High-grade Pancreatic Intraepithelial Neoplasia Research Articles

Uncovering the role of oncogenic HER2 variants as drivers of pancreatic cancer.

772 Background: The therapeutic resistance of pancreatic ductal adenocarcinoma (PDAC) to conventional cytotoxic therapy underscores the need for advances in targeted molecular therapeutics. Previous studies have reported approximately 2% of PDAC patients with HER2 gene amplification. While anti- HER2 cancer therapy has yielded success in other disease sites, it has had minimal reported benefit in PDAC. Multiple HER2 isoforms exist that are generated either by loss of exon 16 ( d16 HER2 ), or through N-terminal truncations ( p95 HER2 ). These isoforms are implicated in differential tumor behavior and response to anti-HER2 therapy in the breast but have not been investigated in PDAC. Herein, we hypothesize that these isoforms may underlie the observed resistance to HER2 therapy in PDAC. Methods: To test for the presence of HER2 structural variants within human PDAC, a tissue microarray was constructed of fifty-one primary tumor samples and two metastatic samples. Multiplex immunohistochemistry (mIHC) was performed for staining of the extracellular N-terminal and intracellular C-terminal HER2 domains. We then utilized our previously described HER2 + cancer rainbow (Crainbow) mouse model in which human wild-type HER2 ( WT HER2 ), d16 HER2 , and p95 HER2 were expressed in the same mouse along with fluorescent protein reporters. HER2 Crainbow mice were crossed with PDX1-Cre recombinase to initiate recombination and expression of each fluorescently labelled HER2 isoform during pancreas development. PDX1-Cre / HER2 Crainbow mice were followed for up to one year and sacrificed to lineage trace each isoform and register HER2 lineages with histopathology. Results: In our human data, four of fifty-three patient samples (three primary and one metastatic) had moderate-to-strong HER2 staining. All samples identified on mIHC as HER2 positive stained primarily for the p95 variant of HER2 , with absence of the N-terminal domain and preservation of the intracellular domain. In our transgenic mice, 65% of the mouse cohort developed pre-malignant low grade pancreatic intraepithelial neoplasia (PanIN) by eight weeks. At one year, 58.3% of mice demonstrated advanced high grade PanIN, and 25% developed invasive disease. Lineage tracing revealed that while early low grade PanIN began as polyclonal lesions, comprised of WT- , d16- , and p95 HER2 expressing cells, advanced PanIN and invasive disease were unanimously monoclonal with one dominant isoform arising. WT HER2 was represented abundantly in low grade disease, but high grade and invasive disease were predominantly driven by d16- and p95-HER2. Conclusions: Our data implicates the oncogenic d16- and p95 HER2 variants, rather than WT HER2 , as the primary drivers of HER2+ PDAC tumorigenesis. In breast p95 HER2 has been described as a known cause of Trastuzumab resistance. Therefore, future efforts will be aimed at characterizing the mechanisms driving therapeutic resistance in p95-HER2 PDAC tumors.

Pancreatic juice cytology for diagnosing invasive pancreatic carcinoma/high-grade pancreatic intraepithelial neoplasia without visible tumors on endoscopic ultrasound

ObjectivesPancreatic ductal adenocarcinoma (PDAC) with a diameter ≤10 mm and high-grade pancreatic intraepithelial neoplasia (HG-PanIN) require pre-operative diagnosis. Most cases present only indirect imaging findings without visible tumors on endoscopic ultrasound (EUS). Therefore, EUS-guided fine-needle aspiration/biopsy is not applicable. An alternative diagnostic method is pancreatic juice cytology (PJC) via endoscopic naso-pancreatic drainage (ENPD-PJC), which is not the standard practice. This study aimed to investigate ENPD-PJC for diagnosing suspected PDAC/HG-PanIN cases without visible tumors on EUS. MethodsData of patients with suspected PDAC/HG-PanIN without visible tumors who underwent PJC were retrospectively evaluated. One PJC sample was collected during endoscopic retrograde pancreatography (ERP-PJC), and 12 samples were collected during ENPD-PJC, 3-hourly for cytological analysis. ERP-PJC, ERP/ENPD-PJC, and ENPD-PJC positivity indicated cytologically positive samples. Patients with positive/negative PJC with follow-up for <4-years were excluded as undiagnosed cases. A non-malignant diagnosis was based on histopathological absence/stable imaging findings for ≥4-years. The primary endpoint was to demonstrate that ERP/ENPD-PJC has a higher diagnostic ability than ERP-PJC. ResultsTwenty-two patients with histopathologically diagnosed PDAC/HG-PanIN and 31 with a non-malignant diagnosis were enrolled. ERP-PJC, ERP/ENPD-PJC, and ENPD-PJC showed sensitivities of 36.4 %, 86.4 %, and 77.3 %, specificities of 93.5 %, 87.1 %, and 93.5 %, and accuracies of 69.8 %, 86.7 %, and 86.7 %, respectively. ERP/ENPD-PJC and ENPD-PJC demonstrated superior sensitivity and accuracy compared to ERP-PJC. A greater occurrence of positive outcomes markedly distinguished true positives from false positives. ConclusionsERP/ENPD-PJC and ENPD-PJC had higher diagnostic accuracies for PDAC/HG-PanIN without visible tumors on EUS. ENPD-PJC is recommended for the diagnosis of these lesions.

