Advances in pancreatic cancer early diagnosis, prevention, and treatment: The past, the present, and the future.

With an overall survival rate of 2–9% at 5 years, pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related deaths in the industrialized world and is predicted to become the second by 2030. Owing to often late diagnosis and rare actionable molecular alterations, PDAC has not yet benefited from the recent therapeutic advances that immune checkpoint inhibitors (ICI) have provided in other cancer types, except in specific subgroups of patients presenting with tumors with high mutational burden (TMB) or microsatellite instability (MSI). The tumor microenvironment (TME) plays a substantial role in therapeutic resistance by facilitating immune evasion. An extracellular stromal protein, βig-h3/TGFβi, is involved in the pathogenesis of PDAC by hampering T cell activation and promoting stiffness of the TME. The study BIGHPANC included 41 patients with metastatic PDAC, and analyzed βig-h3 levels in serum and tumor samples to assess the βig-h3 prognostic value. βig-h3 serum levels are significantly associated with overall survival (HR 2.05, 95%CI 1.07–3.93; p = 0.0301). Our results suggest that βig-h3 serum levels may be considered a prognostic biomarker in patients with metastatic PDAC.

Background: Survival for patients with pancreatic ductal adenocarcinoma (PDAC) remains dismal and the majority of patients succumb to metastatic disease. Even for those with localized PDAC, most will die from metastatic disease despite margin-negative resection and adjuvant therapy. Therefore, these patients must harbor occult metastatic PDAC at presentation. There is a compelling need for the development of preclinical models that efficiently recapitulate occult metastatic liver PDAC to identify molecular and cellular pathways that drive metastatic cellular survival and growth. Towards this aim, we have developed a PDAC model of occult liver metastases using patient-derived xenografts (PDXs) to study the growth of PDAC within the metastatic environment and evaluated the effect of MEK inhibitor therapy on tumor progression. Methods and Results: Extensively characterized patient-derived KRAS-mutant (Tumors 608, 366, and 654) and wild-type (Tumors 738 and 215) PDAC cells were transduced with luciferase and injected into the spleens of athymic, nude mice, allowed to circulate for 10 minutes, after which a splenectomy was performed. To evaluate metastatic cell growth kinetics in the liver, tumor burden was monitored by sequential bioluminescent imaging. All mice exhibited defined phases of survival/dormancy and the proliferative outgrowth; however, differential kinetics were observed for each tumor cell line. To evaluate the effect of MEK inhibition of occult metastatic PDAC cells in the liver, Tumor 608 cells were injected and mice either received the MEK inhibitor trametinib (0.3 mg/kg, daily) or vehicle control beginning 48 hour post-injection. Trametinib significantly reduced metastatic tumor burden, delayed time to proliferative outgrowth, and greatly prolonged survival as compared to control (med. survival: 114 vs. 43 days, p<0.001). In contrast, in an orthotopic model with 250-500 mm3 tumors trametinib led to no inhibition in tumor growth for Tumor 608. To characterize metastatic PDAC cells within the liver after similar treatment conditions, we isolated cells from the liver 48 and 72 hours, 10 and 28 days after splenic injection. Tumor 608 cells were injected and metastatic PDAC cells were retrieved from the liver at the aforementioned timepoints post injection using magnetic column separation with human EpCAM (CD326)-targeted magnetically labeled microbeads. Flow cytometric analyses of retrieved cells revealed that decreased cellular markers of proliferation and increased caspase-3 cleavage correlated with decreased tumor burden observed at these timepoints in mice treated with trametinib. Conclusions: In a model of occult liver metastatic PDAC, patient-derived tumors exhibit different growth kinetics in the liver environment. Furthermore, MEK inhibition with trametinib decreased metastatic cellular proliferation, increased apoptosis, prolonged metastatic tumor outgrowth, and significantly increased survival of mice harboring occult hepatic metastases from PDAC. This efficacy of single agent trametinib is unique to occult metastatic disease and is not seen in orthotopic models of advanced, established pancreatic tumors. This finding illustrates the importance of using specific preclinical models that best recapitulate the clinical aspects of patients who will be evaluated in future clinical trials. Further investigation into the cellular and molecular factors promoting PDAC cell survival within the hepatic microenvironment utilizing this model will lead to development of rational therapeutic strategies for patients with occult metastatic disease. Citation Format: Timothy Newhook, James Lindberg, Sara Adair, J. Thomas Parsons, Todd W. Bauer. In a patient-derived xenograft model of occult hepatic metastasis from pancreatic cancer the MEK inhibitor trametinib delays tumor outgrowth and prolongs survival. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B21.

