Identification of spatially-resolved markers of malignant transformation in Intraductal Papillary Mucinous Neoplasms.

The existing Intraductal Papillary Mucinous Neoplasm (IPMN) risk stratification relies on clinical and histological factors, resulting in inaccuracies and leading to suboptimal treatment. This is due to the lack of appropriate molecular markers that can guide patients toward the best therapeutic options. Here, we assess and confirm subtype-specific markers for IPMN across two independent cohorts of patients using two Spatial Transcriptomics (ST) technologies. Specifically, we identify HOXB3 and ZNF117 as markers for Low-Grade Dysplasia, SPDEF and gastric neck cell markers in borderline cases, and NKX6-2 and gastric isthmus cell markers in High-Grade-Dysplasia Gastric IPMN, highlighting the role of TNFα and MYC activation in IPMN progression and the role of NKX6-2 in the specific Gastric IPMN progression. In conclusion, our work provides a step forward in understanding the gene expression landscapes of IPMN and the critical transcriptional networks related to PDAC progression.

Intraductal Papillary Mucinous Neoplasms in High-Risk Individuals: Incidence, Growth Rate, and Malignancy Risk.

BACKGROUND AND AIMS: In high-risk individuals (HRIs), we aimed to assess the cumulative incidence of intraductal papillary mucinous neoplasms (IPMNs) and compare IPMN growth, neoplastic progression rate, and the value of growth as predictor for neoplastic progression to these in sporadic IPMNs. METHODS: We performed annual surveillance of Dutch HRIs, involving carriers of germline pathogenic variants (PVs) and PV-negative familial pancreatic cancer kindreds. HRIs with IPMNs were compared with Italian individuals without familial risk under surveillance for sporadic IPMNs. RESULTS: A total of 457 HRIs were followed for 48 (range 2-172) months; the estimated cumulative IPMN incidence was 46% (95% confidence interval, 28%-64%). In comparison with 442 control individuals, IPMNs in HRIs were more likely to grow ≥2.5 mm/y (31% vs 7%; P < .001) and develop worrisome features (32% vs 19%; P = .010). PV carriers with IPMNs more often displayed neoplastic progression (n = 3 [11%] vs n = 6 [1%]; P = .011), while familial pancreatic cancer kindreds did not (n = 0 [0%]; P = 1.000). The malignancy risk in a PV carrier with an IPMN was 23% for growth rates ≥2.5 mm/y (n = 13), 30% for ≥5 mm/y (n = 10), and 60% for ≥10 mm/y (n = 5). CONCLUSIONS: The cumulative incidence of IPMNs in HRIs is higher than previously reported in the general population. Compared with sporadic IPMNs, they have an increased growth rate. PV carriers with IPMNs are suggested to be at a higher malignancy risk. Intensive follow-up should be considered for PV carriers with an IPMN growing ≥2.5 mm/y, and surgical resection for those growing ≥5 mm/y.

Integrated Molecular Characterization of Intraductal Papillary Mucinous Neoplasms: An NCI Cancer Moonshot Precancer Atlas Pilot Project.

Intraductal papillary mucinous neoplasms (IPMN) are cystic precursor lesions to pancreatic ductal adenocarcinoma (PDAC). IPMNs undergo multistep progression from low-grade (LG) to high-grade (HG) dysplasia, culminating in invasive neoplasia. While patterns of IPMN progression have been analyzed using multiregion sequencing for somatic mutations, there is no integrated assessment of molecular events, including copy-number alterations (CNA) and transcriptional changes that accompany IPMN progression. We performed laser capture microdissection on surgically resected IPMNs of varying grades of histologic dysplasia obtained from 23 patients, followed by whole-exome and whole-transcriptome sequencing. Overall, HG IPMNs displayed a significantly greater aneuploidy score than LG lesions, with chromosome 1q amplification being associated with HG progression and with cases that harbored co-occurring PDAC. Furthermore, the combined assessment of single-nucleotide variants (SNV) and CNAs identified both linear and branched evolutionary trajectories, underscoring the heterogeneity in the progression of LG lesions to HG and PDAC. At the transcriptome level, upregulation of MYC-regulated targets and downregulation of transcripts associated with the MHC class I antigen presentation machinery as well as pathways related to glycosylation were a common feature of progression to HG. In addition, the established PDAC transcriptional subtypes (basal-like and classical) were readily apparent within IPMNs. Taken together, this work emphasizes the role of 1q copy-number amplification as a putative biomarker of high-risk IPMNs, underscores the importance of immune evasion even in noninvasive precursor lesions, and reinforces that evolutionary pathways in IPMNs are heterogenous, comprised of both SNV and CNA-driven events. SIGNIFICANCE: Integrated molecular analysis of genomic and transcriptomic alterations in the multistep progression of IPMNs, which are bona fide precursors of pancreatic cancer, identifies features associated with progression of low-risk lesions to high-risk lesions and cancer, which might enable patient stratification and cancer interception strategies.