CT findings and clinical effects of high grade pancreatic intraepithelial neoplasia in patients with intraductal papillary mucinous neoplasms.

To investigate the common CT findings of high-grade (HG) PanIN and clinical effects in the remnant pancreas in patients with intraductal papillary mucinous neoplasm (IPMN) of the pancreas. Two hundred fifty-one patients with surgically confirmed IPMNs (118 malignant [invasive carcinoma/high-grade dysplasia] and 133 benign [low-grade dysplasia]) were retrospectively enrolled. The grade of PanIN (233 absent/low-grade and 18 high-grade) was recorded, and all patients underwent serial CT follow-up before and after surgery. Two radiologists analyzed CT findings of high-risk stigmata or worrisome features according to 2017 international consensus guidelines. They also analyzed tumor recurrence on serial follow-up CT after surgery. Statistical analyses were performed to identify significant predictors and clinical impact on postoperative outcomes of HG PanIN. PanIN grade showed a significant association with IPMN grade (p = 0.012). Enhancing mural nodules ≥5 mm, abrupt main pancreatic duct (MPD) changes with distal pancreatic atrophy, increased mural nodule size and MPD diameter were common findings in HG PanIN (P<0.05). In multivariate analysis, abrupt MPD change with distal pancreatic atrophy (odds ratio (OR) 6.59, 95% CI: 2.32-18.72, <0.001) and mural nodule size (OR, 1.05; 95% CI, 1.02-1.08, 0.004) were important predictors for HG PanIN. During postoperative follow-up, HG PanIN (OR, 4.98; 95% CI, 1.22-20.33, 0.025) was significantly associated with cancer recurrence in the remnant pancreas. CT can be useful for predicting HG PanIN using common features, such as abrupt MPD changes and mural nodules. In HG PanIN, extra caution is needed to monitor postoperative recurrence during follow-up.

Abstract B093: Alcoholic chronic inflammation driven CREB mediates acinar-to-ductal reprogramming and promote neoplastic progression