Pancreatic cancer remains one of the deadliest malignancies, with limited therapeutic options and a poor prognosis due to delayed symptom presentation and disease detection. Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 8%, predominantly attributed to oncogenic KRAS mutations and loss-of-function mutations in TP53, SMAD4, and P16. Our lab is interested in studying Transglutaminase 2 (TGM2), a multifunctional enzyme involved in protein cross-linking and cellular signaling, because it is highly correlated with cancer cell survival, malignancy, metastasis, and treatment resistance. Clinically, overexpression of TGM2 is associated with poorer survival in patients with Pancreatic Ductal Adenocarcinoma so it is imperative to understand the mechanism of its action in disease progression. To elucidate the role of TGM2 in PDAC, we studied the targeted deletion of TGM2 in the context of a mouse model of PDAC and saw a significant reduction in PDAC incidence and precursor lesion formation, suggesting that TGM2 has a role in the tumor microenvironment. Using single cell sequencing, we found TGM2 levels to be elevated in certain cell populations, specifically endothelial cells and cancer cells. We discovered high TGM2 levels in TIE2-GFP mouse vessels and subsequently found high TGM2 levels in patient tumor vasculature. Further studies aim to conditionally ablate TGM2 in endothelial cells to assess its mechanistic involvement in PDAC initiation and progression. Additionally, we are studying concurrent conditional TGM2 and SMAD4 deletions in mice to investigate pancreatic cancer precursor lesion formation and are further evaluating the protective mechanisms of a TGM2 deletion in a more aggressive mouse model of PDAC. Our preliminary findings substantiate TGM2 as a critical player in PDAC pathogenesis, opening avenues for targeted therapeutics to ultimately improve patient prognosis. Citation Format: Polina Wright, Azeddine Atfi. Elucidating the role of transglutaminase 2 (TGM2) in pancreatic ductal adenocarcinoma pathogenesis and its therapeutic 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 2896.

4146 Background: Currently, ICI therapy is recommended for patients with metastatic PDAC whose tumors exhibit dMMR, MSI-H, or high TMB (≥10 mutations/Mb). However, due to the low prevalence of high TMB in PDAC (~1%), few studies have evaluated the role of ICI therapy in this subpopulation. This study aimed to compare GA between PDAC patients with high TMB and low TMB ( < 10 mutations/Mb) and to evaluate the effectiveness of ICIs in real-world PDAC patients by TMB status. Methods: This study included PDAC patients who underwent genomic testing using Foundation Medicine tissue comprehensive genomic profiling (CGP) assays. GA were compared between tissue specimens with high and low TMB by chi-squared, adjusted for multiple comparisons. Patient clinical data was obtained by the US-wide de-identified Flatiron Health and Foundation Medicine real-world clinicogenomic pancreatic database (CGDB), originated from ~280 US cancer clinics between 01/2011 and 09/2022. Real-world overall survival (OS) and time to treatment discontinuation (TTD) were compared between patients receiving an ICI (high TMB versus low TMB) and between patients with high TMB (ICI versus other therapies) by Cox models. Results: We included 21,932 patients with PDAC with tissue CGP data available; 98.7% with low TMB and 1.3% with high TMB. Among actionable alterations, patients with high TMB had higher prevalence of mutations in BRCA2 (p < 0.0001), BRAF (p < 0.0001), PALB2 (p < 0.0001), and genes of the mismatch repair pathway ( MSH2, MSH6, MLH1, and PMS2, p < 0.0001), but lower prevalence of KRAS mutations (p < 0.0001). The most common KRAS mutation in both groups of patients was G12D. In CGDB, 51 patients received an ICI (10 with high TMB and 41 with low TMB) and 17 patients with high TMB received other therapies. Among patients receiving an ICI, those with high TMB had more favorable median OS compared to those with low TMB (25.7 versus 5.2 months, hazard ratio (HR) 0.27, 95% confidence interval (CI) 0.09 - 0.76, p = 0.01) and median TTD (20.7 versus 3.0 months, HR 0.33, 95% CI 0.13 - 0.82, p = 0.02). Among patients with high TMB, those receiving an ICI had more favorable OS compared to those receiving other therapies (25.7 versus 6.6 months, HR 0.31, 95% CI 0.10-0.96, p = 0.043), nominally favoring ICI use in this small cohort (n = 10 versus 17). Conclusions: There is no randomized clinical trial evaluating ICI versus other therapies in PDAC. To our knowledge, this is the largest cohort to date comparing ICI effectiveness in PDAC. We observed more favorable OS among PDAC patients with high TMB receiving ICI versus those with low TMB who received an ICI and more favorable to those with high TMB receiving other therapies. This study supports the FDA-approved use of ICIs in patients with PDAC and high TMB and demonstrates the importance of TMB assessment for patients with PDAC.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the USA. The 5-year survival of < 5% has not changed in decades. In contrast to other major cancers, the incidence of PDAC is increasing.Areas covered: The aims of this paper are first to analyze why PDAC is so difficult to treat and, second, to suggest future directions for PDAC therapeutics. The authors provide an article that is based on a comprehensive search through MEDLINE and the clinicalTrials.gov website.Expert opinion: Progress has been made recently. Notably, FOLFIRINOX or nab-paclitaxel plus gemcitabine provide survival benefit over gemcitabine alone, which was previously the mainstay of therapy for PDAC. Most of the current trials are testing combinations of repurposed drugs rather than addressing key targets in the PDAC pathogenesis. It is clear that to really make an impact on this disease, it will be necessary to address three different problems with targeted therapeutics. First, it is important to eradicate PDAC stem cells that result in recurrence. Second, it is important to reduce the peritumoral stroma that provides the tumors with growth support and provides a barrier to access of therapeutic agents. Finally, it is important to address the marked cachexia and metabolic derangement that contribute to morbidity and mortality and further complicate therapeutic intervention.

Many studies focusing on circular RNAs (circRNAs) have recently been published. However, a large number of circRNAs remain to be explored. This study was designed to discover new circRNAs and investigate their potential roles in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). A combination of gene chip analysis and bioinformatic methods was utilized to reveal new circRNAs and their possible mechanisms in PDAC. A circRNA-miRNA-mRNA network was established based on the results of differential analyses and interaction predictions. Promising drugs for treating PDAC were determined by connectivity map (CMap) analysis. Expression profile data were collected from the Gene Expression Omnibus database, and integration of differentially expressed circRNAs (DECs) from two gene chips using the RobustRankAggreg method revealed 10 DECs. The microRNA (miRNA) response elements of these 10 DECs were predicted. The predicted miRNAs and differentially expressed miRNAs were intersected, and 12 overlapping miRNAs were acquired. Next, 2908 miRNA target mRNAs and 1187 differentially expressed genes (DEGs) in PDAC were identified and combined, revealing 118 overlapping mRNAs. A protein-protein interaction network was constructed with the 118 mRNAs, and four hub genes (CDH1, SERPINE1, IRS1 and FYN) were identified. Using Gene Expression Profiling Interactive Analysis, survival analyses were conducted for the four hub genes, and SERPINE1 and FYN were found to be significantly associated with PDAC patient survival. Functional enrichment analysis indicated that these four hub genes are closely associated with certaincancer-related biological functions and pathways. In addition, CMap analysis based on the four hub genes was performed to screen potential therapeutic agents for PDAC, and three bioactive chemicals (celastrol, 5109870 and MG-132) were discovered. The results of this study further our understanding of the pathogenesis and treatment of PDAC from the perspective of the circRNA-related competing endogenous RNA network.