Co-occurring IPMN and pancreatic cancer: the same or different? An overview from histology to molecular pathology.

Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is one of the most well-established precursors of pancreatic cancer. Its progression to acquire invasiveness is a complex process, based on the accumulation of morphological and genetic alterations. Recent advances in DNA sequencing also showed that co-occurring IPMNs and pancreatic cancers could be totally independent, further complicating our understanding of this complex scenario. The distinction between IPMN and related pancreatic cancer vs IPMN and co-occurring-but not related-pancreatic cancer is a challenging task in routine diagnostic activity, but may have important implications for precision oncology. Of note, recent multiregional sequencing-based studies focused not only on IPMN multi-step tumourigenesis, but also on the divergent intratumoural heterogeneity of this neoplasm. Globally considered, there are three different situations in which co-occurring IPMNs and invasive carcinomas can be found in the same pancreata, indicated with different terminologies: (1) IPMN-associated carcinoma: this definition indicates a carcinoma arising from an IPMN and can be also defined as IPMN-derived carcinoma, sequential or likely related; (2) independent IPMN and invasive carcinoma: the two lesions are not related, and this situation is defined as concomitant, de novo or likely independent; (3) branch-off pathway, where an invasive carcinoma and an adjacent IPMN develop divergently in a forked fashion from a common ancestral clone. In this review, we aim at clarifying the most important nomenclature/definitions of these different situations, also providing an overview of the molecular state-of-the-art and of the clinical implications of this complex landscape.

Spatial Transcriptomics of Intraductal Papillary Mucinous Neoplasms of the Pancreas Identifies NKX6-2 as a Driver of Gastric Differentiation and Indolent Biological Potential.

Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). The most common subtype of IPMNs harbors a gastric foveolar-type epithelium, and these low-grade mucinous neoplasms are harbingers of IPMNs with high-grade dysplasia and cancer. The molecular underpinning of gastric differentiation in IPMNs is unknown, although identifying drivers of this indolent phenotype might enable opportunities for intercepting progression to high-grade IPMN and cancer. We conducted spatial transcriptomics on a cohort of IPMNs, followed by orthogonal and cross-species validation studies, which established the transcription factor NKX6-2 as a key determinant of gastric cell identity in low-grade IPMNs. Loss of NKX6-2 expression is a consistent feature of IPMN progression, while reexpression of Nkx6-2 in murine IPMN lines recapitulates the aforementioned gastric transcriptional program and glandular morphology. Our study identifies NKX6-2 as a previously unknown transcription factor driving indolent gastric differentiation in IPMN pathogenesis. SIGNIFICANCE: Identification of the molecular features driving IPMN development and differentiation is critical to prevent cancer progression and enhance risk stratification. We used spatial profiling to characterize the epithelium and microenvironment of IPMN, which revealed a previously unknown link between NKX6-2 and gastric differentiation, the latter associated with indolent biological potential. See related commentary by Ben-Shmuel and Scherz-Shouval, p. 1768. This article is highlighted in the In This Issue feature, p. 1749.

Patients with deleterious germline mutations: A heterogeneous population for pancreatic cancer screening?