Abstract Introduction: Alcoholic chronic pancreatitis (ACP) is a progressive fibroinflammatory disease of the pancreas often characterized by extensive acinar cell injury leading to acinar-to-ductal metaplasia (ADM). However, the impact of ADM associated with ACP on the acceleration of oncogenic mutant Kras (Kras*)-driven tumorigenesis remains poorly understood. In this study, we aimed to unravel the underlying mechanism by which ACP-induced dysfunction and with Kras*, leads to the development of irreversible ADM and premalignant lesions that establish a tumor-permissive microenvironment for the emergence of high-grade pancreatic intraepithelial neoplasia (PanINs). Methods: ACP mice model was developed in acinar cell-specific Ptf1aCreERTM/+, Ptf1aCreERTM/+; LSL-KrasG12D/+ (KC), and genetic ablation of cyclic AMP response element-binding protein 1 (CREB) in KC (KCC−/−) mice crossed with or without R26R-EYFP reporter mice, and these mice were fed with alcohol-enriched or regular diet along with repetitive caerulein administration. Lineage tracing studies and acinar explants were employed to investigate the fate of adult acinar cells. ACP-induced mice were subsequently euthanized either 3 days or 21 days after ACP induction, the pancreas was collected for immunohistochemical analysis and examined for ADM, PanINs, fibrosis, and inflammation. To gain insights into the molecular mechanisms; kinase array analysis, acinar cell explant culture, Western blot and qPCR-based studies were conducted. Additionally, single-cell RNA sequencing (scRNA-seq) was employed to investigate cell-specific transcriptomic changes focusing on distinct cellular compartments. Results: Lineage tracing studies showed that ACP-induced ADM stems from direct transdifferentiating adult acinar cells to a ductal phenotype in Kras* mice. ACP induction with Kras* cooperatively provided a sustained Kras - dependent oncogenic program in inflammation-induced metaplastic progenitor cells. The upregulation of CREB accompanied this process of malignant transformation. CREB activation persists even after ACP withdrawal in KC mice compared to Ptf1aCreERTM/+ mice without mutant Kras. Acinar-specific CREB ablation attenuated advanced PanIN lesion development, impeding tumor progression in Kras* mice. Additionally, it reduced inflammation-induced tissue injury and facilitated acinar cell recovery. ScRNA-seq analysis revealed gene enrichment favoring acinar cell homeostasis and downregulation of ductal gene signatures in KCC−/− mice, further emphasizing CREB's pivotal role in modulating these cellular processes. Conclusion: Our findings demonstrate that Kras* sustains an irreversible ADM and PanIN through continuous activation of CREB. Additionally, we have identified that CREB mediates an oncogenic transcriptional program that drives tumor progression in the context of inflammation-induced disease. Importantly, our study reveals that ACP synergistically cooperates with Kras*-driven activated CREB, promoting the progression of ADM toward high-grade PanINs. Citation Format: Siddharth Mehra, Supriya Srinivasan, Anna Bianchi, Austin Dosch, Samara Singh, Vanessa Garrido, Iago De Castro Silva, Varunkumar Krishnamoorthy, Sudhakar Jinka, Jashodeep Datta, Nipun B. Merchant, Nagaraj Nagathihalli. Alcoholic chronic inflammation driven CREB mediates acinar-to-ductal reprogramming and promote neoplastic progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B093.

Pancreatic intraepithelial neoplasia detection and duct pathology grading using FT-IR imaging and machine learning

Pancreatic intraepithelial neoplasia (PanIN) is manifested by noninvasive lesions in the epithelium of smaller pancreatic ducts. Generally, cancer development risk from low-grade PanIN is minor, whereas, invasive pancreatic ductal adenocarcinoma (PDAC) development is highly related to high-grade PanINs. Therefore, in the case of high-grade PanIN detection, additional surgical resection may be recommended. However, even the low-grade PanINs can indicate possible progression to PDAC. The definition of PanIN is constantly changing and there is a need for new tools to better characterize and understand its behavior. We have recently developed a comprehensive pancreatic cancer classification model with biopsies collected from over 600 biopsies from 250 patients. Here, we take the next step and employ Infrared (IR) spectroscopy to build the first classification model for PanINs detection. Furthermore, we created a Partial Least Squares Regression (PLSR) model to characterize ducts from benign to cancerous. This model was then used to predict and grade PanINs accordingly to their malignancy level.

Preoperative Endoscopic Minor Papilla Sphincterotomy for Pancreas Divisum in a Patient with Pancreatic Cancer Who Underwent Laparoscopic Distal Pancreatectomy.

Pancreas divisum (PD) is a congenital anomaly that occurs due to failure of fusion of the dorsal and ventral pancreatic ductal systems.1-3 In PD, pancreatic juice drains mainly through the minor papilla via the dorsal duct, leading to high intraductal pressure, which can cause pancreatitis.1-3 We report a case of PD that underwent preoperative decompression using endoscopic minor papilla sphincterotomy (EMPS) before laparoscopic distal pancreatectomy (LDP) for pancreatic cancer.3 METHODS: The patient was a 74-year-old woman with pancreatic tail cancer, measuring 35 mm in size, in PD with an entirely dilated dorsal duct, implying high, intraductal pressure caused by minor papillary dysfunction. We performed EMPS to prevent postoperative pancreatitis and pancreatic fistula before LDP using a left-posterior approach, as previously described.4 As the pancreatic transection margin was positive for high-grade pancreatic intraepithelial neoplasia on intraoperative pathology, an additional resection of the pancreatic head to the right side of the portal vein was performed after the liberation of the gastroduodenal artery with both the dorsal and ventral pancreatic ducts ligated and divided. The operative time was 421 min, and blood loss was 70 mL. The postoperative course was uneventful, with no evidence of pancreatitis or pancreatic fistula. The patient was discharged on postoperative Day 10. Postoperative computed tomography revealed reduced dilatation of the dorsal duct. Preoperative EMPS may be effective in preventing pancreatic fistula after LDP in patients with PD.