4145 Background: Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death. Outcomes remain poor, due to irresectability at diagnosis for many and sub-optimal responses to systemic therapy. Cytotoxic chemotherapy remains the standard of care. High microsatellite instability (MSI-H) predicts response to immune checkpoint inhibition (ICI) in many cancers. Detecting high MSI is rare in PDAC (incidence &lt;2%), but case reports demonstrate potential therapeutic benefit with ICI. Here, we present multi-institutional data to characterize the clinical behavior of MSI-H PDAC, with special attention to response to ICI. Methods: Cases of MSI-H PDAC were obtained by reviewing data of all PDAC patients from our tertiary cancer center who had undergone genomic sequencing by one commercially available platform. The resulting cohort was supplemented with MSI-H PDAC cases identified by GI oncology specialists at multiple institutions. De-identified patient data were compiled and analyzed. Results: 15 MSI-H PDAC patients were identified. 20% had stage II disease at diagnosis, 27% stage III, and 53% stage IV. 73% of patients received ICI (n=11); 40% as 1st line and 33% as 2nd line. These patients demonstrated 100% overall response rate; 45% complete response (1 pathologic CR, 4 radiographic CR) and 55% partial response. No patient that received ICI had lost response or died after a median follow-up of 18 months (range 6-89 mos). 1 patient had oligoprogression of a single hepatic lesion after 7 mos that was then irradiated; this patient retained radiographic CR for 17 subsequent mos (ongoing). In this cohort, we observe poor responses to cytotoxic chemotherapy. In total, 12 regimens were trialed among 9 patients. Overall response rate was 0%. 42% achieved disease stability, with median duration of response of 2 mos; only 2 cases maintained disease stability for &gt;5 mos. 4 patients did not receive ICI; all patients died, with a median survival of 7.5 mos. Conclusions: MSI-H PDAC represents a rare but important subtype of PDAC with unique clinical behavior. Given its rarity, large-scale analyses and trials are unlikely to be performed, making case series such as ours crucial. In our cohort, we observe impressive, durable responses to ICI, along with very poor responses to cytotoxic chemotherapy. Our data argues for consideration of ICI in any patient presenting with MSI-H PDAC, including in the first-line and neoadjuvant settings.

Pancreatic ductal adenocarcinoma (PDAC) is the fifth leading cause of cancer-related death in Western countries. The incidence of PDAC has increased over the last 40 years and is projected to be the second leading cause of cancer death by 2030. Despite aggressive treatment regimens, prognosis for patients diagnosed with PDAC is very poor; PDAC has the lowest 5-year survival rate for any form of cancer in the United States (US). PDAC is very rarely detected in early stages when surgical resection can be performed. Only 20% of cases are suitable for surgical resection; this remains the only curative treatment when combined with adjuvant chemotherapy. Treatment regimens excluding surgical intervention such as chemotherapeutic treatments are associated with adverse effects and genetherapy strategies also struggle with lack of specificity and/or efficacy. The lack of effective treatments for this disease highlights the necessity for innovation in treatment options for patients diagnosed with early- to late-phase PDAC and immuno-oncolytic viruses (OVs) have been of particular interest since 2006 when the first oncolytic virus was approved as a therapy for nasopharyngeal cancers in China. Interest resurged in 2015 when T-Vec, an oncolytic herpes simplex virus, was approved in the United States for treatment of advanced melanoma. While many vectors have been explored, few show promise as treatment for pancreatic cancer, and fewer still have progressed to clinical trial evaluation. This review outlines recent strategies in the development of OVs targeting treatment of PDAC, current state of preclinical and clinical investigation and application.

