Pancreatic Ductal Adenocarcinoma Cohort Research Articles

CircFARP1 enables cancer-associated fibroblasts to promote gemcitabine resistance in pancreatic cancer via the LIF/STAT3 axis

BackgroundCancer-associated fibroblasts (CAFs) are critically involved in gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC). However, the underlying mechanism by which CAFs promote chemotherapy resistance remains unexplored. Here, we explored the role of circRNAs in CAF-induced GEM resistance in PDAC.MethodscircRNA sequencing and quantitative real-time PCR (qRT–PCR) were utilized to screen CAF-specific circRNAs. The effects of CAF circFARP1 expression on GEM resistance in tumor cells were assessed in vitro and in vivo. RNA-seq, RNA pulldown, RNA immunoprecipitation, and luciferase reporter assays were used to screen the downstream target and underlying mechanism of circFARP1.ResultscircFARP1 (hsa_circ_0002557), a CAF-specific circRNA, was positively correlated with GEM chemoresistance and poor survival in an advanced PDAC cohort. Silencing or overexpressing circFARP1 in CAFs altered the ability of CAFs to induce tumor cell stemness and GEM resistance via leukemia inhibitory factor (LIF). Mechanistically, we found that circFARP1 directly binds with caveolin 1 (CAV1) and blocks the interaction of CAV1 and the E3 ubiquitin-protein ligase zinc and ring finger 1 (ZNRF1) to inhibit CAV1 degradation, which enhances LIF secretion. In addition, circFARP1 upregulated LIF expression by sponging miR-660-3p. Moreover, high circFARP1 levels were positively correlated with elevated serum LIF levels in PDAC and poor patient survival. Decreasing circFARP1 levels and neutralizing LIF significantly suppressed PDAC growth and GEM resistance in patient-derived xenograft models.ConclusionsThe circFARP1/CAV1/miR-660-3p/LIF axis is critical for CAF-induced GEM resistance in PDAC. Hence, circFARP1 may be a potential therapeutic target for PDAC.

Soluble AXL is a novel blood marker for early detection of pancreatic ductal adenocarcinoma and differential diagnosis from chronic pancreatitis.

SummaryBackgroundEarly diagnosis is crucial for patients with pancreatic ductal adenocarcinoma (PDAC). The AXL receptor tyrosine kinase is proteolytically processed releasing a soluble form (sAXL) into the blood stream. Here we explore the use of sAXL as a biomarker for PDAC.MethodsAXL was analysed by immunohistochemistry in human pancreatic tissue samples. RNA expression analysis was performed using TCGA/GTEx databases. The plasma concentrations of sAXL, its ligand GAS6, and CA19-9 were studied in two independent cohorts, the HMar cohort (n = 59) and the HClinic cohort (n = 142), including healthy controls, chronic pancreatitis (CP) or PDAC patients, and in a familial PDAC cohort (n = 68). AXL expression and sAXL release were studied in PDAC cell lines and murine models.FindingsAXL is increased in PDAC and precursor lesions as compared to CP or controls. sAXL determined in plasma from two independent cohorts was significantly increased in the PDAC group as compared to healthy controls or CP patients. Patients with high levels of AXL have a lower overall survival. ROC analysis of the plasma levels of sAXL, GAS6, or CA19-9 in our cohorts revealed that sAXL outperformed CA19-9 for discriminating between CP and PDAC. Using both sAXL and CA19-9 increased the diagnostic value. These results were validated in murine models, showing increased sAXL specifically in animals developing PDAC but not those with precursor lesions or acinar tumours.InterpretationsAXL appears as a biomarker for early detection of PDAC and PDAC–CP discrimination that could accelerate treatment and improve its dismal prognosis.FundingThis work was supported by grants PI20/00625 (PN), RTI2018-095672-B-I00 (AM and PGF), PI20/01696 (MG) and PI18/01034 (AC) from MICINN-FEDER and grant 2017/SGR/225 (PN) from Generalitat de Catalunya.