BACKGROUND AND OBJECTIVES: Modest data exist on the benefits of screening and surveillance for pancreatic cancer (PC) in high-risk individuals. Intraductal papillary mucinous neoplasms (IPMN) are known precursors to PC. We hypothesized that patients with high-risk deleterious germline mutations have a higher prevalence of IPMN. METHODS: All patients undergoing prospective screening at a single institution from 2013 to 2019 were reviewed. RESULTS: Of 1166 patients screened, 358 (31%) possessed germline mutations and/or family history of PC (mutations n = 201/358, 56%, family history n = 226/358, 63%) (median follow-up 2.7 years). IPMN was found in 127 patients (35.5%). The prevalence of IPMN in mutation carriers (18%) was higher than in the general population (p < 0.01). Germline mutation was an independent predictor of IPMN (odds ratio [OR] = 3.2; p < 0.01), while family history was not (p = 0.22). IPMN prevalence was distributed unevenly between mutation types (67%-Peutz-Jeghers; 43%-HNPCC, 24%-BRCA2; 17%-ATM; 9%-BRCA1; 0%-CDKN2A and PALB2). CONCLUSION: In this series, 18% of mutation carriers harbored IPMN, higher than the general population. Germline mutation, but not a family history of PC, was independently associated with IPMN. This prevalence varied across mutation subtypes, suggesting not all mutation carriers develop precancerous lesions. Genetic testing for patients with a positive family history may improve screening modalities for this high-risk population.

Digital spatial profiling of intraductal papillary mucinous neoplasms: Toward a molecular framework for risk stratification.

The histopathologic heterogeneity of intraductal papillary mucinous neoplasms (IPMN) complicates the prediction of pancreatic ductal adenocarcinoma (PDAC) risk. Intratumoral regions of pancreaticobiliary (PB), intestinal (INT), and gastric foveolar (GF) epithelium may occur with either low-grade dysplasia (LGD) or high-grade dysplasia (HGD). We used digital spatial RNA profiling of dysplastic epithelium (83 regions) from surgically resected IPMN tissues (12 patients) to differentiate subtypes and predict genes associated with malignancy. The expression patterns of PB and GF lesions diverged from INT, suggesting that PB and GF arise from a common lineage. Transcriptional dysregulation within PB lesions mirrored that of PDAC, whereas INT and GF foci did not. Tumor necrosis factor/nuclear factor κB (TNF-NFκB) and cell cycle (cycling S and cycling G2-M) programs occurred with relative prominence in PB and INT subtypes, respectively. Together, this study delineates markers of high-risk IPMN and insights into malignant progression.

Utility of promoter hypermethylation in malignant risk stratification of intraductal papillary mucinous neoplasms.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs), a type of cystic pancreatic cancer (PC) precursors, are increasingly identified on cross-sectional imaging and present a significant diagnostic challenge. While surgical resection of IPMN-related advanced neoplasia, i.e., IPMN-related high-grade dysplasia or PC, is an essential early PC detection strategy, resection is not recommended for IPMN-low-grade dysplasia (LGD) due to minimal risk of carcinogenesis, and significant procedural risks. Based on their promising results in prior validation studies targeting early detection of classical PC, DNA hypermethylation-based markers may serve as a biomarker for malignant risk stratification of IPMNs. This study investigates our DNA methylation-based PC biomarker panel (ADAMTS1, BNC1, and CACNA1G genes) in differentiating IPMN-advanced neoplasia from IPMN-LGDs. METHODS: Our previously described genome-wide pharmaco-epigenetic method identified multiple genes as potential targets for PC detection. The combination was further optimized and validated for early detection of classical PC in previous case-control studies. These promising genes were evaluated among micro-dissected IPMN tissue (IPMN-LGD: 35, IPMN-advanced neoplasia: 35) through Methylation-Specific PCR. The discriminant capacity of individual and combination of genes were delineated through Receiver Operating Characteristics curve analysis. RESULTS: As compared to IPMN-LGDs, IPMN-advanced neoplasia had higher hypermethylation frequency of candidate genes: ADAMTS1 (60% vs. 14%), BNC1 (66% vs. 3%), and CACGNA1G (25% vs. 0%). We observed Area Under Curve (AUC) values of 0.73 for ADAMTS1, 0.81 for BNC1, and 0.63 for CACNA1G genes. The combination of the BNC1/ CACNA1G genes resulted in an AUC of 0.84, sensitivity of 71%, and specificity of 97%. Combining the methylation status of the BNC1/CACNA1G genes, blood-based CA19-9, and IPMN lesion size enhanced the AUC to 0.92. CONCLUSION: DNA-methylation based biomarkers have shown a high diagnostic specificity and moderate sensitivity for differentiating IPMN-advanced neoplasia from LGDs. Addition of specific methylation targets can improve the accuracy of the methylation biomarker panel and enable the development of noninvasive IPMN stratification biomarkers.

