P4Kα in PDAC and suggests that PI5P4Kα inhibition has therapeutic potential in pancreatic cancer. Citation Format: Gurpreet Kaur Arora, Ryan Loughran, Kyanh Ly, Cheska Marie Galapate, Alicia Llorente, Taylor R Anderson, Chantal Pauli, David A Scott, Yoav Altman, Cosimo Commisso, Brooke M Emerling. PI5P4Kα regulates cell fitness through iron homeostasis in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C044.

TAK1 Promotes an Immunosuppressive Tumor Microenvironment through Cancer-Associated Fibroblast Phenotypic Conversion in Pancreatic Ductal Adenocarcinoma.

We aim to clarify the precise function of Transformed growth factor-beta 1 activated kinase-1 (TAK1) in cancer-associated fibroblasts (CAFs) within human pancreatic ductal adenocarcinoma (PDAC) by investigating its role in cytokine-mediated signaling pathways. The expression of TAK1 in pancreatic cancer was confirmed by TCGA data and human pancreatic cancer specimens. CAFs from freshly resected PDAC specimens were cultured and used in a three-dimensional model for direct and indirect co-culture with PDAC tumors to investigate TAK1 function. Additionally, organoids from KPC (LSL-K-RasLSLG12D/+; LSL-p53R172H/+; Pdx1-Cre) mice were mixed with CAFs and injected subcutaneously into C57BL/6 mice to explore in vivo functional interactions of TAK1. TCGA data revealed significant upregulation of TAK1 in PDAC, associating with a positive correlation with the T-cell exhaustion signature. Knockdown of TAK1 in CAFs decreased the iCAF signature and increased the myCAF signature both in vitro and in vivo. The absence of TAK1 hindered CAF proliferation, blocked several inflammatory factors via multiple pathways associated with immunosuppression, and hindered EMT, outgrowth in vitro in spheroid co-cultures with PDAC cells. Additionally, TAK1 inhibitor restrained tumor growth, increased CD4+ and CD8+ T cell abundance, and reduced immunosuppressive cells present in vivo. Blocking the TAK1+CAF phenotype leads to the conversion of protumorigenic CAFs to antitumorigenic CAFs. This highlights TAK1 as a potential therapeutic target, particularly in CAFs, and represents a novel avenue for combined immunotherapy in PDAC.

Prognostic significance of involvement of the circumferential resection margin/surface in patients with pancreatic head cancer: A prospective evaluation of pancreatoduodenectomy specimens using the 0 and 1 mm rules

Background/ObjectivesThe prognostic significance of circumferential resection margin (CRM) or circumferential surface (CS) in pancreatic head cancer is controversial. We investigated the survival outcomes according to CRM or CS involvement in pancreatoduodenectomy specimens of pancreatic ductal adenocarcinoma (PDAC). MethodsA total of 102 pancreatoduodenectomy specimens after upfront surgery for PDAC between 2014 and 2018 were prospectively collected. The superior mesenteric vein/portal vein or superior mesenteric artery margins were classified as CRM, and the anterior or posterior surfaces as CS. Survival outcomes and recurrence were compared according to the CRM/CS status, which was categorized into R10mm, R11mm, and R0 (≥1 mm) by the 0 and 1 mm rules. ResultsFor CRM, R10mm had significantly lower overall survival (OS) (P < 0.001) and disease-free survival (P < 0.001) rates than R11mm and R0, with no difference between R11mm and R0. For CS, R0 had a significantly higher OS rate (P < 0.001) than R10mm and R11mm, with no difference between R10mm and R11mm. In multivariable analysis, R10mm CRM was an independent risk factor for OS (hazard ratio 2.410, P = 0.003) and DFS (hazard ratio 5.019, P < 0.001). When CRM/CS were analyzed separately, only the R10mm superior mesenteric artery margin was significantly associated with local recurrence (P = 0.012). ConclusionsThe results suggest that CRM involvement defined by the 0 mm rule is more appropriate than the 1 mm rule for predicting survival outcomes, but CS involvement defined by the 0 or 1 mm rules is not prognostically significant.