An Inflammatory Response Related Gene Signature Associated with Survival Outcome and Gemcitabine Response in Patients with Pancreatic Ductal Adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors with an extremely low 5-year survival rate. Accumulating evidence has unveiled that inflammatory response promotes tumor progression, enhances angiogenesis, and causes local immunosuppression. Herein, we aim to develop an inflammatory related prognostic signature, and found it could be used to predict gemcitabine response in PDAC. Methods: PDAC cohorts with mRNA expression profiles and clinical information were systematically collected from the four public databases. An inflammatory response related genes (IRRGs) prognostic signature was constructed by LASSO regression analysis. Kaplan–Meier survival analysis, receiver operating characteristic analysis, principal component analysis, and univariate and multivariate Cox analyses were carried out to evaluate effectiveness, and reliability of the signature. The correlation between gemcitabine response and risk score was evaluated in the TCGA-PAAD cohort. The GDSC database, pRRophetic algorithm, and connectivity map analysis were used to predict gemcitabine sensitivity and identify potential drugs for the treatment of PDAC. Finally, we analyzed differences in frequencies of gene mutations, infiltration of immune cells, as well as biological functions between different subgroups divided by the prognostic signature. Results: We established a seven IRRGs (ADM, DCBLD2, EREG, ITGA5, MIF, TREM1, and BTG2) signature which divided the PDAC patients into low- and high-risk groups. Prognostic value of the signature was validated in 11 PDAC cohorts consisting of 1337 PDAC patients from 6 countries. A nomogram that integrated the IRRGs signature and clinicopathologic factors of PDAC patients was constructed. The risk score showed positive correlation with gemcitabine resistance. Two drugs (BMS-536924 and dasatinib) might have potential therapeutic implications in high-risk PDAC patients. We found that the high-risk group had higher frequencies of KRAS, TP53, and CDKN2A mutations, increased infiltration of macrophages M0, neutrophils, and macrophages M2 cells, as well as upregulated hypoxia and glycolysis pathways, while the low-risk group had increased infiltration of CD8+ T, naïve B, and plasma and macrophages M1 cells. Conclusion: We constructed and validated an IRRGs signature that could be used to predict the prognosis and gemcitabine response of patients with PDAC, as well as two drugs (BMS-536924 and dasatinib) may contribute to PDAC treatment.

Survival Outcome and Prognostic Factors for Pancreatic Acinar Cell Carcinoma: Retrospective Analysis from the German Cancer Registry Group.

Simple SummaryLess than 1% of all pancreatic malignancies are acinar cell carcinomas. Based on data from the German Cancer Registry Group, we performed a comparative analysis of characteristics and prognostic factors of pancreatic acinar cell carcinoma and the most common type of pancreatic cancer—pancreatic ductal adenocarcinoma. Compared to pancreatic ductal adenocarcinoma, patients with pancreatic acinar cell carcinoma were younger at the time of diagnosis and the percentage of males was higher. The prognosis of patients with pancreatic acinar carcinoma was better than that of patients with pancreatic ductal adenocarcinoma. Surgical resection was the strongest positive prognostic factor for pancreatic acinar cell carcinoma. The study shows that pancreatic acinar cell carcinoma has features distinct from pancreatic ductal adenocarcinoma. Radical resection should be advocated, whenever feasible. Background: Pancreatic acinar cell carcinoma (PACC) is a distinct type of pancreatic cancer with low prevalence. We aimed to analyze prognostic factors and survival outcome for PACC in comparison to pancreatic ductal adenocarcinoma (PDAC), based on data from the German Cancer Registry Group. Methods: Patients with PACC and PDAC were extracted from pooled data of the German clinical cancer registries (years 2000 to 2019). The distribution of demographic parameters, tumor stage and therapy modes were compared between PACC and PDAC. The Kaplan–Meier method and Cox regression analysis were used to delineate prognostic factors for PACC. Propensity score matching was used to compare survival between PACC and PDAC. Results: There were 233 (0.44%) patients with PACC out of 52,518 patients with pancreatic malignancy. Compared to PDAC, patients with PACC were younger (median age 66 versus 70, respectively, p < 0.001) and the percentage of males was higher (66.1% versus 53.3%, respectively, p < 0.001). More patients were resected with PACC than with PDAC (56.2% versus 38.9%, respectively, p < 0.001). The estimated overall median survival in PACC was 22 months (95% confidence interval 15 to 27), compared to 12 months (95% confidence interval 10 to 13) in the matched PDAC cohort (p < 0.001). Surgical resection was the strongest positive prognostic factor for PACC after adjusting for sex, age, and distant metastases (hazard ratio 0.34, 95% confidence interval 0.22 to 0.51, p < 0.001). There was no survival benefit for adjuvant therapy in PACC. Conclusions: PACC has overall better prognosis than PDAC. Surgical resection is the best therapeutic strategy for PACC and should be advocated even in advanced tumor stages.

PD-1/PD-L1–Associated Immunoarchitectural Patterns Stratify Pancreatic Cancer Patients into Prognostic/Predictive Subgroups

Immunotherapy, including PD-1/PD-L1 agonists, has shown limited efficacy in pancreatic ductal adenocarcinoma (PDAC). We examined the PD-1/PD-L1 expression and immunoarchitectural features by automated morphometric analysis using multiplex immunofluorescence and 118 microsatellite-stable, treatment-naïve, surgically resected PDACs (study cohort). Five microsatellite-instable cases were stained in parallel (MSI cohort). Molecular analysis was additionally performed. An independent PDAC cohort (n = 226) was immunostained for PD-L1 and used as a validation cohort. PD-L1 expression on tumor cells (TC) and/or immune cells (IC) was present in 32% and 30% of the study and validation cohorts, respectively, and assigned into one of four patterns: "adaptive-1" (TC: 0, IC > 1%), "adaptive-2" (TC > 1% to < 25%, IC > 1%), "constitutive" (TC ≥ 25%, IC: 0), and "combined" (TC ≥ 25%, IC > 1%). "Constitutive" tumors were characterized by reduced numbers of all ICs and poor outcome. In contrast, "adaptive-1" tumors exhibited abundant T cells, including high counts of cytotoxic CD3+CD8+ and PD-1+CD3+CD8+ cells, but low counts of PD-L1+CD3+CD8+ cells and associated with the best outcome. "Adaptive-2" tumors displayed higher proportions of PD-L1+CD3+CD8+ T cells and tumor-associated macrophages (CD68+ and CD68+CD206+) compared with "adaptive-1" tumors. In the "combined" pattern, extensive PD-L1 expression on TCs was accompanied by increased numbers of T cells and improved overall survival. ICs were closer to PD-L1- than to PD-L1+ PDAC cells. TP53 and PIK3CA alterations tended to be more frequent in PD-L1+ tumors. The 5 MSI cases were PD-L1- The distinct PD-1/PD-L1-associated immunoarchitectural patterns underpin the heterogeneity of the immunologic responses and might be used to inform patient outcomes and therapeutic decisions in pancreatic cancer.