A pilot study to evaluate tissue- and plasma-based DNA driver mutations in a cohort of patients with pancreatic intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions to pancreatic ductal adenocarcinoma that are challenging to manage due to limited imaging, cytologic, and molecular markers that accurately classify lesions, grade of dysplasia, or focus of invasion preoperatively. The objective of this pilot study was to determine the frequency and type of DNA mutations in a cohort of surgically resected, pathologically confirmed IPMN, and to determine if concordant mutations are detectable in paired pretreatment plasma samples. Formalin-fixed paraffin-embedded (FFPE) tissue from 46 surgically resected IPMNs (31 low-grade, 15 high-grade) and paired plasma from a subset of 15 IPMN cases (10 low-grade, 5 high-grade) were subjected to targeted mutation analysis using a QIAseq Targeted DNA Custom Panel. Common driver mutations were detected in FFPE from 44 of 46 (95.6%) IPMN cases spanning all grades; the most common DNA mutations included: KRAS (80%), RNF43 (24%), and GNAS (43%). Of note, we observed a significant increase in the frequency of RNF43 mutations from low-grade to high-grade IPMNs associated or concomitant with invasive carcinoma (trend test, P = 0.01). Among the subset of cases with paired plasma, driver mutations identified in the IPMNs were not detected in circulation. Overall, our results indicate that mutational burden for IPMNs is a common occurrence, even in low-grade IPMNs. Furthermore, although blood-based biopsies are an attractive, noninvasive method for detecting somatic DNA mutations, the QIAseq panel was not sensitive enough to detect driver mutations that existed in IPMN tissue using paired plasma in the volume we were able to retrieve for this retrospective study.

Molecular heterogeneity and commonalities in pancreatic cancer precursors with gastric and intestinal phenotype.

OBJECTIVE: Due to the limited number of modifiable risk factors, secondary prevention strategies based on early diagnosis represent the preferred route to improve the prognosis of pancreatic ductal adenocarcinoma (PDAC). Here, we provide a comparative morphogenetic analysis of PDAC precursors aiming at dissecting the process of carcinogenesis and tackling the heterogeneity of preinvasive lesions. DESIGN: Targeted and whole-genome low-coverage sequencing, genome-wide methylation and transcriptome analyses were applied on a final collective of 122 morphologically well-characterised low-grade and high-grade PDAC precursors, including intestinal and gastric intraductal papillary mucinous neoplasms (IPMN) and pancreatic intraepithelial neoplasias (PanIN). RESULTS: Epigenetic regulation of mucin genes determines the phenotype of PDAC precursors. PanIN and gastric IPMN display a ductal molecular profile and numerous similarly regulated pathways, including the Notch pathway, but can be distinguished by recurrent deletions and differential methylation and, in part, by the expression of mucin-like 3. Intestinal IPMN are clearly distinct lesions at the molecular level with a more instable genotype and are possibly related to a different ductal cell compartment. CONCLUSIONS: PDAC precursors with gastric and intestinal phenotype are heterogeneous in terms of morphology, genetic and epigenetic profile. This heterogeneity is related to a different cell identity and, possibly, to a different aetiology.

Blood-Based Diagnosis and Risk Stratification of Patients with Pancreatic Intraductal Papillary Mucinous Neoplasm (IPMN).