Targeting B cells in pancreatic adenocarcinoma: does RESOLVE resolve the question?

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. At present, surgical resection is the only curative treatment for PDAC, but the majority of PDAC patients show early recurrence even after curative resection. There is a possibility of having been distant micrometastasis at early stage of carcinogenesis in PDAC. The control of PDAC metastatic progression leads to improve the prognosis of PDAC patients. Recently, it is reported that a verified mediator of distant metastasis, Metadherin (MTDH), promotes metastasis in experimental models of breast cancer, and its high expression is associated with poor prognosis in a large spectrum of cancer types. Recent evidences imply that the epithelial-mesenchymal transition (EMT) and putative cancer stem cell (CSC) functions orchestrates to play crucial roles in cancer progression. In this study, we aim to investigate the function of MTDH in PDAC progression concomitant with undergoing EMT/MET plasticity or involving putative CSC functions. Experimental methods: We investigated MTDH expression in 7 human and 6 mouse PDAC cell lines and elucidated the roles of MTDH in PDAC progression by in vitro experiments for the loss of function using siMTDH and shMTDH. In immunohistochemical (IHC) staining of MTDH in resected PDAC tissues, we analyzed the correlation between MTDH expression and the clinico-pathological parameters as well as clinical outcome of the 82 PDAC patients. Results: Western blot analyses showed higher MTDH protein expression in metastatic PDAC compared to primary PDAC cell lines. In metastatic cell lines, MTDH expression has a positive correlation with E-cadherin expression. These data suggest that MTDH might be related to mesenchymal-epithelial transition (MET) and play a crucial role in metastatic cascade of PDAC. Conversely, MTDH has a negative correlation with E-cadherin in primary PDAC cell lines. It is implicated that MTDH might play inverse functions in primary site and metastatic site. Next, to examine what is the role of MTDH in the EMT/MET process, we evaluated the expression pattern of EMT markers and MTDH in response to transient TGF-β1 treatment. We found that E-cadherin expression decreases at 8 to 24 hours after treatment (EMT initiation), and then comes back to increase of the expression at 4 days after treatment (MET induction). While MTDH and E-cadherin expression shows an inverse relation during EMT initiation, these two expressions have a positive correlation during MET period (from 7 days after treatment). Additionally, we found EMT induction by TGF-β1 is attenuated by knockdown of MTDH, These results suggest MTDH might contribute to the phenotypic switch of PDAC cells via the EMT/MET process. MTDH knockdown significantly impaired the ability of pancreatosphere formation in PDAC cells and sensitized PDAC cells to gemcitabine. In FACS analyses, we also found that the percentage of CD133-positive cancer stem cell subpopulation was significantly reduced in MTDH knockdown metastatic PDAC cells. In anoikis assay, (this assay evaluate the resistance for apoptosis after losing contact from extracelluar matrix), MTDH knockdown reduced anoikis resistance in PDAC cells. These data indicate that MTDH might foster putative CSC functions in PDAC cells. In IHC staining, high MTDH expression indicated significantly higher incidence of hematogenous metastasis (p=0.02). Furthermore, High expression group showed poorer prognosis of PDAC patients compared to Low expression group in Kaplan Meier analysis (p=0.029). Conclusions: MTDH might promote PDAC metastasis to undergo EMT in primary PDAC and MET in metastatic PDAC along with.putative CSC functions. The regulation of MTDH might develop the new strategy for PDAC treatment, and lead to improve the outcome of patients. Citation Format: Kensuke Suzuki, Shigetsugu Takano, Hideyuki Yoshitomi, Shingo Kagawa, Hiroaki Shimizu, Masayuki Otsuka, Katunori Furukawa, Tukasa Takayashiki, Satoshi Kuboki, Daisuke Suzuki, Nozomu Sakai, Hiroyuki Nojima, Masaru Miyazaki.{Authors}. The elucidation for functional roles of Metadherin in metastatic cascade of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B29.