A systematic review of the prevalence of germline pathogenic variants in patients with pancreatic cancer.

The genetics of pancreatic ductal adenocarcinoma (PDAC) is complex with patients reported to harbor germline pathogenic variants (PVs) in many different genes. PDAC patients with familial pancreatic cancer (FPC) are more likely to carry germline PVs but there is no consensus main gene involved in FPC. We performed a systematic review of publications from PubMed and Scopus reporting PVs in patients with FPC, sporadic pancreatic cancer (SPC) and unselected cohorts of PDAC patients undergoing genetic testing and calculated a cumulative prevalence of PVs for each gene evaluated across these three groups of patients. When available, variants in the selected publications were reclassified according to the American College of Medical Genetics and Genomics classification system and used for prevalence calculations if classified as pathogenic or likely pathogenic. We observed an increased prevalence of PVs in FPC compared to SPC or unselected PDAC patients for most of the 41 genes reported. The genes with the highest prevalence of carriers of PVs in FPC were ATM, BRCA2, and CDKN2A. BRCA2 and ATM showed the highest prevalence of PVs in both SPC and unselected PDAC cohorts. Several genes with the highest prevalence of PVs are involved in breast and ovarian cancer suggesting strong overlap with underlying genetics in these disorders but no single gene was predominant. More research is needed to further understand the risk of PDAC associated with these many diverse genes.

Abstract 2372: Mechanisms of microRNA-21 dysregulation in pancreatic ductal adenocarcinoma (PDAC)

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with tumorigenesis largely driven by a nearly ubiquitous activating KRAS (mKRAS) mutation. Thus far efforts to target mKRAS have been largely unsuccessful due to compensatory mechanisms and associated toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that represent a novel avenue to target KRAS effector function through post-transcriptional regulation. We recently described dysregulation of miRNA-21 (miR-21) in transgenic KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice as a cell- and compartment-specific regulator of tumor epithelial cells. Here we report on the use of human models to elucidate the underlying mechanisms responsible for cellular phenotypes defined by KPC modeling. Experimental Procedures: The Cancer Genome Atlas (TCGA) was leveraged to computationally identify putative targets of miR-21 and additional pathways of interest for transcriptional and functional experiments. Concurrently, de-novo patient-derived organoid (PDO) models were generated from core biopsies and surgical resection specimens and expanded in Matrigel for current experiments. miR-21 expression in five PDO cell lines is profiled using qPCR. Stable knock-down of miR-21 is explored using lentiviral constructs in PDOs and cancer associated fibroblasts (CAFs) for use in a co-culture models to examine the mechanisms of action of miR-21 and investigating messenger RNA targets (mRNA) of miR-21. We have simultaneously used TCGA to examine targets of miR-21 and identify pathways of interest for further transcriptional and functional experiments. Results: Analysis of TCGA PDAC cohort examined 48 putative targets of miR-21. Decreases in the target mRNA levels of these 48 targets were heterogenous. Prominently, an inverse relationship was identified between miR-21 expression and the expression of one of its target mRNAs: programmed cell death protein 4 (PDCD4). Gene set enrichment analysis is underway to identify additional biologic targets of interest. Evaluation of miR-21 expression in PDO and matched CAF cell lines demonstrated robust and repeatable miR-21 identification as well as stability of knocked down miR-21 expression following transduction with a lentiviral miR-21 inhibitor construct. Conclusions: We previously demonstrated that miR-21 appears to be an early and reliable molecular marker of pancreatic neoplasia. The lack of global inverse relationship between miR-21 and target messenger RNA expression suggests more complexity to this regulation or bias from TCGA cohort, as the majority of patients in this cohort have early stage disease. These findings warrant additional mechanistic evaluation of specific targets such as PDCD4, which may have significant biologic importance and insight into our previously described phenotype. PDOs are powerful models for these mechanistic studies. Citation Format: Jacquelyn W. Zimmerman, Richard A. Burkhart, Elana J. Fertig, Elizabeth M. Jaffee. Mechanisms of microRNA-21 dysregulation in pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2372.

