The biological and clinical significance of the intratumoral microbiota in pancreatic ductal adenocarcinoma (PDAC) remains poorly defined. Using oncogenic Kras-driven mouse models of pancreatic tumorigenesis and human pancreatic tissue from patients with non-metastatic PDAC treated with neoadjuvant (n = 62) or adjuvant therapy (n = 101), along with samples from intraductal papillary mucinous neoplasms (IPMN, n = 24), chronic pancreatitis (CP, n = 33), and healthy donors (n = 9), we determined tissue lipopolysaccharide (LPS), 16S rRNA sequencing, high-resolution quantitative multiplex immunofluorescence of autophagy markers (LAMP2, LC3B) and LPS, 16S rRNA fluorescence in situ hybridization (FISH) staining, and TLR4 signaling. In KrasCre mice, we identified significant bacterial accumulation in the pancreas, indicated by elevated LPS, increased FISH signals, enhanced TLR4 signaling, and activation of autophagy. Administration of exogenous LPS further amplified autophagy and TLR4 signaling, supporting a mechanistic link between microbial sensing and autophagy. In human tissue, both adjuvant and neoadjuvant PDAC samples showed significantly increased bacterial α- and β-diversity compared to controls, with similar enrichment in CP but not in IPMN, but with differed diversity patterns across the disease. Increased LPS levels along with elevated autophagy markers and abundant FISH-positive bacteria confirmed intratumoral colonization. Across four independent transcriptomic datasets PDAC samples showed elevated expression of bacterial-associated pathways (including TLR4) and autophagy-related genes. Clinically, higher autophagic activity of LAMP2_LC3B and increased densities of FISH-positive cells were independently associated with prolonged survival in both adjuvant and neoadjuvant or combined PDAC patients. Collectively, these findings link intratumoral microbial enrichment to enhanced autophagy/xenophagy and improved patient outcomes, suggesting that effective microbial handling within the tumor microenvironment favorably influences PDAC progression.

KRAS-dependent acinar-to-ductal metaplasia (ADM) is a fundamental step in the development of pancreatic ductal adenocarcinoma (PDAC), but the involvement of cell death pathways remains unclear. Here, we show that key regulators of programmed cell death (PCD) become upregulated during KRAS-driven ADM, thereby priming transdifferentiated cells to death. Using transgenic mice and primary cell and organoid cultures, we show that transforming growth factor (TGF)-β-activated kinase 1 (TAK1), a kinase regulating cell survival and inflammatory pathways, prevents the elimination of transdifferentiated cells through receptor-interacting protein kinase 1 (RIPK1)-mediated apoptosis and necroptosis, enabling PDAC development. Accordingly, pharmacological inhibition of TAK1 induces PCD in patient-derived PDAC organoids. Importantly, cell death induction via TAK1 inhibition does not appear to elicit an overt injury-associated inflammatory response. Collectively, these findings suggest that TAK1 supports cellular plasticity by suppressing spontaneous PCD activation during ADM, representing a promising pharmacological target for the prevention and treatment of PDAC.

Background: A dilated main pancreatic duct (MPD) ≥ 5 mm can be observed in main-duct IPMNs (MD-IPMN) and chronic pancreatitis (CP); however, distinguishing between the two differently treated diseases can be difficult. Cell-free (cf) DNA in MPD fluid obtained by EUS-guided FNA might help to distinguish MD-IPMN from CP. Methods: All patients with a dilated MPD ≥ 5 mm on EUS during the period of 1 June 2017 to 30 April 2024 were prospectively analysed in this single-centre study, with EUS-guided MPD fluid aspiration performed for suspected MD-IPMN or CP in patients who were suitable for surgery. Twenty-two known gastrointestinal cancer genes, including GNAS and KRAS, were analysed by deep targeted (dt) NGS. The results were correlated with resected tissue, biopsy, and long-term follow-up. Results: A total of 164 patients with a dilated MPD were identified, of which 30 (18.3%) underwent EUS-guided FNA, with 1 patient having a minor complication (3.3%). Twenty-two patients (mean MPD diameter of 12.4 (7–31) mm) with a definitive, mostly surgically confirmed diagnosis were included in the analysis. Only a fish-mouth papilla, which was present in 3 of 12 (25%) MD-IPMNs, could reliably differentiate between the two diseases, with history, symptoms, diffuse or segmental MPD dilation, presence of calcifications on imaging, cytology, and CEA in the ductal fluid failing to achieve differentiation. However, GNAS mutations were found exclusively in 11 of the 12 (91.6%) patients with MD-IPMN (p < 0.01), whereas KRAS mutations were identified in both diseases. Conclusions: GNAS testing by dtNGS in aspirated fluid from dilated MPD obtained by EUS-guided FNA may help differentiate MD-IPMN from CP for surgical resection.