Pancreatic Cancer: Novel Approaches to Diagnosis and Therapy

Background: Pancreatic ductal adeno-carcinoma (PDAC) ranks among the most lethal of human malignancies. We have demonstrated that ATDC is overexpressed in PDAC and promotes pancreatic cancer growth by activation of Wnt/β-catenin signaling (Cancer Cell, 2009). However, the role of ATDC during PDAC initiation and progression is incompletely understood. Hence, we developed a new transgenic mouse model to evaluate the impact of ATDC overexpression on PDAC formation. Methods: To genetically engineer a mouse ubiquitously overexpressing ATDC, a Flag-ATDC construct was subcloned into a pCAGGS vector containing a CMV enhancer/chicken β-actin (CAG) promoter. ATDC mice were bred onto a FVB/n background. Mice were genotyped by multiplex PCR. ATDC, β-catenin, Zeb1 and PTEN were measured by immunohistochemical staining. Results: ATDC overexpression resulted in acinar atypia with a long latency but was not sufficient to induce pancreatic intraepithelial neoplasia (PanIN) formation. We then analyzed the impact of ATDC overexpression in the context of KrasG12D by crossing of ATDC mice with LSL-KrasG12D;p48-Cre (KC) mice to generate ATDC;LSL-KrasG12D;p48-Cre (AKC) triple transgenic mice. KC mice developed PanINs with a long latency, and PDAC was not observed until mice reached an age of 15 months or greater. However, ATDC overexpression combined with KrasG12D accelerated PanIN progression and resulted in the development of invasive and widely metastatic PDAC with a high penetrance. 45% of AKC (0% KC) mice exhibited PanIN2or 3 as early as 2 months of age. 85% of AKC mice at 6-8 months of age developed highly invasive and metastatic PDAC, whereas none of the age-matched KC mice displayed those lesions. The evolution of PDAC in AKC mice recapitulated the histopathological manifestations of human PDAC, possessing a proliferative stromal component (Ki67 and collagen positive) and glandular architecture (CK19 and E cadherin positive) with a propensity for some tumors to advance to a poorly differentiated state (CK19, E cadherin and mucin negative). ATDC and β-catenin were upregulated in these primary and metastatic PDAC lesions. Furthermore, epithelial-to-mesenchymal transition (EMT) markers, ZEB1 and vimentin, were significantly upregulated in invasive PDAC and liver metastasis from AKC mice. In 2 separate cell lines developed from the AKC primary tumors, increased ATDC expression was found to not only correlate with upregulated β-catenin signaling and expression of the EMT markers ZEB1 and vimentin, but also loss of PTEN expression. Downregulation of PTEN expression by ATDC was determined to be due to ATDC-mediated methylation of the PTEN promoter. Loss of PTEN expression was observed in pancreatic tumors arising in the AKC mice. Conclusions: Here we demonstrate that ATDC, when coupled with oncogenic Kras, accelerates PanIN progression and the development of invasive pancreatic cancer that mimics human PDAC. ATDC may mediate these effects by alterations in the β-catenin and PTEN signaling pathways. This novel mouse model provides a platform for an improved understanding the pathogenesis of PDAC and for development of targeted treatment strategies. Citation Format: Lidong Wang, Huibin Yang, Filip Bednar, Yaqing Zhang, Jacob Leflein, Taylor Detzler, Gina M. Ney, Marina Pasca di Magliano, Diane M. Simeone. ATDC synergizes with oncogenic Kras to induce invasive pancreatic adeno-carcinoma in transgenic mice. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B49.