Genomic relevance of FGF14 and associated genes on the prognosis of pancreatic cancer.

BackgroundFibroblast (FGFs) and insulin (IGF) growth factor pathways are among 10 most recurrently altered genomic pathways in pancreatic ductal adenocarcinoma (PDAC). However, the prognostic and therapeutic relevance of FGF and IGF pathways in PDAC is largely unknown.MethodsWe investigated the relationship between fibroblast and insulin pathway gene expression and clinicopathological features in three independent transcriptomic cohorts of 532 PDAC patients. Furthermore, we have examined the coexpressed genes specific to the prognostic marker identified from these cohorts. Statistical tests including Fisher-exact\\Chi-square, Kaplan–Meier, Pearson Correlation and cox regression analyses were performed. Additionally, pathway analysis of gene-specific co-expressed genes was also performed.ResultsThe dysregulation of six genes including FGF9, FGF14, FGFR1, FGFR4, IGF2BP2 and IGF2BP3 were significantly associated with different clinical characteristics (including grade, stage, recurrence and nodes) in PDAC cohorts. 11 genes (including FGF9, FGF13, FGF14, FGF17, FGFR1, FGFRL1, FGFBP3, IGFBP3, IGF2BP2, IGF2BP3 and IGFBPL1) showed association with overall survival in different PDAC cohorts. Interestingly, overexpression of FGF14 was found associated with better overall survival (OS) in all three cohorts. Of note, multivariate analysis also revealed FGF14 as an independent prognostic marker for better OS in all three cohorts. Furthermore, FMN2 and PGR were among the top genes that correlated with FGF14 in all 3 cohorts. Of note, overexpression of FMN2 and PGR was found significantly associated with good overall survival in PDAC patients, suggesting FMN2 and PGR can also act as potential markers for the prediction of prognosis in PDAC patients.ConclusionFGF14 may define a distinct subset of PDAC patients with better prognosis. Moreover, FGF14-based sub-classification of PDAC suggests that FMN2 and PGR can be employed as good prognostic markers in PDAC and this classification may lead to new therapeutic approaches.

Single-cell analysis of pancreatic ductal adenocarcinoma identifies a novel fibroblast subtype associated with poor prognosis but better immunotherapy response

The current pathological and molecular classification of pancreatic ductal adenocarcinoma (PDAC) provides limited guidance for treatment options, especially for immunotherapy. Cancer-associated fibroblasts (CAFs) are major players of desmoplastic stroma in PDAC, modulating tumor progression and therapeutic response. Using single-cell RNA sequencing, we explored the intertumoral heterogeneity among PDAC patients with different degrees of desmoplasia. We found substantial intertumoral heterogeneity in CAFs, ductal cancer cells, and immune cells between the extremely dense and loose types of PDACs (dense-type, high desmoplasia; loose-type, low desmoplasia). Notably, no difference in CAF abundance was detected, but a novel subtype of CAFs with a highly activated metabolic state (meCAFs) was found in loose-type PDAC compared to dense-type PDAC. MeCAFs had highly active glycolysis, whereas the corresponding cancer cells used oxidative phosphorylation as a major metabolic mode rather than glycolysis. We found that the proportion and activity of immune cells were much higher in loose-type PDAC than in dense-type PDAC. Then, the clinical significance of the CAF subtypes was further validated in our PDAC cohort and a public database. PDAC patients with abundant meCAFs had a higher risk of metastasis and a poor prognosis but showed a dramatically better response to immunotherapy (64.71% objective response rate, one complete response). We characterized the intertumoral heterogeneity of cellular components, immune activity, and metabolic status between dense- and loose-type PDACs and identified meCAFs as a novel CAF subtype critical for PDAC progression and the susceptibility to immunotherapy.

An Eight-Gene Hypoxia Signature Predicts Survival in Pancreatic Cancer and Is Associated With an Immunosuppressed Tumor Microenvironment.

Intratumoral hypoxia is a widely established element of the pancreatic tumor microenvironment (TME) promoting immune escape, tumor invasion, and progression, while contributing to treatment resistance and poor survival. Despite this critical role, hypoxia is underrepresented in molecular signatures of pancreatic ductal adenocarcinoma (PDA) and concurrent investigations into the hypoxia-immune status are lacking. In this work a literature-based approach was applied to derive an eight-gene hypoxia signature that was validated in fourteen cancer cell lines and in a cohort of PDA. The eight-gene hypoxia signature was significantly associated with overall survival in two distinct PDA datasets and showed independent prognostic value in multivariate analysis. Comparative analysis of tumors according to their hypoxia score (high versus low) determined that tumors with high hypoxia were significantly less enriched in cytotoxic T-cells, and cytolytic activity. In addition, they had lower expression of cytokines and tumor inflammatory markers, pointing to the signature’s ability to discern an immune “cold”, hypoxic TME. Combining the signature with an immune metric highlighted a worse survival probability in patients with high hypoxia and low immune reactivity, indicating that this approach could further refine survival estimates. Hypoxia as determined by our signature, was significantly associated with certain immune checkpoint inhibitors (ICI) biomarkers, suggesting that the signature reflects an aspect of the TME that is worth pursuing in future clinical trials. This is the first work of its kind in PDA, and our findings on the hypoxia-immune tumor contexture are not only relevant for ICI but could also guide combinatorial hypoxia-mediated therapeutic strategies in this cancer type.