Background Despite some recent advances, pancreatic ductal adenocarcinoma (PDAC) remains a growing oncological challenge. New drugs capable of targeting more than one oncogenic pathway may be one way to improve patient outcomes. This study characterizes the effectiveness of Metavert a first-in-class dual inhibitor of GSK3-β and histone deacetylase in treating PDAC as a single agent or in combination with standard cytotoxics. Methods Thirty-six Patient-Derived Organoids (hPDOs) characterised by RNASeq and whole exome sequencing were treated with Metavert alone or in combination with standard cytotoxics. Transcriptomic signatures (TS) representing sensitivity to Metavert alone or sensitivity to Metavert + irinotecan (IR) were evaluated in 47 patient samples, chemo-naïve in 26 and post-chemotherapy in 21 (gemcitabine=5; FOLFIRINOX=14, both=2) with companion multiplexed immunofluorescence and RNASeq data. Results Metavert combined with gemcitabine, irinotecan, 5FU, oxaliplatin, and paclitaxel was synergistic in the hPDOs. Basal-subtype hPDOs were more sensitive to Metavert alone whereas the Metavert+IR combination exhibited synergy in Classical-subtype hPDOs with increased apoptosis and autophagy. hPDO-derived TS evaluated in PDAC tissues demonstrated that Metavert-TSHi samples were enriched for mRNA splicing and DNA repair processes; they were associated with Basal-like tissues but also with GATA6+ve-chemo-naïve samples and were higher following gemcitabine but not FOLFIRINOX treatment. In contrast, Metavert+IR-TSHI samples were enriched for TP53 pathways; they were associated with Classical-like pretreatment samples and with GATA6+ve/KRT17+ve hybrid cell types following FOLFIRINOX, but not gemcitabine treatment, and were unrelated to transcriptional subtypes. Conclusions Metavert as a single agent and in combination with irinotecan offers novel strategies for treating pancreatic cancer.

Distinguishing primary liver cancer (PLC), namely hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), from liver metastases is of crucial clinical importance. Histopathology remains the gold standard, but differential diagnosis may be challenging. While absent in most epithelial, the expression of the adherens junction glycoprotein N-cadherin is commonly restricted to neural and mesenchymal cells, or carcinoma cells that undergo the phenomenon of epithelial-to-mesenchymal transition (EMT). However, we recently established N- and E-cadherin expression as hallmarks of normal hepatocytes and cholangiocytes, which are also preserved in HCC and iCCA. Therefore, we hypothesized that E- and/or N-cadherin may distinguish between carcinoma derived from the liver vs carcinoma of other origins. We comprehensively evaluated E- and N-cadherin in 3359 different tumors in a multicenter study using immunohistochemistry and compared our results with previously published 882 cases of PLC, including 570 HCC and 312 iCCA. Most carcinomas showed strong positivity for E-cadherin. Strong N-cadherin positivity was present in HCC and iCCA. However, except for clear cell renal cell carcinoma (23.6% of cases) and thyroid cancer (29.2%), N-cadherin was only in some instances faintly expressed in adenocarcinomas of the gastrointestinal tract (0%-0.5%), lung (7.1%), pancreas (3.9%), gynecological organs (0%-7.4%), breast (2.2%) as well as in urothelial (9.4%) and squamous cell carcinoma (0%-5.6%). As expected, N-cadherin was detected in neuroendocrine tumors (25%-75%), malignant melanoma (46.2%) and malignant mesothelioma (41%). In conclusion, N-cadherin is a useful marker for the distinction of PLC vs liver metastases of extrahepatic carcinomas (P < .01).