PURPOSE: Intraductal papillary mucinous neoplasm (IPMN) is a precursor of pancreatic ductal adenocarcinoma. Low-grade dysplasia has a relatively good prognosis, whereas high-grade dysplasia and IPMN invasive carcinoma require surgical intervention. However, diagnostic distinction is difficult. We aimed to identify biomarkers in peripheral blood for accurate discrimination. EXPERIMENTAL DESIGN: Sera were obtained from 302 patients with IPMNs and 88 healthy donors. For protein biomarkers, serum samples were analyzed on microarrays made of 2,977 antibodies. A support vector machine (SVM) algorithm was applied to define classifiers, which were validated on a separate sample set. For microRNA biomarkers, a PCR-based screen was performed for discovery. Biomarker candidates confirmed by quantitative PCR were used to train SVM classifiers, followed by validation in a different sample set. Finally, a combined SVM classifier was established entirely independent of the earlier analyses, again using different samples for training and validation. RESULTS: Panels of 26 proteins or seven microRNAs could distinguish high- and low-risk IPMN with an AUC value of 95% and 94%, respectively. Upon combination, a panel of five proteins and three miRNAs yielded an AUC of 97%. These values were much better than those obtained in the same patient cohort by using the guideline criteria for discrimination. In addition, accurate discrimination was achieved between other patient subgroups. CONCLUSIONS: Protein and microRNA biomarkers in blood allow precise diagnosis and risk stratification of IPMN cases, which should improve patient management and thus the prognosis of IPMN patients. See related commentary by Löhr and Pantel, p. 1387.

Single-cell sequencing reveals heterogeneity between pancreatic adenosquamous carcinoma and pancreatic ductal adenocarcinoma with prognostic value.

Pancreatic adenosquamous carcinoma (ASPC) is a rare subtype of pancreatic cancer with lethal malignancy, and few studies have focused on the heterogeneity of ASPC. Here, we performed a single-cell sequencing procedure on pancreatic tumor tissue from an ASPC patient and a patient with high-grade intraductal papillary mucinous neoplasm (IPMN). Through the combined analysis of single-cell sequencing data from five pancreatic ductal adenocarcinoma (PDAC) patients, one IPMN patient, and one ASPC patient in a public database, we identified 11 main types of cells, including macrophages, B cells, cancer stem cells, ductal cells, fibroblasts, endo/stellate cells, neutrophils, acinar cells, T cells, natural killer (NK) cells, dendritic cells, and mast cells. Then, the different characteristics and differentiation paths of the immune microenvironment among IPMN, ASPC, and PDAC in macrophages, T cells, and cancer-associated fibroblasts (CAFs) were identified through multiple bioinformatics analyses. Two novel special cancer-associated fibroblasts were identified as nCAFs and imCAFs. Then, cancer cells in duct cells were identified using the infercnv software. Two ASPC-specific subgroups of cancer cells with squamous cell features were identified. Finally, the identified specific CAFs and cancer cells were mapped to TCGA-PAAD cohort through the cibersoftx software. All of these identified subgroups were calculated to have a significant prognostic value in pancreatic cancer patients. These findings will promote the clinical application of single-cell sequencing data of pancreatic cancer and deepen our understanding of ASPC.

Investigation of -PRKACA/-PRKACB fusion genes in oncocytic tumors of the pancreatobiliary and other systems.

Intraductal oncocytic papillary neoplasms (IOPNs) of the pancreatobiliary system are tumors comprising oncocytic cells, in which three types of fusion genes involving -PRKACA/-PRKACB were recently identified. IOPNs infrequently combine with other histological subtypes of pancreatic intraductal papillary mucinous neoplasms (IPMNs) and intraductal papillary neoplasms of the bile duct (IPNBs). This study aimed to confirm the sensitivity/specificity of the fusion genes for IOPNs and to examine their significance in other oncocytic lesions. An RT-PCR, followed by DNA sequencing, was undertaken to examine the fusions in 18 histologically diagnosed IOPNs, including four combined IOPNs. Moreover, in two IOPN cases, invasive carcinomatous lesions were separately examined on their fusion status. Oncocytic thyroidal (n = 10), renal (n = 10), and salivary gland (n = 3) lesions and IPMNs (n = 9)/IPNBs (n = 4) with focal oncocytic changes were examined as controls. Fluorescence in situ hybridization using PRKACA break-apart probes was conducted for the combined IOPN cases. Target sequencing of KRAS exon2/3 and GNAS exon 8/9 was performed for IOPN cases. Fusions were detected in all IOPN cases including invasive lesions/none of the control cases. The fusion event was confirmed also in non-IOPN component in one of the four combined cases. Regarding mutation events, 5.6%/0% of IOPNs were KRAS-mt/GNAS-mt, respectively, and both components of combined IOPNs were all KRAS-wt/GNAS-wt. In conclusion, our study confirmed the sensitivity and specificity of these fusions for IOPNs. Here, we analyzed the roles of these fusion genes in combined IOPNs, proposing the possibility of IOPN development via IPMNs/IPNBs. Further studies with more combined cases are warranted.