Genomic profiling of KRAS wide-type pancreatic ductal adenocarcinomas identifies targetable genetic alterations.

4121 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and the 5-year survival of PDAC patients is below 10%. The oncogenic KRASmutations account for about 90% of PDAC cases. Unfortunately, there is no FDA-approved targeted therapy for KRAS mutations. Therefore, the genomic profiling of KRAS wide-type PDACs can provide invaluable sights to the etiology of these patients and offer them the opportunity of precision therapy trials. Methods: To characterize actionable targets in 521 Chinese PDAC patients, deep sequencing of a 831-gene panel (OncoPanscan, Genetronhealth) was applied to assess somatic mutations of their tumor tissues including SNV, insertions/deletions, CNV and re-arrangements, as well as possible pathogenic germline variants of paired genomic DNA sample. Results: There were 89% (463/521) of patients in our PDAC cohort harbored KRAS mutations. Among the remaining 58 patients in KRAS wild-type subgroup, 33% (19/58) carried activating mutations in the RTK/Ras/MAPK pathway. Targetable BRAF mutations were seen in five (9%) patients: V600E (1/5), G464V (1/5), N486_P490del (2/5) and BRAF fusion (1/5). The frequency of BRAF mutations was 9% (5/58) in KRAS wild-type PDACs but only 0.4% (2/463) in KRAS-mutated PDACs (P &lt; 0.001). We found one classic EGFR activing mutation (L747_A750delinsP) and one MAP2K1 activating mutation (F53_Q58delinsL), which can be targeted by EGFR-TKIs and MEK inhibitor trametinib, respectively. An oncogenic ERBB3 mutation (V104L) was seen in one patient, who was eligible for HER2-targeted therapy clinical trials. We also found STK11/TSC2 inactivating mutations and a dominant-negative mutation of PTEN (R130Q) which could be targeted by mTOR inhibitor everolimus and AKT inhibitor capivasertib, respectively. Additionally, we observed a patient with high level amplification of MET and another patient with the NCOA4-RET fusion gene which can be targeted by MET inhibitor carbozantinib. Interestingly, we also identified two patients with inactivating mutations in ELF3 (one frameshift and one in splicing-site), which is a driver gene of ampullary carcinoma. Lastly, two patients carried deleterious germline mutations in BRCA1 and PALB2, which may be targeted with PARP inhibitors. Overall, at least 29% (17/58) KRAS wide-type patients harbored potentially actionable genomic alterations to currently used anticancer drugs. Conclusions: The mutational landscape of our PDAC cohort provided compelling evidence that targetable driver mutations accounted for a significant portion of KRAS wide-type tumors. Our findings demonstrated that genomic profiling of PDAC patients can enable physicians to optimize their clinical management and enroll them into genomically matched clinical trials.

CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2 and promotes growth and metastasis of pancreatic cancer

BackgroundBoth aberrant alternative splicing and m6A methylation play complicated roles in the development of pancreatic cancer (PC), while the relationship between these two RNA modifications remains unclear.MethodsRNA sequencing (RNA-seq) was performed using 15 pairs of pancreatic ductal adenocarcinoma (PDAC) tissues and corresponding normal tissues, and Cdc2-like kinases 1 (CLK1) was identified as a significantly upregulated alternative splicing related gene. Real-time quantitative PCR (qPCR) and western blotting were applied to determine the CLK1 levels. The prognostic value of CLK1 was elucidated by Immunohistochemistry (IHC) analyses in two independent PDAC cohorts. The functional characterizations and mechanistic insights of CLK1 in PDAC growth and metastasis were evaluated with PDAC cell lines and nude mice. SR-like splicing factors5250-Ser (SRSF5250-Ser) was identified as an important target phosphorylation site by phosphorylation mass spectrometry. Through transcriptome sequencing, Methyltransferase-like 14exon10 (METTL14exon10) and Cyclin L2exon6.3 skipping were identified as key alternative splicing events regulated by the CLK1-SRSF5 axis. RIP assays, RNA-pulldown and CLIP-qPCR were performed to confirm molecular interactions and the precise binding sites. The roles of the shift of METTL14exon 10 and Cyclin L2exon6.3 skipping were surveyed.ResultsCLK1 expression was significantly increased in PDAC tissues at both the mRNA and protein levels. High CLK1 expression was associated with poor prognosis. Elevated CLK1 expression promoted growth and metastasis of PC cells in vitro and in vivo. Mechanistically, CLK1 enhanced phosphorylation on SRSF5250-Ser, which inhibited METTL14exon10 skipping while promoted Cyclin L2exon6.3 skipping. In addition, aberrant METTL14exon 10 skipping enhanced the N6-methyladenosine modification level and metastasis, while aberrant Cyclin L2exon6.3 promoted proliferation of PDAC cells.ConclusionsThe CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2, which promotes growth and metastasis and regulates m6A methylation of PDAC cells. This study suggests the potential prognostic value and therapeutic targeting of this pathway in PDAC patients.