Exploring the prognostic value of circular RNAs in pancreatic ductal adenocarcinoma using genome-wide expression profiling

Background/ObjectivesMedian survival of pancreatic ductal adenocarcinoma (PDAC) is around eight months and new prognostic tools are needed. Circular RNAs (circRNAs) have gained interest in different types of cancer. However, only a few studies have evaluated their potential in PDAC. We aimed to identify the most differentially expressed circRNAs in PDAC compared to controls and to explore their potential as prognostic markers. MethodsUsing frozen specimens with PDAC and controls, we performed RNA sequencing and identified 20,440 unique circRNAs. A custom code set of capture- and reporter probes for NanoString nCounter analysis was designed to target 152 circRNAs, based on abundancy, differential expression and a literature study. Expression of these 152 circRNAs was examined in 108 formalin-fixed and paraffin-embedded surgical PDAC specimens and controls. The spatial expression of one of the most promising candidates, ciRS-7 (hsa_circ_0001946), was evaluated by chromogenic in situ hybridization (CISH) using multi-punch tissue microarrays (TMAs) and digital imaging analysis. ResultsBased on circRNA expression profiles, we identified different PDAC subclusters. The 30 most differentially expressed circRNAs showed log2 fold changes from −3.43 to 0.94, where circNRIP1 (hsa_circ_0004771), circMBOAT2 (hsa_circ_0007334) and circRUNX1 (hsa_circ_0002360) held significant prognostic value in multivariate analysis. CiRS-7 was absent in PDAC cells but highly expressed in the tumor microenvironment. ConclusionsWe identified several new circRNAs with biomarker potential in surgically treated PDAC, three of which showed an independent prognostic value. We also found that ciRS-7 is absent in cancer cells but abundant in tumor microenvironment and may hold potential as marker of activated stroma.

Abstract 1134: Dissecting pancreatic cancer tumor-immune microenvironment crosstalk using spatial transcriptomics

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) often fails to respond to immune therapies due to various factors, including the role of Epithelial to Mesenchymal Transition (EMT) plasticity in conferring broad resistance to diverse therapies. However, the relationship between cancer cell heterogeneity and the tumor immune microenvironment remains unclear. To address this, we utilized single cell RNA-seq and spatial transcriptomics to uncover the landscape of these cell-cell interactions in human PDAC. Here, we applied to specimens from a clinical trial testing Losartan, an indirect TGF-beta inhibitor with FOLFIRINOX chemotherapy and nivolumab (anti-PD1) in a randomized multi-institutional clinical trial for PDAC (NCT03563248). Methods: Using the NanoString GeoMx Digital Spatial Profiler, we selected multiple regions of interest in formalin-fixed paraffin-embedded (FFPE) human PDAC specimens. Immunofluorescent antibody-guided isolation of RNA and protein from cancer cells (pan-cytokeratin), cancer-associated fibroblasts (alpha-SMA), and immune cells (CD45) were performed. Utilizing the whole transcriptome assay (WTA; 18,000+ protein-coding genes) and a new IO Proteome Atlas (IPA; 500+ plex proteins), we ventured to understand the relationship between tumor cells and the surrounding microenvironment. Results: PDAC cells, CAFs, and immune cells were successfully characterized using NanoString GeoMx in clinical trial specimens. Analysis revealed associations between cancer cell plasticity, TGF-beta signaling mesenchymal subtypes in PDAC cells. These TGF-beta enriched mesenchymal PDAC cells were enriched in Arm 1 (FOLFIRINOX) compared to Arm 2 (FOLFIRINOX+losartan). Immune deconvolution analysis identified variations in immune infiltrates, showing an anti-correlation between macrophages and T-cells. The IPA analysis revealed lower MHC I protein levels in short compared to long term survivors in Arm 3. Conclusions: Spatial transcriptomics and proteomics reveal insights into the spatial relationship between PDAC tumor cell EMT plasticity, CAFs, and immune infiltrates. This enables the discovery of novel immune response biomarkers and potential therapeutic avenues to target tumor and microenvironment interactions. Trial Registration: DF/HCC protocol 18-179: Losartan and Nivolumab in Combination With FOLFIRINOX and SBRT in Localized Pancreatic Cancer. NCT03563248 Ethics Approval: All studies presented were approved by the Dana-Farber/Harvard Cancer Center IRB protocols 18-179. Citation Format: Amaya Pankaj, Michael J. Raabe, Bidish K. Patel, Evan R. Lang, Joshua R. Kocher, Katherine H. Xu, Linda T. Nieman, William L. Hwang, Alec C. Kimmelman, David P. Ryan, Theodore S. Hong, Martin J. Aryee, David T. Ting. Dissecting pancreatic cancer tumor-immune microenvironment crosstalk using spatial transcriptomics [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 1134.

The radiological morphology of mesopancreas: A new variable to predict a positive vascular margin after pancreatoduodenectomy for pancreatic ductal adenocarcinoma?