Neoadjuvant folinic acid, fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) and chemoradiation have been used to downstage borderline and locally advanced pancreatic ductal adenocarcinoma (PDAC). Whether neoadjuvant therapy-induced tumor immune response contributes to the improved survival is unknown. Therefore, we evaluated whether neoadjuvant therapy induces an immune response towards PDAC. Clinicopathological variables were collected for surgically resected PDACs at the Massachusetts General Hospital (1998-2016). Neoadjuvant regimens included FOLFIRINOX with or without chemoradiation, proton chemoradiation (25 Gy), photon chemoradiation (50.4 Gy), or no neoadjuvant therapy. Human leukocyte antigen (HLA) class I and II expression and immune cell infiltration (CD4+, FoxP3+, CD8+, granzyme B+ cells, and M2 macrophages) were analyzed immunohistochemically and correlated with clinicopathologic variables. The antitumor immune response was compared among neoadjuvant therapy regimens. All statistical tests were 2-sided. Two hundred forty-eight PDAC patients were included. The median age was 64 years and 50.0% were female. HLA-A defects were less frequent in the FOLFIRINOX cohort (P = .006). HLA class II expression was lowest in photon and highest in proton patients (P = .02). The FOLFIRINOX cohort exhibited the densest CD8+ cell infiltration (P < .001). FOLFIRINOX and proton patients had the highest CD4+ and lowest T regulatory (FoxP3+) cell density, respectively. M2 macrophage density was statistically significantly higher in the treatment-naïve group (P < .001) in which dense M2 macrophage infiltration was an independent predictor of poor overall survival. Neoadjuvant FOLFIRINOX with or without chemoradiation may induce immunologically relevant changes in the tumor microenvironment. It may reduce HLA-A defects, increase CD8+ cell density, and decrease T regulatory cell and M2 macrophage density. Therefore, neoadjuvant FOLFIRINOX therapy may benefit from combinations with checkpoint inhibitors, which can enhance patients' antitumor immune response.

The key epigenetic regulator DNA methyltransferase 1 (DNMT1) is a scientifically validated target in p53-null chemorefractory cancers like pancreatic ductal adenocarcinoma (PDAC) since DNMT1-depletion effects cancer cell cycle exits by p53-independent epithelialization. DNMT1 can be depleted by the pyrimidine nucleoside analog pro-drugs decitabine (Dec) or 5-azacytidine (5Aza). However, PDAC clinical trials with Dec/5Aza disappointed. In pre-clinical and clinical analyses, we found resistance was caused by configurations of pyrimidine metabolism in PDAC cells that forestall Dec or 5Aza processing into DNMT1-depleting nucleotide: high expression of cytidine deaminase (CDA) that rapidly catabolizes Dec/5Aza; and suppression of deoxycytidine kinase (DCK) and uridine kinase 2 (UCK2) that rate limit Dec/5Aza pro-drug processing respectively. Accordingly, combination of Dec with a CDA clinical inhibitor, tetrahydrouridine (THU), enabled DNMT1-depletion and PDAC cytoreduction in vitro and in Dec/gemcitabine-refractory PDAC pre-clinical in vivo models. We then conducted a pilot clinical trial in 13 patients with chemorefractory PDAC given oral THU ~10 mg/kg/day combined with decitabine ~0.2 mg/kg/day, for 5 consecutive days, then twice weekly. This Phase 2 was based on several PK/PD studies in human subjects showing potent non-cytotoxic DNMT1-targeting in myeloid cells. Yet again, there were no meaningful clinical responses in the patients. A reason for this was a surprising lack of neutropenia, the most sensitive indicator of systemic DNMT1-targeting. Upon measuring plasma CDA enzyme activity, we found a &amp;gt;10-fold increase in patients with metastatic vs resectable PDAC. Thus, CDA activity is increased not only locally but also systemically in metastatic PDAC, suggesting a need for higher THU doses. We have also observed DCK downregulation, necessary for Dec/gemcitabine uptake and processing, as a cause of PDAC resistance to Dec/gemcitabine. To counter this mechanism, we discovered that 5Aza upregulates DCK as an