Pancreatic cancer (PDAC) is characterized by infiltrative, spiculated tumor growth into the surrounding non-neoplastic tissue. Clinically, its diagnosis is often established by magnetic resonance imaging (MRI). At the invasive margin, tumor buds can be detected by histology, an established marker associated with poor prognosis in different types of tumors. We analyzed PDAC by determining the degree of tumor spiculation on T2-weighted MRI using a 3-tier grading system. The grade of spiculation was correlated with the density of tumor buds quantified in histological sections of the respective surgical specimen according to the guidelines of the International Tumor Budding Consensus Conference (n = 28 patients). 64

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily that controls bile acid (BA) homeostasis, has also been proposed as a tumor suppressor for breast and liver cancer. However, its role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis remains controversial. We recently found that FXR attenuates acinar cell autophagy in chronic pancreatitis resulting in reduced autophagy and promotion of pancreatic carcinogenesis. Feeding Kras-p48-Cre (KC) mice with the BA chenodeoxycholic acid (CDCA), an FXR agonist, attenuated pancreatic intraepithelial neoplasia (PanIN) progression, reduced cell proliferation, neoplastic cells and autophagic activity, and increased acinar cells, elevated pro-inflammatory cytokines and chemokines, with a compensatory increase in the anti-inflammatory response. Surprisingly, FXR-deficient KC mice did not show any response to CDCA, suggesting that CDCA attenuates PanIN progression and decelerate tumorigenesis in KC mice through activating pancreatic FXR. FXR is activated in pancreatic cancer cell lines in response to CDCA in vitro. FXR levels were highly increased in adjuvant and neoadjuvant PDAC tissue compared to healthy pancreatic tissue, indicating that FXR is expressed and potentially activated in human PDAC. These results suggest that BA exposure activates inflammation and suppresses autophagy in KC mice, resulting in reduced PanIN lesion progression. These data suggest that activation of pancreatic FXR has a protective role by reducing the growth of pre-cancerous PDAC lesions in response to CDCA and possibly other FXR agonists.

Supplementary Table S2 shows sequencing coverage metrics.

Introduction: Resection margin status is a well-established prognosticator in pancreatic cancer. The prognostic impact of IPMN dysplasia at the pancreatic transection margin in IPMN-associated carcinoma (IPMN-Ca) remains unclear, hence institutional practices on additional resections vary.Methods: Patients undergoing partial pancreatectomy or attempted partial pancreatectomy converted to total pancreatectomy for IPMN-Ca between 04/2002 and 12/2018 were identified. Final pathology of the definitive pancreatic transection margin was identified. The association between the presence of IPMN dysplasia at the margin and overall survival (OS) was assessed.Results: Of 302 patients with IPMN-Ca, 181 (59.9%) patients received partial pancreatoduodenectomy, 61 (20.2%) distal pancreatectomy, and 60 (19.9%) were converted to total pancreatectomy. Median OS was 98.6 months in R0 (>= 1 mm), 39.3 months in R1 (<1 mm), and 22.0 months in R1(direct) resected pa-tients, respectively (p < 0.0001). No IPMN dysplasia at the definitive margin was present in 103 (34.1%), low-grade in 131 (43.4%), and high-grade/R1 in 8 (2.6%) patients. Low-grade dysplasia or total pancre-atectomy were not associated with shorter OS compared to dysplasia-free margin across the entire cohort. Sensitivity analyses confirmed a lack of prognostic relevance of low-grade IPMN dysplasia at the pancreatic margin in R0 resected IPMN-Ca and in R0 resected UICC stage IA/IB IPMN-Ca.Conclusions: Low-grade IPMN at the transection margin is not associated with shorter overall survival after partial pancreatectomy for IPMN-Ca. Additional resections for low-grade dysplasia, up to total pancreatectomy do not result in a survival benefit and should be omitted. Due to limited sample size, high-grade dysplasia could not be analyzed.(c) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Supplementary Figure 4 from Up-regulation of L1CAM in Pancreatic Duct Cells Is Transforming Growth Factor β1– and Slug-Dependent: Role in Malignant Transformation of Pancreatic Cancer