615 Background: The most frequently invaded surgical margins on pancreatoduodenectomy (PD) specimens of pancreatic ductal adenocarcinoma (PDAC) are vascular margins, especially the superior mesenteric artery (SMA), also called mesopancreatic margin. Considering embryology, it may be hypothesized that PDAC cells tend to infiltrate the retroperitoneum through the contents of mesopancreas, justifying the frequent SMA positive margin. Because the radiological aspect of mesopancreas has been poorly studied, the aim of this original research was to assess the mesopancreatic infiltration on diagnostic imaging, corroborate with the corresponding margin pathology and evaluate the impact on survival in PDAC patients who underwent PD. Methods: From 2015 to 2021, all patients who underwent PD for PDAC with curative intent were reviewed, excluding patients lost to follow-up, who died postoperatively or within the first year for non-oncological reason, and those with unavailable preoperative imaging. Surgical margins of pathological specimens were reassessed. Blinded reviewing of preoperative radiographic images was conducted. According to qualitative assessment, the mesopancreas tissue was defined as normal fat (NF), fat stranding (FS) or solid infiltration (SI). Results: 133 patients were included in the study, including 51 (38%) who received neoadjuvant therapy. The tumor location was into the head or the uncinate process in 54% and 46%, respectively. At diagnosis, PDAC were classified as resectable (51%), borderline resectable (28%) and locally advanced (9%) according to the NCCN classification. FS or SI in the mesopancreas was present in 45 (34%) and 18 (14%) patients, respectively. Tumor size on imaging, tumor location, chronic obstructive pancreatitis, vascular contacts and NCCN resectability status were predictive factors of mesopancreas infiltration (p&lt;0.001). Median overall and disease-free survivals were significantly lower in case of SI compared to NF and FS in the mesopancreas. When comparing patients with mesopancreatic FS at diagnosis who received neoadjuvant therapy (n=20) to those who underwent upfront surgery (n=25), no significant impact was observed in survivals. R0 resection was obtained in 36%; in all patients with R1 resection, a vascular margin was involved. Tumor size at pathology, SMA margin and resection status were factors that were significantly influenced by the radiological infiltration of the mesopancreas. Conclusions: The SI of mesopancreas on diagnostic imaging was associated with a poor prognosis, but not FS, in patients who underwent PD for PDAC. SMA margin and resection status were correlated with the radiological texture of the mesopancreas, which suggests to further explore underlying mechanisms related to tumoral invasion of vascular margin and mesopancreas.

Abstract B065: Biomarker approach to define tumor subtype in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumors with a predicted five-year survival rate in 2023 of 12%. Building on prior observations that molecular subtypes correlate with survival and response to treatment, our goal has been to understand the patterns of expression and prognostic correlations of three reciprocally expressed proteins previously identified as surrogate biomarkers of PDAC subtypes. Keratin 17 (K17) and Keratin 5 (K5) have both been identified as biomarkers of the basal subtype whereas GATA6, a transcription factor necessary for normal pancreatic development, is a surrogate marker of the classical subtype associated with longer-term survival. Here, we explore the expression of these three diagnostic biomarkers in PDAC and their specificity to tumor cells. Methods: Immunohistochemical analyses of K17 (KDx Diagnostics), GATA6 (R&amp;D Systems), and KT5 (Leica) were performed on archival primary PDAC specimens from Stony Brook University Hospital (n= 30), by routine immunoperoxidase methods on serial sections. IHC stained slides were scanned using an Olympus VS200 slide scanner and images were imported into QuPath for digital analysis. Regions of interest (ROIs) were defined by the presence of tumor cells and were identified by histological assessment of representative sections. Digital scoring was performed on identified ROIs using the integrated "positive cell detection" function in QuPath and classified into three different stain intensities (1+, 2+, 3+). The threshold for each intensity is as follows: 1+, 0.2; 2+, 0.4; 3+, 1.0. According to the number of detections of each intensity, tumor cells were categorized into negative (0 and 1+) or positive (2+ and 3+). The percentage positive of analyzed biomarker expression within the tumor ROI was calculated by dividing the total number of positive cells by the total number of detected cells. Results: K17 was detected in both well and poorly differentiated PDAC components and in a few benign proliferative ducts of chronic pancreatitis but no staining was detected in stromal cells, benign ductal epithelium, or acinar cells. K5 was detected in a smaller proportion of cells from glandular components of the tumor as well as in most diffusely infiltrative single cells, benign ductal, and acinar cells but not in stromal cells. By contrast, GATA6 was detected in the nuclei of most cancer epithelial cells and was also detected in benign ductal cells, acinar cells, and stromal cells. The proportion of K17 expressing cells exceeded that of K5 positive cells in the great majority of ROIs and across cases. Conclusions: Keratin 17 detects a higher proportion of tumor cells than K5 and shows the lowest level of expression in benign cell types. By contrast, GATA6 IHC shows variable patterns of expression related to K17 or K5 expression and is expressed in the highest proportion of tumor cells, including both well-differentiated, diffusely infiltrative components, and benign tissue components. Citation Format: Carson Ho, Michael Horowitz, Lyanne Oblein, Shayan Sarkar, Soma Kobayashi, Karen Bai, Natalia Marchenko, Luisa Escobar-Hoyos, Kenneth Shroyer. Biomarker approach to define tumor subtype in pancreatic ductal adenocarcinoma [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 B065.