Deciphering the Prognostic Implications of the Components and Signatures in the Immune Microenvironment of Pancreatic Ductal Adenocarcinoma

Background: The treatment modalities for pancreatic ductal adenocarcinoma (PDAC) are limited and unsatisfactory. Although many novel drugs targeting the tumor microenvironment, such as immune checkpoint inhibitors, have shown promising efficacy for some tumors, few of them significantly prolong the survival of patients with PDAC due to insufficient knowledge on the tumor microenvironment.Methods: A single-cell RNA sequencing (scRNA-seq) dataset and seven PDAC cohorts with complete clinical and bulk sequencing data were collected for bioinformatics analysis. The relative proportions of each cell type were estimated using the gene set variation analysis (GSVA) algorithm based on the signatures identified by scRNA-seq or previous literature.Results: A meta-analysis of 883 PDAC patients showed that neutrophils are associated with worse overall survival (OS) for PDAC, while CD8+ T cells, CD4+ T cells, and B cells are related to prolonged OS for PDAC, with marginal statistical significance. Seventeen cell categories were identified by clustering analysis based on single-cell sequencing. Among them, CD8+ T cells and NKT cells were universally exhausted by expressing exhaustion-associated molecular markers. Interestingly, signatures of CD8+ T cells and NKT cells predicted prolonged OS for PDAC only in the presence of “targets” for pyroptosis and ferroptosis induction. Moreover, a specific state of T cells with overexpression of ribosome-related proteins was associated with a good prognosis. In addition, the hematopoietic stem cell (HSC)-like signature predicted prolonged OS in PDAC. Weighted gene co-expression network analysis identified 5 hub genes whose downregulation may mediate the observed survival benefits of the HSC-like signature. Moreover, trajectory analysis revealed that myeloid cells evolutionarily consisted of 7 states, and antigen-presenting molecules and complement-associated genes were lost along the pseudotime flow. Consensus clustering based on the differentially expressed genes between two states harboring the longest pseudotime span identified two PDAC groups with prognostic differences, and more infiltrated immune cells and activated immune signatures may account for the survival benefits.Conclusion: This study systematically investigated the prognostic implications of the components of the PDAC tumor microenvironment by integrating single-cell sequencing and bulk sequencing, and future studies are expected to develop novel targeted agents for PDAC treatment.

Abstract LT005: Tumor-intrinsic gain of function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment that promotes resistance to immunotherapy

Abstract KRASG12D mutations in tumor cells can modify the immune microenvironment of pancreatic ductal adenocarcinoma (PDAC), though the effects of secondary genetic alterations have been unexplored. After oncogenic KRAS, TP53 is the most frequently mutated gene in PDAC, however the immune effects of neomorphic TP53 mutations have not been defined. We sought to evaluate the non-cell-autonomous role of mutant p53 in modulating the immune microenvironment of pancreatic cancer by comparing the immune profiles generated by KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) genetically engineered to express the Trp53R172H gain-of-function mutation to their p53 wildtype control. We found orthotopically implanted tumors derived from KrasG12D;Trp53R172H PDEC had a distinct immune profile in comparison to KrasG12D/+;Trp53+/+ tumors, characterized by an influx of intratumoral CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 (Th1) cells. Analysis of publicly available human PDAC cohorts revealed enrichment of genes in neutrophil-related pathways in TP53-mutated tumors. Knockdown of CXCL2, a neutrophil chemoattractant, in the tumor epithelial compartment of KrasG12D;Trp53R172H PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing KrasG12D;Trp53R172H PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link mutant p53 to the presence of intratumoral neutrophils in pancreatic cancer and suggests that tumor genotypes could inform patient selection for immunotherapy. Citation Format: Despina Siolas, Emily Vucic, Emma Kurz, Cristina Hajdu, Dafna Bar-Sagi. Tumor-intrinsic gain of function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment that promotes resistance to immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT005.

Metabolic profiling of a cohort of pancreatic ductal adenocarcinoma (PDAC) patients of african ancestry