Mechanisms of development and progression of pancreatic neoplasms.

Pancreatic ductal adenocarcinoma (PDAC) develops via dysplastic changes in the epithelia graded as low- and high-grade with accumulation of molecular alterations. Constitutive activation of mitogen-activated protein kinase (MAPK) contributed by attenuation of DUSP6 plays a key role in sustaining PDAC. Active MAPK induces various molecules that function as effectors to sustain PDAC. AURKA and SON are downstream effectors that contribute substantially to the proliferation and survival of PDAC cells and are potentially useful as therapeutic targets. Active MAPK also promote microRNAs that modulate the proliferation of PDAC cells and are useful as diagnostic markers. Familial pancreatic cancer kindreds in Japan show various germline mutations supposed to increase a pancreatic cancer risk. Intraductal papillary mucinous neoplasms (IPMNs) consist of dilated ducts lined by papillary neoplastic epithelia of various shapes and varying grades of atypia. Various papillae of IPMNs are classified into four subtypes that are associated with clinicopathological features, including patient prognosis. GNAS is a specific driver gene for the development of IPMN through gain-of-function mutations. Tracing of molecular alterations has elucidated the mechanism of progression of IPMN from dysplasia to carcinoma, as well as one type of papillae. Intraductal tubulopapillary neoplasms belong to a distinct class of pancreatic neoplasms.

Multidimensional Immunophenotyping of Intraductal Papillary Mucinous Neoplasms Reveals Novel T Cell and Macrophage Signature.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMN) are the only radiographically identifiable precursor to pancreatic adenocarcinoma, yet little is known about how these lesions progress to cancer. Inflammation has been associated with dysplastic progression; however, the cause and composition of this inflammation remains poorly characterized. We sought to comprehensively profile immune cell infiltration using parallel spatial transcriptomic and flow cytometric techniques. METHODS: Twelve patients with resected IPMN exhibiting both high-grade dysplasia (HGD) and low-grade dysplasia (LGD) were selected for spatial transcriptomics (NanoString GeoMx). Immune (CD45+), epithelial (PanCK+), and stromal (SMA+) compartments were analyzed separately using the GeoMx NGS Pipeline. An additional 11 patients resected for IPMN of varying degrees of dysplasia underwent immunophenotyping using flow cytometry (DURAClone IM). RESULTS: Spatial transcriptomics revealed that T cells represent the dominant immune cell within IPMN stroma, which was confirmed by flow cytometry (56%). Spatial profiling found that the T-cell infiltrate was significantly higher in regions of LGD compared with HGD (62% vs. 50%, p = 0.038). Macrophages were the only other immune cell type with > 10% abundance, yet conversely, were generally more abundant in regions of HGD compared to LGD (19% vs. 11%, p = 0.058). Correspondingly, immune cells within regions of HGD demonstrated transcriptional upregulation of genes associated with macrophage activity including secretion (CXCL1) and phagocytosis (C1QA, C1S, C4B). CONCLUSIONS: IPMN immune infiltrate is primarily composed of T cells and macrophages. Regions of HGD appear to be relatively deplete of T cells and show a trend toward macrophage enrichment compared with regions of LGD.

Intraductal tubulopapillary neoplasm (ITPN) of the pancreas: a distinct entity among pancreatic tumors.