Abstract C008: MUC16 expression in pancreatic ductal adenocarcinoma patient samples after neoadjuvant chemotherapy

Abstract Purpose Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with low five-year survival rates. Currently, surgical resection is the only potentially curative treatment available to patients. However, few patients are eligible for surgery without receiving prior therapy due to disease progression. Of those that receive upfront surgery, up to 60% develop recurrent disease within 5 years. Therefore, there is a need to increase the rate of complete surgical resections and the number of eligible patients for surgery. In the last decade, neoadjuvant therapy or treatment prior to surgery has increased for patients diagnosed with borderline resectable and locally advanced PDAC. Often, this therapy consists of chemotherapy alone or chemoradiotherapy treatment intended to downstage the tumor and reduce micrometastatic disease. Fluorescence-guided surgery (FGS) is a potential tool to increase the rate of complete surgical resections by specifically targeting a protein biomarker upregulated in the tumor with a fluorescent dye conjugated probe. Previous in vitro and in vivo research from our lab has indicated that the transmembrane mucin, MUC16, is a promising target for FGS in PDAC. However, protein expression has the potential to deviate after chemotherapy treatment. Therefore, this research aimed to determine the effect of neoadjuvant chemotherapy treatment (NCT) on the FGS-targetable biomarker, MUC16, in PDAC patient specimens. Experimental Procedures Deidentified PDAC and matched adjacent patient specimens receiving the NCT regimen or surgery-first treatment were obtained from UNMC Pathology and the UNMC Paraffin Tissue Bank. Deidentified normal pancreas specimens were received from the Normal Organ Recovery program, UNMC. MUC16 expression was investigated in each specimen. The presence of PDAC tumor and MUC16 stain expression patterns were confirmed by a specimen-blinded, board-certified pathologist. The expression of MUC16 between the NCT, surgery-first, and normal pancreas specimens was compared via statistical analysis. Results MUC16 was expressed in both the NCT and surgery-first specimens. There was a significant difference in MUC16 expression between the NCT specimens and both the normal pancreas (P = 0.0450) and surgery-first (P = 0.0494) specimens. In matched adjacent tissue, MUC16 expression was found to be higher in the NCT (P = 0.0003) and surgery-first (P = 0.1446) tumor specimens allowing for distinct tumor margins; however, this difference was not significant in the surgery-first specimens. Conclusions MUC16 expression retention in NCT PDAC specimens may indicate that MUC16 is a stable biomarker after chemotherapy treatment. Distinct MUC16 expression between tumor and matched adjacent specimens indicates the ability to achieve distinguishable tumor margins allowing for complete tumor resection. Therefore, MUC16 remains targetable for FGS in chemotherapy-treated patients. This has the potential to broaden the benefiting patient population and increase the number of patients that may undergo a complete surgical resection. Citation Format: Kathryn M. Muilenburg, Evie G. Ehrhorn, Madeline T. Olson, Carly C. Isder, Kelsey A. Klute, Geoffrey A. Talmon, Mark A. Carlson, Quan P. Ly, Aaron M. Mohs. MUC16 expression in pancreatic ductal adenocarcinoma patient samples after neoadjuvant chemotherapy [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 C008.

Abstract A038: Non-canonical MHC class I-associated antigens in pancreatic cancer