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is deadly cancer with dysregulated metabolism being one of its key features, affecting several processes such as immune response. Studying metabolism in PDAC especially in under-studied population groups could lead to the identification of biomarkers and in better understanding of the immune response. We aimed to identify dysregulated metabolites in PDAC progression and their possible role in the immune response. Method: Blood samples were collected from 34 PDAC, 6 CP, and 6 healthy volunteers, all of African ancestry (M190681). Nuclear Magnetic Resonance (NMR) spectroscopy and Reactive Oxygen Species (ROS) analyses were used to identify metabolites and quantify ROS, respectively. The R studio V1.3 was used for statistical analysis and a p-value of <0.05 was considered significant. Results: Preliminary results from the study showed that tyrosine (p-value:0.001), histidine (p-value:0.004), acetate (p-value:0.002) and 3-hydroxybutyrate (p-value:0.018) levels were elevated as the disease progressed. Total Bilirubin exceeded the normal threshold of 5-21 μmol/L for the PDAC patients. Mortality occurred earlier for patients with inorganic phosphates (p-value: 0.229) higher than 1.42 mmol/L and anion gap (p-value: 0.002) higher than the threshold 9-16 mmol/L. Furthermore, patients having C-reactive protein (CRP) < 200mg/L and metabolic value of GlycA < 1.1 survived longer than 150 days. ROS was elevated in resectable (p-value:0.003) and Locally advanced (p-value:0.008) PDAC when compared with HC. Conclusion: Dysregulated metabolism is associated with increased ROS which has been shown to be linked to immunosuppression. The combination of dysregulated metabolites, ROS, and clinical parameters may have potential utility in disease stratification, management, and future therapeutic targeting.

Transcriptome profiling reveals liver metastasis-associated genes in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies worldwide. However, the mechanisms underlying the acquisition of the metastatic potential in PDAC has not been well understood. In this study, we identified a total of 154 genes upregulated in primary tissues of PDAC with liver metastasis using the Genome Cancer Atlas (TCGA) and GSE151580 cohorts. The epithelial-mesenchymal transition and glycolysis were enriched by the liver metastasis-related genes, indicating that the liver metastasis-related genes might be functionally relevant to liver metastasis in PDAC. Moreover, we also found that the liver metastasis-related genes were primarily regulated at epigenetic level. Particularly, SFN, a cell cycle checkpoint protein, and KRT19, a marker gene for ductal cells, were predicted to be regulated by multiple methylation sites at the promoter. Clinically, we for the first time defined a liver metastasis score (LMS), which was derived from liver metastasis-related genes, and closely associated with clinical characteristics such as disease type and tumor grade, in PDAC. Furthermore, we also divided the samples into high and low LMS groups using three cohorts with long-term follow-up (TCGA, GSE71729, and E-MTAB-6134), which exhibited significantly different prognostic outcomes across three PDAC cohorts, suggesting that the LMS might be a good indicator for risk stratification in PDAC. Furthermore, we also found that the liver metastasis-related genes were primarily expressed in malignant ductal cells by integrative analysis of the bulk and single-cell gene expression data. Moreover, the malignant ductal cells and M0 macrophages were highly correlated with LMS, indicating that the two cell types might function as tumor-promoting cells in PDAC. In summary, the systematic analysis for the first time defined an LMS score to evaluate the risk of liver metastasis in PDAC and revealed that malignant ductal cells might promote PDAC liver metastasis, which greatly improves our understanding of the liver metastasis-related genes, their underlying mechanisms, and association with prognosis in PDAC.

Immune profiling and immunotherapeutic targets in pancreatic cancer.

BackgroundImmunotherapeutic approaches for pancreatic ductal adenocarcinoma (PDAC) are less successful as compared to many other tumor types. In this study, comprehensive immune profiling was performed in order to identify novel, potentially actionable targets for immunotherapy.MethodsFormalin-fixed paraffin embedded (FFPE) specimens from 68 patients were evaluated for expression of 395 immune-related markers (RNA-seq), mutational burden by complete exon sequencing of 409 genes, PD-L1 expression by immunohistochemistry (IHC), pattern of tumor infiltrating lymphocytes (TILs) infiltration by CD8 IHC, and PD-L1/L2 copy number by fluorescent in situ hybridization (FISH).ResultsThe seven classes of actionable genes capturing myeloid immunosuppression, metabolic immunosuppression, alternative checkpoint blockade, CTLA-4 immune checkpoint, immune infiltrate, and programmed cell death 1 (PD-1) axis immune checkpoint, discerned 5 unique clinically relevant immunosuppression expression profiles (from most to least common): (I) combined myeloid and metabolic immunosuppression [affecting 25 of 68 patients (36.8%)], (II) multiple immunosuppressive mechanisms (29.4%), (III) PD-L1 positive (20.6%), (IV) highly inflamed PD-L1 negative (10.3%); and (V) immune desert (2.9%). The Wilcoxon rank-sum test was used to compare the PDAC cohort with a comparison cohort (n=1,416 patients) for the mean expressions of the 409 genes evaluated. Multiple genes including TIM3, VISTA, CCL2, CCR2, TGFB1, CD73, and CD39 had significantly higher mean expression versus the comparison cohort, while three genes (LAG3, GITR, CD38) had significantly lower mean expression.ConclusionsThis study demonstrates that a clinically relevant unique profile of immune markers can be identified in PDAC and be used as a roadmap for personalized immunotherapeutic decision-making strategies.