adaptive response to 5Aza-mediated decrease in dCTP, while Dec upregulates UCK2 (that mediates 5Aza uptake) as an adaptive response to Dec mediated reductions in dTTP. Thus, we alternated Dec with 5Aza in an in vivo model of gemcitabine-resistant PDAC, to exploit their mutual cross-priming, together with THU to inhibit CDA: median vehicle control tumor measurements 972 mm3(range 726-1267.5); median THU-Dec/THU-5Aza 16 mm3 (range 0-87.5); P&amp;lt;0.00001). A non-cytotoxic, epithelial-differentiation based mechanism was confirmed by significant increases in pancreatic epithelial markers while apoptosis markers were unchanged. In sum, metabolism-based resistance to Dec/5Aza can be countered by clinically relevant modifications to treatment, such as alternating doses of THU/Dec and THU/5Aza, for non-cytotoxic p53-independent therapy, a modality distinct from chemoradiation. Citation Format: Rita Tohme, Francis Enane, Caroline Schuerger, Xiaorong Gu, Melissa Fishel, John Pink, Daniel Lindner, Davendra Sohal, Yogen Saunthararajah. Advancing non-cytotoxic DNMT1-targeting to treat chemorefractory pancreatic cancer [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 1088.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies, driven by accumulating genetic alterations and sustained by a fibrotic, immunosuppressive tumor microenvironment. We previously identified Transgultaminase 2 (TGM2), a stress-inducible enzyme involved in extracellular matrix remodeling and cell survival, as a critical downstream effector of oncogenic KRAS signaling. In the KrasG12D mouse model (KC), genetic deletion of Tgm2 almost completely protected mice from developing PDAC, establishing TGM2 as a key mediator of tumor initiation. PDAC progression, however, depends on additional genetic alterations in key tumor suppressor genes, such as TP53, SMAD4, and p16INK4A. That said, we next asked whether it could also contribute to PDAC progression. To address this, we initially examined TGM2 expression in representative KrasG12D-based mouse models carrying individual deletions in Trp53 (KPC), Smad4 (KSC), or p16Ink4a (KIC). TGM2 expression was markedly elevated in KPC mice, modestly increased in KIC mice, and unchanged in KSC mice. To functionally assess TGM2’s role in more aggressive disease resembling human PDAC, we made use of the KPC model to evaluate the impact of deleting Tgm2 on disease progression. Interestingly, while Tgm2 deletion in KPC mice led to a modest increase in survival, it did not prevent the development of aggressive PDAC. Histological analysis revealed that tumors from TGM2-deficient KPC mice (TKPC) retained the altered architecture and metastatic behavior characteristic of KPC tumors, indicating that TGM2 is dispensable for PDAC progression once the disease has advanced beyond the initiation stage. Mechanistically, the lack of TGM2 upregulation in KSC mice suggested a potential link between TGF-β signaling and TGM2 expression. Transcriptomic analyses of KC tumors using public datasets revealed that the upregulation in TGM2 expression strongly correlates with increased TGF-β signaling, and in vitro assays confirmed that TGF-β induces TGM2 expression in a Smad4-dependent manner, as shown using both wild-type and SMAD4-deleted human PDAC cells, Panc1. These findings were corroborated in primary tumor cell lines derived from KSC mice, providing compelling evidence that TGF-β signaling induces TGM2 expression, which in turn contributes to the initiation of PDAC in the KC mouse model of PDAC. Together, these data provide the first mechanistic evidence that TGM2 functions as a context-dependent effector in PDAC pathogenesis, being essential to tumor initiation but bypassed in advanced disease driven by loss of key tumor suppressors. As such, this discover could open up new opportunities for developing innovative therapeutic strategies to curb this devastating disease. Citation Format: Polina Wright, Parash Parajuli, Deanna Ward, Emma Cullen, Muhammad Akbar, Azeddine Atfi. Context-dependent role of TGM2 in Pancreatic Ductal Adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A012.