Abstract Translation of the ostensibly non-coding genome in cancer can generate novel peptides capable of presentation by major histocompatibility complex class I (MHC-I, HLA-I in humans). These non-canonical peptide sources can broaden the landscape of potentially targetable antigens in low-to-intermediate mutational burden malignancies, typified by pancreatic ductal adenocarcinoma (PDA). However, non-canonical MHC-I-associated peptides (ncMAPs) have yet to be explored in pancreatic cancer. We purified the malignant compartment from low tumor cellularity PDA specimens using patient-derived organoids (PDOs) and developed a personalized proteogenomics pipeline coupled with high-depth immunopeptidomics to deeply characterize the repertoire of HLA:peptide targets presented specifically on malignant cells. We demonstrate that ncMAPs are abundant on PDA and predominate over mutation-derived neoepitopes in the detectable immunopeptidome. While emerging evidence in other tumor types has implicated dysregulated translation of non-canonical open reading frames (ncORFs), it is currently unknown to what extent these translation products are truly cancer-restricted and how effectively the resulting ncMAPs can elicit a cytolytic T lymphocyte (CTL) response. To investigate the cancer-specificity of non-canonical peptides, we developed a translation-centric analysis pipeline that examines ncORF expression across a range of healthy tissues, including healthy thymus. We provide evidence that ~30% of ncMAPs exhibit cancer-restricted translation patterns, and a substantial subset of these are shared among pancreatic cancer patients with the appropriate HLA. To investigate immunogenicity, we employed a highly sensitive ex vivo platform to prime and expand antigen-specific T cells. We demonstrate that PDA-restricted ncMAPs are highly immunogenic, on par with or even exceeding the immunogenicity observed with mutation-derived peptides or tumor-associated antigens assayed using the same platform. These findings uncover a critical role for dysregulated translation in pancreatic cancer as a potential source for recurrent cancer-restricted epitopes capable of recognition by cytotoxic T cells. We envision that this novel class of antigens will accelerate ongoing efforts to treat pancreatic cancer patients with vaccines and cell-based therapies. Citation Format: Zackery A. Ely, Zachary J. Kulstad, Eva K. Verzani, Jennifer G. Abelin, Sudarsana Addepalli, Karl R. Clauser, Marta Casarrubios, Kevin S. Kapner, Miles P. Agus, Connor J. Hennessey, Sine R. Hadrup, Susan Klaeger, Jennifer Su, Alex M. Jaeger, Brian M. Wolpin, Srivatsan Raghavan, Philip D. Greenberg, Andrew J. Aguirre, Steven A. Carr, Tyler Jacks, William A. Freed-Pastor. Non-canonical MHC class I-associated antigens 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 A038.

Autotaxin-lysolipid signaling suppresses a CCL11-eosinophil axis to promote pancreatic cancer progression.

Lipids and their modifying enzymes regulate diverse features of the tumor microenvironment and cancer progression. The secreted enzyme autotaxin (ATX) hydrolyzes extracellular lysophosphatidylcholine to generate the multifunctional lipid mediator lysophosphatidic acid (LPA) and supports the growth of several tumor types, including pancreatic ductal adenocarcinoma (PDAC). Here we show that ATX suppresses the accumulation of eosinophils in the PDAC microenvironment. Genetic or pharmacologic ATX inhibition increased the number of intratumor eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop, wherein ATX-LPA signaling negatively regulates the activity of the AP-1 transcription factor c-Jun, in turn suppressing the expression of the potent eosinophil chemoattractant CCL11 (eotaxin-1). Eosinophils were identified in human PDAC specimens, and rare individuals with high intratumor eosinophil abundance had the longest overall survival. Together with recent findings, this study reveals the context-dependent, immune-modulatory potential of ATX-LPA signaling in cancer.

REV7 is involved in outcomes of platinum-based chemotherapy in pancreatic cancer by controlling the DNA damage response.

REV7 is a multifunctional protein implicated in various biological processes, including DNA damage response. REV7 expression in human cancer cells affects their sensitivity to DNA-damaging agents. In the present study, we investigated the significance of REV7 in pancreatic ductal adenocarcinoma (PDAC). REV7 expression was immunohistochemically examined in 92 resected PDAC specimens and 60 endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNAB) specimens of unresectable PDAC treated with platinum-based chemotherapy, and its association with clinicopathologic features was analyzed. Although REV7 expression was not significantly associated with the progression of primary tumors (T-factor and Stage) in either resected or unresectable PDAC, decreased levels of REV7 expression in EUS-FNAB specimens of unresectable PDAC were significantly associated with better outcomes of platinum-based chemotherapy and a favorable prognosis. REV7-deficient PDAC cell lines showed suppressed cell growth and enhanced sensitivity to cisplatin invitro. Tumor-bearing mice generated using REV7-deficient PDAC cell lines also showed enhanced sensitivity to cisplatin invivo. RNA sequencing analysis using WT and REV7-deficient PDAC cell lines revealed that REV7 inactivation promoted the downregulation of genes involved in the DNA repair and the upregulation of genes involved in apoptosis. Our results indicate that decreased expression of REV7 is associated with better outcomes of platinum-based chemotherapy in PDAC by suppressing the DNA damage response. It is also suggested that REV7 is a useful biomarker for predicting the outcome of platinum-based chemotherapy and the prognosis of unresectable PDAC and is a potential target for PDAC treatment.