Supplementary Figure Legends 1-6 from Up-regulation of L1CAM in Pancreatic Duct Cells Is Transforming Growth Factor β1– and Slug-Dependent: Role in Malignant Transformation of Pancreatic Cancer

Objective: To investigate the outcome of conversion surgery in patients with metastatic pancreatic cancer (mPDAC) and to identify patients who may benefit from this approach. Background: The role of conversion surgery in patients with mPDAC and exceptional response to chemotherapy remains unclear. Methods: Patients who underwent surgical exploration for mPDAC following chemotherapy between 2006 and 2019 were included. Data on demographics, oncologic treatment, pathology, and postoperative outcomes were analyzed. Univariate and multivariate survival analyses were performed. Results: Some 173 patients received preoperative chemotherapy and underwent surgical exploration. Ninety-three patients underwent resection of the primary tumor and metastatic sites, 80 patients underwent exploration only. In the resection subgroup, 45 patients had complete pathological response of metastases (ypM0) and 48 patients had residual metastases (ypM1). ypM0 status was associated with lower carcinoembryonic antigen levels and lower ypN stage. Overall survival after resection was 25.5 months in ypM0, 10.7 months in ypM1, and 8.1 months in patients without resection (P<0.001). Additional adjuvant chemotherapy was significantly associated with prolonged survival in resected patients (29.0 vs 14.8 mo, P=0.024) as well as in ypM0 (29.1 vs 19.2 mo, P=0.047). Multivariable analysis identified conversion surgery, carbohydrate antigen 19-9 (CA19-9) and time of resection as independent prognostic markers for the entire cohort. CA19-9, ypM0 and adjuvant treatment were independent predictors of survival in the resection subgroup. Conclusion: In patients with mPDAC and ypM0 status after chemotherapy, surgical resection is associated with encouraging survival. mPDAC patients with exceptional response to chemotherapy may be candidates for exploration and for resection in ypM0. Adjuvant chemotherapy may provide an additional survival advantage.

Supplementary Figure S1 shows circos plots including 17 PDACs of the patient cohort and a PDCM.

In this study, we aimed to find genes with a key function in the development of molecular subtypes in pancreatic ductal adenocarcinoma (PDAC). Through transcriptome analysis, we discovered that the endogenous serine/cysteine proteinase inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) was distinctively upregulated in the basal-like/squamous subtype, known as an aggressive subtype, and this upregulation associated with decreased patient survival in both a test (N=123) and a validation cohort (N=84). In additional investigations of the tumor metabolome and transcriptome using PDAC patient tumors and cell lines, SERPINB3 and the basal-like/squamous subtype showed a robust relationship with upregulated levels of amino acids (e.g., hydroxyproline) whereas SERPINB3 promoted a gene signature indicative of the basal-like/squamous subtype. Additional mechanistic studies revealed that SERPINB3 and hydroxyproline promoted the migration/invasion of PDAC cells. Moreover, SERPINB3 also promoted metastasis in an orthotopic mouse model of PDAC through stromal factors that increased tumor microvessel density. To conclude, inhibiting SERPINB3 function may attenuate disease progression of the basal-like/squamous subtype in PDAC through changes to the tumor stroma and tumor metabolism. Citation Format: Yuuki Ohara, Wei Tang, Liu Huaitian, Shouhui Yang, Peijun He, Helen Cawley, Paloma Valenzuela, Lin Zhang, Jochen Gaedcke, B. Michael Ghadimi, Matthias M. Gaida, Frank Bergmann, H. Richard Alexander, Nader Hanna, Stefan Ambs, S. Perwez Hussain. SERPINB3 promotes the aggressive basal-like/squamous subtype and correlates with poor prognosis in pancreatic ductal adenocarcinoma through metabolic reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6225.

Supplementary Figure S3 illustrates genomic breakpoints of fusions in KRASwt tumors.

Supplementary Figure S2 shows experimental data demonstrating the proliferative capacity of the ATP1B1-NRG1 fusion.

Supplementary Table S1 lists potentially clinically relevant somatic alterations in the PDAC patient cohort.