A new prognosis prediction model combining TNM stage with MAP2K4 and JNK in postoperative pancreatic cancer patients

Mitogen-activated protein kinase kinase 4 (MAP2K4) is a tumor suppressor in many cancers. However, its roles and action mechanisms in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Here, we analyzed MAP2K4 and its downstream kinases (c-Jun N-terminal kinase (JNK) and p38) using immunohistochemical staining and their prognostic significances using univariate and multivariate Cox proportional hazards regression analysis in our PDAC cohort. Then, we validated MAP2K4/JNK/p38 mRNA levels and prognostic significances using The Cancer Genome Atlas (TCGA) database. Finally, we evaluated the effects of MAP2K4 on the proliferation and invasion of PDAC cells. MAP2K4, JNK, and p38 proteins were expressed in 97.3 % (72/74), 95.6 % (65/68), and 88.6 % (62/70) of the samples, respectively, and their levels in tumor tissues were significantly higher than those in normal ducts. MAP2K4 protein expression was lower in male patients (p = 0.028). In our PDAC cohort, advanced TNM stage, low MAP2K4, and high JNK protein levels were significant prognostic factors for poor overall survival (OS) based on a univariate survival analysis (p = 0.006, p < 0.001, and p = 0.004, respectively). N stage and MAP2K4 and JNK protein levels were independent prognostic factors for OS based on multivariate analysis. We then built a prognosis prediction nomogram combining the standard TNM staging system with MAP2K4 and JNK expression that had a Harrell’s C-index of 0.645. The new prognosis prediction model effectively stratified the resected patients with PDAC, from both our cohort and TCGA database, into low- and high-risk groups. Finally, MAP2K4 overexpression inhibited pancreatic cancer cell proliferation and migration in vitro. This study shows that reduced protein and mRNA levels of MAP2K4 found in PDAC patients, coupled to in vitro effects observed, support the tumor suppressor role of MAP2K4 in PDAC. Importantly, combining MAP2K4 and JNK expression with the TNM staging system results in a better prediction of postoperative survival of patients with PDAC.

Tumor Size Differences Between Preoperative Endoscopic Ultrasound and Postoperative Pathology for Neoadjuvant-Treated Pancreatic Ductal Adenocarcinoma Predict Patient Outcome

The assessment of therapeutic response after neoadjuvant treatment and pancreatectomy for pancreatic ductal adenocarcinoma (PDAC) has been an ongoing challenge. Several limitations have been encountered when employing current grading systems for residual tumor. Considering endoscopic ultrasound (EUS) represents a sensitive imaging technique for PDAC, differences in tumor size between preoperative EUS and postoperative pathology after neoadjuvant therapy were hypothesized to represent an improved marker of treatment response. For 340 treatment-naïve and 365 neoadjuvant-treated PDACs, EUS and pathologic findings were analyzed and correlated with patient overall survival (OS). A separate group of 200 neoadjuvant-treated PDACs served as a validation cohort for further analysis. Among treatment-naïve PDACs, there was a moderate concordance between EUS imaging and postoperative pathology for tumor size (r= 0.726, P < .001) and AJCC 8th edition T-stage (r= 0.586, P < .001). In the setting of neoadjuvant therapy, a decrease in T-stage correlated with improved 3-year OS rates (50% vs 31%, P < .001). Through recursive partitioning, a cutoff of ≥47% tumor size reduction was also found to be associated with improved OS (67% vs 32%, P < .001). Improved OS using a ≥47% threshold was validated using a separate cohort of neoadjuvant-treated PDACs (72% vs 36%, P < .001). By multivariate analysis, a reduction in tumor size by ≥47% was an independent prognostic factor for improved OS (P= .007). The difference in tumor size between preoperative EUS imaging and postoperative pathology among neoadjuvant-treated PDAC patients is an important prognostic indicator and may guide subsequent chemotherapeutic management.

Abstract PR-005: Tumor intrinsic p53 mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy

Abstract Intratumoral genetic alterations can cause remodeling of local immune cell populations through distinct cellular mechanisms. After oncogenic KRAS, TP53 is the gene most frequently mutated in pancreatic ductal adenocarcinoma (PDAC). These mutations in TP53 are neomorphic and confer phenotypes distinct from those due to homozygous TP53 deletion. We sought to evaluate the non-cell-autonomous role of mutant p53 protein in the immune microenvironment by studying KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) engineered using CRISPR/Cas gene editing to express the Trp53R172H gain-of-function mutation. We found distinct immune profiles associated with orthotopically implanted tumors derived from KrasG12D;Trp53R172H PDEC, characterized by an influx of intratumoral CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 (Th1) cells. Analysis of publicly available human PDAC cohorts revealed enrichment of genes in neutrophil-related pathways in TP53-mutated tumors. Knockdown of CXCL2, a neutrophil chemoattractant, in the tumor epithelial compartment of KrasG12D;Trp53R172H PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing KrasG12D;Trp53R172H PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link mutant p53 to the presence of intratumoral neutrophils in pancreatic cancer and suggests that tumor genotypes could inform patient selection for immunotherapy. Citation Format: Despina Siolas, Emily Vucic, Emma Kurz, Cristina Hajdu, Dafna Bar-Sagi. Tumor intrinsic p53 mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PR-005.