High overall copy number variation burden by genome-wide methylation profiling holds negative prognostic value in surgically treated pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer with an overall 5-year survival of around 10 %. New prognostic tools to stratify patients are needed. Our main aim was to evaluate the prognostic value of overall copy number variation (CNV) burden in surgically treated PDAC. DNA extracted from 108 surgical PDAC specimens was examined to collect data on the genome-wide DNA methylation status of >850,000 CpG sites in promoter, gene body, and enhancer regions (Illumina Infinium Methylation EPIC BeadChip Kit). CNV profiles were obtained and all PDACs were stratified into one of three groups: Low, moderate, or high overall CNV burden. Tumors histologically showing a dominant conventional and/or tubulopapillary pattern in 60 %–100 % and 0–59 % were categorized as Group A and Group B as per Kalimuthu. We also performed targeted next-generation sequencing (NGS) and immunohistochemistry. High overall CNV burden held independent negative prognostic value with poor survival (HR 4.01 (95%CI 1.96–8.19), p = 0.00014) and was more frequent in Group B (p = 0.0003). Most frequent chromosomal arm-level aberrations were gains of 8q (29 %) and 1q (19 %) and losses of 17p (55 %), 18q (43 %), 6q (37 %), 9p (36 %), 6p (26 %), 19p (26 %), and 8p (25 %). Most frequent mutations found were in KRAS (95 %), TP53 (62 %), CDKN2A (24 %), SMAD4 (23 %), ATM (9 %), ARID1A (7 %), RNF43 (7 %), GNAS (6 %), and KDM6A (6 %). Group A PDACs showed more frequently KRAS variants other than Gly12Val and Gly12Asp (p = 0.012). Our data indicate that overall CNV burden using genome-wide methylation profiling may be a useful prognostic tool in surgically treated PDAC. Importantly, our approach, using data from genome-wide methylation profiling for analysis of overall CNV burden, can be performed on formalin-fixed and paraffin embedded PDAC tissues. Future studies should examine the prognostic value of overall CNV burden in unresectable PDAC.

LncRNA CCAT1 participates in pancreatic ductal adenocarcinoma progression by forming a positive feedback loop with c-Myc.

Long noncoding RNAs (lncRNAs) play fundamental roles in cancer development; however, the underlying mechanisms for a large proportion of lncRNAs in pancreatic ductal adenocarcinoma (PDAC) have not been elucidated. The expression of colon cancer-associated transcript-1 (CCAT1) in PDAC specimens and cell lines was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The function of CCAT1 was examined in vitro and in vivo. The interactions among CCAT1, miR-24-3p and c-Myc were determined by bioinformatics analysis, RNA immunoprecipitation (RIP), dual-luciferase reporter assay, and rescue experiments. CCAT1 was significantly increased in PDAC, positively correlated with PDAC progression and predicted a worse prognosis. Furthermore, CCAT1 enhanced Adenosine triphosphate (ATP) production to facilitate PDAC cell proliferation, colony formation and motility in vitro and tumor growth in vivo. CCAT1 may serve as an miR-24-3p sponge, thereby counteracting its repression by c-Myc expression. Reciprocally, c-Myc may act as a transcription factor to alter CCAT1 expression by directly targeting its promoter region, thus forming a positive feedback loop with CCAT1. Collectively, these results demonstrate that a positive feedback loop of CCAT1/miR-24-3p/c-Myc is involved in PDAC development, which may serve as a biomarker and therapeutic target for PDAC.

Clinical Significance of Phosphorylated Sphingosine Kinase 1 Expression in Pancreatic Ductal Adenocarcinoma.

Sphingosine-1-phosphate (S1P) is a pleiotropic, bioactive, lipid mediator, produced by sphingosine kinase 1 (SphK1). In this study, we evaluated the expression of phosphorylated SphK1 (pSphK1) in patients with pancreatic ductal adenocarcinoma (PDAC) and investigated its clinical significance. A total of 111 patients who underwent curative-intent resection for PDAC were enrolled. We investigated pSphK1 (Ser-225) expression in surgically resected specimens of PDAC using immunohistochemistry. The patients were divided into two groups according to pSphK1 immunoreactive expression: a pSphK1-high group (n=63) and a pSphK1-low group (n=48). Logistic regression analyses revealed that lymphatic invasion (p=0.007) was a significantly independent factor associated with high pSphK1 immunoreactive expression. The pSphK1-high group showed significantly worse disease-specific survival (DSS) than the pSphK1-low group (5-year DSS rate, 19.6% vs. 58.7%; p=0.001). High pSphK1 immunoreactive expression (hazard ratio=2.547; 95% confidence interval= 1.434-4.527; p=0.001) was an independent prognostic factor for DSS. High pSphK1 expression is independently associated with lymphatic invasion and unfavorable prognosis in PDAC patients. Thus, the SphK1-S1P axis may be important in mechanisms of tumor progression, such as lymphatic invasion, in PDAC patients.