Dilatation of the main pancreatic duct of unknown origin: causes and risk factors of pre-malignancy or malignancy.

A dilatation of the main pancreatic duct (MPD) is mainly due to obstructive causes (pancreatic tumor, chronic pancreatitis) or intraductal papillary mucinous neoplasm (IPMN). This study aims to assess the risk of pre-malignancy or malignancy in case of MPD dilatation with no visible mass nor obstructive calcification on computed tomography scan (CT-scan) in a population operated for it. All patients operated on from November 2015 to December 2019 in our center for a significant dilatation of the MPD without visible obstructive cause on CT-scan were included. Preoperative work-up included at least CT-scan, magnetic resonance imaging (MRI), and endoscopic ultrasonography (EUS). Primary endpoint was the final pathological diagnosis. Secondary endpoints were predictive factors of malignancy. 101 patients were included, mean age 68years-old. Final pathological data were pancreatic adenocarcinoma (n = 2), IPMN with high-grade dysplasia (n = 37), high-grade Pancreatic Intraepithelial Neoplasia (PanIN) (n = 2) (total of pre-malignant or malignant lesions: n = 41), neuroendocrine tumor (n = 6), IPMN with low-grade dysplasia (n = 45), low-grade PanIN (n = 5), chronic pancreatitis (n = 3), and benign stenosis (n = 1). On preoperative explorations, the median diameter of MPD was 7mm [3-35]. MRI and/or EUS showed intraductal material, nodule, or cyst in 22, 32, and 52 patients, respectively; 22 patients without nodule visible on MRI or EUS had still a pre-malignant or malignant lesion. In multivariate analysis, predictive factors forpre-malignancy or malignancy were symptoms before surgery (p = 0.01), MPD dilatation without downstream stenosis (p = 0.046), and the presence of nodule (p = 0.009). A dilatation of the MPD without detectable mass or obstructive calcification on CT-scan was associated with a pre-malignant or malignant lesion in 41 patients. Symptoms before surgery, MPD dilatation without duct narrowing, and the presence of nodules on MRI/EUS were associated with the risk of pre-malignancy ormalignancy.

Abstract C037: KMT2D loss in pancreatic cancer cells leads to an immunosuppressive tumor microenvironment by upregulating the interferon and inflammatory response pathways

Abstract Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemoradiation and immune therapies, which is at least in part driven by its immunosuppressive tumor microenvironment (TME) consisting of dense cancer-associated fibroblasts (CAF) and highly suppressed immune cells. Lysine methyltransferase 2D (KMT2D), also known as MLL4 in mice, has been identified as one of the most frequently altered epigenetic regulators in PDAC. Most importantly, KMT2D mutation was found in more than 80% of neoadjuvant-treated PDAC, suggesting that KMT2D loss may be associated with treatment resistance. In human PDAC, low KMT2D protein level is correlated with worse survival. However, it is unknown whether KMT2D loss in PDAC cells modulates the TME. This project aimed to elucidate the role of KMT2D in remodeling the immune and stromal environment in PDAC. We first screened for changes in the immune cell population by performing immunohistochemistry (IHC) stains with human PDAC samples. We found that primary human tumors with low KMT2D expression had increased immunosuppressive tumor-associated neutrophils. xCell digital dissection analysis with The Cancer Genome Atlas (TCGA) data revealed that KMT2D-low PDAC had decreased effector T cell signature and enriched immunosuppressive Treg cell signature. Single-cell RNA sequencing analysis showed that KMT2D-low PDAC had increased suppressive myeloid cells and decreased CD8 T cells. We then generated Ptf1a-Cre; LSL-KrasG12D; Mll4+/+ (KCM+/+) and Ptf1a-Cre; LSL-KrasG12D; Mll4f/f (KCMf/f) mice to model pancreas-specific loss of Mll4 in the background of Kras mutation, a classic PDAC genetic mouse model. All KCM+/+ mice younger than 12 weeks had normal pancreas or low-grade pancreatic intraepithelial neoplasia (PanIN). In contrast, 66% of KCMf/f mice developed high-grade PanIN lesions or PDAC by 12 weeks. Immunohistochemistry staining showed that infiltration of immunosuppressive myeloid cells (tumor-associated macrophages and neutrophils) increased, whereas the presence of CD8+ T cells decreased in Mll4-deficient pancreata. Additionally, myofibroblastic CAFs, defined as PDPN+aSMA+ CAFs, decreased in KCMf/f mice pancreata compared to KCM+/+ pancreata. Mechanistically, we found that KMT2D loss in PDAC cells upregulates the interferon and inflammatory response pathways, including many cytokines and chemokines. These results suggest that KMT2D is a critical tumor suppressor, partly by promoting an immunogenic and tumor-suppressive TME. Cytokines in the interferon and inflammatory response pathways may serve as potential therapeutic targets, especially for treatment-resistant PDACs and KMT2D-deficient PDACs. Citation Format: Hong S. Kim, Jing Yang, Shuang Lu, Marina Pasca di Magliano, Kai Ge, Jiaqi Shi. KMT2D loss in pancreatic cancer cells leads to an immunosuppressive tumor microenvironment by upregulating the interferon and inflammatory response pathways [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C037.