AIMS: Intraductal tubulopapillary neoplasm (ITPN) of the pancreas is a recently recognized pancreatic tumor entity. Here we aimed to determine the most important features with a systematic review coupled with an integrated statistical approach. METHODS AND RESULTS: PubMed, SCOPUS, and Embase were searched for studies reporting data on pancreatic ITPN. The clinicopathological, immunohistochemical, and molecular data were summarized. Then a comprehensive survival analysis and a comparative analysis of the molecular alterations of ITPN with those of pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) from reference cohorts (including the International Cancer Genome Consortium- ICGC dataset and The Cancer Genome Atlas, TCGA program) were conducted. The core findings of 128 patients were as follows: (i) Clinicopathological parameters: pancreatic head is the most common site; presence of an associated adenocarcinoma was reported in 60% of cases, but with rare nodal metastasis. (ii) Immunohistochemistry: MUC1 (>90%) and MUC6 (70%) were the most frequently expressed mucins. ITPN lacked the intestinal marker MUC2; unlike IPMN, it did not express MUC5AC. (iii) Molecular landscape: Compared with PDAC/IPMN, the classic pancreatic drivers KRAS, TP53, CDKN2A, SMAD4, GNAS, and RNF43 were less altered in ITPN (P < 0.001), whereas MCL amplifications, FGFR2 fusions, and PI3KCA mutations were commonly altered (P < 0.001). (iv) Survival analysis: ITPN with a "pure" branch duct involvement showed the lowest risk of recurrence. CONCLUSION: ITPN is a distinct pancreatic neoplasm with specific clinicopathological and molecular characteristics. Its recognition is fundamental for its clinical/prognostic implications and for the enrichment of potential targets for precision oncology.

Deficient Rnf43 potentiates hyperactive Kras-mediated pancreatic preneoplasia initiation and malignant transformation.

BACKGROUND: Largely due to incidental detection, asymptomatic pancreatic cystic lesions (PCLs) have become prevalent in recent years. Among them, intraductal papillary mucinous neoplasm (IPMN) infrequently advances to pancreatic ductal adenocarcinoma (PDAC). Conservative surveillance versus surgical intervention is a difficult clinical decision for both caregivers and PCL patients. Because RNF43 loss-of-function mutations and KRAS gain-of-function mutations concur in a subset of IPMN and PDAC, their biological significance and therapeutic potential should be elucidated. METHODS: Pancreatic Rnf43 knockout and Kras activated mice (Rnf43-/-; KrasG12D) were generated to evaluate their clinical significance in pancreatic pre-neoplastic initiation and malignant transformation. RESULTS: Loss of Rnf43 potentiated the occurrence and severity of IPMN and PDAC in oncogenic Kras mice. The Wnt/ß-catenin signaling pathway was activated in pancreatic KrasG12D and Rnf43 knockout mice and the PORCN inhibitor LGK974 blocked pancreatic IPMN initiation and progression to PDAC accordingly. CONCLUSIONS: Rnf43 is a tumor suppressor in the prevention of pancreatic malignant transformation. This genetically reconstituted autochthonous pancreatic Rnf43-/-; KrasG12D preclinical cancer model recapitulates the pathological process from pancreatic cyst to cancer in humans and can be treated with inhibitors of Wnt/ß-catenin signaling. Since the presence of RNF43 and KRAS mutations in IPMNs predicts future development of advanced neoplasia from PCLs, patients with these genetic anomalies warrant surveillance, surgery, and/or targeted therapeutics such as Wnt/ß-catenin inhibitors.

Relevance of gene mutations and methylation to the growth of pancreatic intraductal papillary mucinous neoplasms based on pyrosequencing.

We aimed to assess some of the potential genetic pathways for cancer development from non-malignant intraductal papillary mucinous neoplasm (IPMN) by evaluating genetic mutations and methylation. In total, 46 dissected regions in 33 IPMN cases were analyzed and compared between malignant-potential and benign cases, or between malignant-potential and benign tissue dissected regions including low-grade IPMN dissected regions accompanied by malignant-potential regions. Several gene mutations, gene methylations, and proteins were assessed by pyrosequencing and immunohistochemical analysis. RASSF1A methylation was more frequent in malignant-potential dissected regions (p = 0.0329). LINE-1 methylation was inversely correlated with GNAS mutation (r = - 0.3739, p = 0.0105). In cases with malignant-potential dissected regions, GNAS mutation was associated with less frequent perivascular invasion (p = 0.0128), perineural invasion (p = 0.0377), and lymph node metastasis (p = 0.0377) but significantly longer overall survival, compared to malignant-potential cases without GNAS mutation (p = 0.0419). The presence of concordant KRAS and GNAS mutations in the malignant-potential and benign dissected regions were more frequent among branch-duct IPMN cases than among the other types (p = 0.0319). Methylation of RASSF1A, CDKN2A, and LINE-1 and GNAS mutation may be relevant to cancer development, IPMN subtypes, and cancer prognosis.