Pancreatic ductal adenocarcinoma with acinar-to-ductal metaplasia-like cancer cells shows increased cellular proliferation

Background/objectivesAcinar-to-ductal metaplasia (ADM) has been shown to contribute to the development of pancreatic ductal adenocarcinoma (PDAC) in genetically engineered mouse models, but little is known about whether acinar cell plasticity contributes to carcinogenesis in human PDAC. We aimed to assess whether cancer cells that stain positive for amylase and CK19 (ADM-like cancer cells) are present in human resected PDAC and to investigate their role in tumor progression. MethodsWe immunohistochemically investigated the presence of ADM-like cancer cells, and compared the clinical and histological parameters of PDAC patients with and without ADM-like cancer cells. ResultsADM-like cancer cells were detected in 16 of 60 (26.7%) PDAC specimens. Positive staining for anterior gradient protein 2 (AGR2) was observed in 14 of 16 (87.5%) PDAC specimens with ADM-like cancer cells. On the other hand, the intensity of AGR2 expression (negative, low/moderate or high) was lower in PDAC with ADM-like cancer cells (9/7) than in PDAC without these cells (11/33) (P = 0.032). The presence of ADM-like cancer cells was significantly correlated with increased cell proliferation (P = 0.012) and tended to be associated with MUC1 expression (P = 0.067). ConclusionsThese results indicated that acinar cells may act as the origin of human PDAC, and that their presence may be useful for the stratification of human PDAC to predict prognosis.

Perineural invasion detection in pancreatic ductal adenocarcinoma using artificial intelligence

Perineural invasion (PNI) refers to the presence of cancer cells around or within nerves, raising the risk of residual tumor. Linked to worse prognosis in pancreatic ductal adenocarcinoma (PDAC), PNI is also being explored as a therapeutic target. The purpose of this work was to build a PNI detection algorithm to enhance accuracy and efficiency in identifying PNI in PDAC specimens. Training used 260 manually segmented nerve and tumor HD images from 6 scanned PDAC cases; Analytical performance analysis used 168 additional images; clinical analysis used 59 PDAC cases. The algorithm pinpointed key areas of tumor-nerve proximity for pathologist confirmation. Analytical performance reached sensitivity of 88% and 54%, and specificity of 78% and 85% for the detection of nerve and tumor, respectively. Incorporating tumor-nerve distance in clinical evaluation raised PNI detection from 52 to 81% of all cases. Interestingly, pathologist analysis required an average of only 24 s per case. This time-efficient tool accurately identifies PNI in PDAC, even with a small training cohort, by imitating pathologist thought processes.

A deep learning model using hyperspectral image for EUS-FNA cytology diagnosis in pancreatic ductal adenocarcinoma.

Endoscopic ultrasonography-guided fine-needle aspiration/biopsy (EUS-FNA/B) is considered to be a first-line procedure for the pathological diagnosis of pancreatic cancer owing to its high accuracy and low complication rate. The number of new cases of pancreatic ductal adenocarcinoma (PDAC) is increasing, and its accurate pathological diagnosis poses a challenge for cytopathologists. Our aim was to develop a hyperspectral imaging (HSI)-based convolution neural network (CNN) algorithm to aid in the diagnosis of pancreatic EUS-FNA cytology specimens. HSI images were captured of pancreatic EUS-FNA cytological specimens from benign pancreatic tissues (n = 33) and PDAC (n = 39) prepared using a liquid-based cytology method. A CNN was established to test the diagnostic performance, and Attribution Guided Factorization Visualization (AGF-Visualization) was used to visualize the regions of important classification features identified by the model. A total of 1913 HSI images were obtained. Our ResNet18-SimSiam model achieved an accuracy of 0.9204, sensitivity of 0.9310 and specificity of 0.9123 (area under the curve of 0.9625) when trained on HSI images for the differentiation of PDAC cytological specimens from benign pancreatic cells. AGF-Visualization confirmed that the diagnoses were based on the features of tumor cell nuclei. An HSI-based model was developed to diagnose cytological PDAC specimens obtained using EUS-guided sampling. Under the supervision of experienced cytopathologists, we performed multi-staged consecutive in-depth learning of the model. Its superior diagnostic performance could be of value for cytologists when diagnosing PDAC.

18F] AlF-NOTA-FAPI-04 PET/CT can predict treatment response and survival in patients receiving chemotherapy for inoperable pancreatic ductal adenocarcinoma.

We investigated whether uptake of [18F] AlF-NOTA-FAPI-04 on positron emission tomography/computed tomography (PET/CT) could predict treatment response and survival in patients with pancreatic ductal adenocarcinoma (PDAC). We prospectively evaluated 47 patients with histopathologically confirmed primary PDAC who provided pretreatment [18F] AlF-NOTA-FAPI-04 scans to detect fibroblast activation protein (FAP) on the tumor surface by uptake of [18F] AlF-NOTA-FAPI-04. PDAC specimens were immunohistochemically stained with cancer-associated fibroblast (CAF) markers. We obtained a second PET scan after one cycle of chemotherapy to study changes in FAPI uptake variables from before to during treatment. Correlations between baseline PET variables and CAF-related immunohistochemical markers were assessed with Spearman's rank test. Cox regression and Kaplan-Meier methods were used to assess relationships between disease progression and potential predictors. Receiver operating characteristic (ROC) curve analysis was used to define the optimal cut-off points for distinguishing patients according to good response vs. poor response per RECIST v.1.1. The FAPI PET variables maximum and mean standardized uptake values (SUVmax, SUVmean), metabolic tumor volume (MTV), and total lesion FAP expression (TLF) were positively correlated with CAF markers (FAP, α-smooth muscle actin, vimentin, S100A4, and platelet-derived growth factor receptor α/β, all P < 0.05). MTV was associated with survival in patients with inoperable PDAC (all P < 0.05). Cox multivariate regression showed that MTV was associated with overall survival (MTV hazard ratio [HR] = 1.016, P = 0.016). Greater changes from before to during chemotherapy in SUVmax, MTV, and TLF were associated with good treatment response (all P < 0.05). ΔMTV, ΔTLF, and ΔSUVmax had larger areas under the curve than ΔCA19-9 for predicting treatment response. Kaplan-Meier analysis showed that the extent of change in MTV and TLF from before to after treatment predicted progression-free survival, with cut-off values (based on medians) of - 4.95 for ΔMTV (HR = 8.09, P = 0.013) and - 77.83 for ΔTLF (HR = 4.62, P = 0.012). A higher baseline MTV on [18F] AlF-NOTA-FAPI-04 scans was associated with poorer survival in patients with inoperable PDAC. ΔMTV was more sensitive for predicting response than ΔCA19-9. These results are clinically meaningful for identifying patients with PDAC who are at high risk of disease progression.

Dual epigenetic changes in diabetes mellitus-associated pancreatic ductal adenocarcinoma correlate with downregulation of E-cadherin and worsened prognosis.

Diabetes mellitus (DM) is a risk factor for pancreatic ductal adenocarcinoma (PDAC) that promotes the promoter methylation of CDH1. It is still unclear whether DM can exert other epigenetic effects, such as altering microRNA (miR) expression, in PDAC. The expression of miR-100-5p is known to be changed in DM patients and can suppress the expression of E-cadherin. In this study, the correlation between DM status and dual epigenetic changes was evaluated in PDAC specimens from patients who underwent radical surgical resection. A total of 132 consecutive patients with PDAC were clinicopathologically evaluated. E-cadherin and nuclear β-catenin expression was measured using immunohistochemistry. DNA and miRs were extracted from the main tumor site on formalin-fixed paraffin-embedded tissue sections. TaqMan miR assays were applied to assess miR-100-5p expression. Bisulfite modification was conducted on the extracted DNA, which was then subjected to methylation-specific polymerase chain reaction. Immunohistochemistry revealed that decreased E-cadherin expression and increased nuclear β-catenin expression were significantly associated with DM and poor tumor cell differentiation. The presence of long-duration DM (≥3 years) was a significant factor contributing to CDH1 promoter methylation (p < 0.01), while miR-100-5p expression was proportionally correlated with the preoperative HbA1c level (R = 0.34, p < 0.01), but not the duration of DM. The subjects with high miR-100-5p expression and CDH1 promoter methylation showed the highest level of vessel invasion and prevalence of tumor size ≥30 mm. PDAC subjects with dual epigenetic changes showed poorer overall survival (OS) than those with a single epigenetic change. miR-100-5p expression ≥4.13 and CDH1 promoter methylation independently predicted poor OS and disease-free survival (DFS) in the multivariate analysis. OS and DFS worsened in DM subjects with both HbA1c ≥ 6.5% and DM duration ≥3 years. Thus, DM is associated with two modes of epigenetic change by independent mechanisms and worsens prognosis.