The Impact of Clinical and Pathological Features on Intraductal Papillary Mucinous Neoplasm Recurrence After Surgical Resection: Long-Term Follow-Up Analysis.

OBJECTIVE: This study aimed to identify risk factors for recurrence after pancreatic resection for intraductal papillary mucinous neoplasm (IPMN). SUMMARY BACKGROUND DATA: Long-term follow-up data on recurrence after surgical resection for IPMN are currently lacking. Previous studies have presented mixed results on the role of margin status in risk of recurrence after surgical resection. METHODS: A total of 126 patients that underwent resection for noninvasive IPMN were followed for a median of 9.5 years. Dedicated pathological and radiological reviews were performed to correlate clinical and pathological features (including detailed pathological features of the parenchymal margin) with recurrence after surgical resection. In addition, in a subset of 32 patients with positive margins, we determined the relationship between the margin and original IPMN using driver gene mutations identified by next-generation sequencing. RESULTS: Family history of pancreatic cancer and high-grade IPMN was identified as risk factors for recurrence in both uni- and multivariate analysis (adjusted hazard ratio 3.05 and 1.88, respectively). Although positive margin was not significantly associated with recurrence in our cohort, the size and grade of the dysplastic focus at the margin were significantly correlated with recurrence in margin-positive patients. Genetic analyses showed that the neoplastic epithelium at the margin was independent from the original IPMN in at least 9 of 32 cases (28%). The majority of recurrences (74%) occurred after 3 years, and a significant minority (32%) occurred after 5 years. CONCLUSION: Sustained postoperative surveillance for all patients is indicated, particularly those with risk factors such has family history and high-grade dysplasia.

MNX1-HNF1B Axis Is Indispensable for Intraductal Papillary Mucinous Neoplasm Lineages.

BACKGROUND & AIMS: Chromatin architecture governs cell lineages by regulating the specific gene expression; however, its role in the diversity of cancer development remains unknown. Among pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMN) with an associated invasive carcinoma (IPMNinv) arise from 2 distinct precursors, and their fundamental differences remain obscure. Here, we aimed to assess the difference of chromatin architecture regulating the transcriptional signatures or biological features in pancreatic cancers. METHODS: We established 28 human organoids from distinct subtypes of pancreatic tumors, including IPMN, IPMNinv, and PDAC. We performed exome sequencing (seq), RNA-seq, assay for transposase-accessible chromatin-seq, chromatin immunoprecipitation-seq, high-throughput chromosome conformation capture, and phenotypic analyses with short hairpin RNA or clustered regularly interspaced short palindromic repeats interference. RESULTS: Established organoids successfully reproduced the histology of primary tumors. IPMN and IPMNinv organoids harbored GNAS, RNF43, or KLF4 mutations and showed the distinct expression profiles compared with PDAC. Chromatin accessibility profiles revealed the gain of stomach-specific open regions in IPMN and the pattern of diverse gastrointestinal tissues in IPMNinv. In contrast, PDAC presented an impressive loss of accessible regions compared with normal pancreatic ducts. Transcription factor footprint analysis and functional assays identified that MNX1 and HNF1B were biologically indispensable for IPMN lineages. The upregulation of MNX1 was specifically marked in the human IPMN lineage tissues. The MNX1-HNF1B axis governed a set of genes, including MYC, SOX9, and OLFM4, which are known to be essential for gastrointestinal stem cells. High-throughput chromosome conformation capture analysis suggested the HNF1B target genes to be 3-dimensionally connected in the genome of IPMNinv. CONCLUSIONS: Our organoid analyses identified the MNX1-HNF1B axis to be biologically significant in IPMN lineages.