MiR-4653-3p overexpression is associated with a poor prognosis of pancreatic ductal adenocarcinoma via HIPK2 downregulation

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignant tumor. Several upregulated and downregulated microRNAs (miRNAs) are associated with invasiveness, tumorigenesis, and prognosis of PDAC. Herein, using in situ hybridization, we evaluated miR-4653-3p expression and pancreatic intraepithelial neoplasia (PanIN) and the association between miR-4653-3p expression and clinicopathological factors in PDAC patients. The miR-4653-3p target was also identified. Ninety PDAC cases, including 30 each with normal pancreatic ducts, low-grade PanINs, and high-grade PanINs, were evaluated. miR-4653-3p expression increased in the order—normal pancreatic duct, low-grade PanIN, high-grade PanIN, and PDAC—with no expression detected in normal pancreatic duct. High expression significantly correlated with advanced pathological T stage, lymph node metastasis, advanced Union for International Cancer Control stage, perineural invasion, venous involvement, and shorter overall and disease-specific survival. Homeodomain Interacting Protein Kinase 2 (HIPK2) was identified as a miR-4653-3p target based on mRNA microarray analysis and database screening. In MIA PaCa-2 cells, miR-4653-3p significantly downregulated HIPK2 expression. HIPK2 expression, unlike that of miR-4653-3p, decreased in the order—normal pancreatic duct, low-grade PanIN, high-grade PanIN, and PDAC. Low HIPK2 expression was associated with shorter overall and disease-specific survival in PDAC patients. Thus, miR-4653-3p associates with tumorigenesis and worse prognosis, partly by reducing HIPK2 expression.

Focal pancreatic parenchyma atrophy is a harbinger of pancreatic cancer and a clue to the intraductal spreading subtype

Background/ObjectivesRadiological evidence of focal pancreatic parenchymal atrophy (FPPA) may presage early pancreatic ductal adenocarcinoma (PDAC) development. We aimed to clarify the incidence of FPPA and the clinicopathological features of PDAC with FPPA before diagnosis. MethodsData on endoscopic ultrasound-guided fine-needle biopsies and surgical samples from 170 patients with pancreatic cancer histologically diagnosed between 2014 and 2019 were extracted from the pathology database of Komagome Hospital and Juntendo University hospital and retrospectively evaluated together with 51 patients without PDAC. ResultsFPPA was identified in 47/170 (28%) patients before PDAC diagnosis and in 2/51 (4%) patients in the control group (P < 0.01). The median duration from FPPA detection to diagnosis was 35 (interquartile range [IQR]:16–63) months. In 24/47 (51%) patients with FPPA, the atrophic area resolved.The lesion was in the head and body/tail in 7/40 and 67/56 of the patients with (n = 47) and without FPPA (n = 123), respectively (P < 0.001). Histopathologically confirmed non-invasive lesions in the main pancreatic duct and a positive surgical margin in the resected specimens occurred in 53% vs. 21% (P = 0.078) and 29% vs. 3% (P = 0.001) of the groups, respectively. The PDAC patients with FPPA accompanied by a malignant pancreatic resection margin had high-grade pancreatic intraepithelial neoplasia. ConclusionsFPPA occurred in 28% of the PDAC group at 35 months prediagnosis. The FPPA area resolved before PDAC onset. Benchmarking previous images of the pancreas with the focus on FPPA may enable prediction of PDAC. PDAC with FPPA involves widespread high-grade pancreatic intraepithelial neoplasia requiring a wide surgical margin for surgical excision.

Endoscopic approach in the diagnosis of high-grade pancreatic intraepithelial neoplasia.

Early diagnosis of pancreatic ductal adenocarcinoma (PDAC) is essential for improving prognosis; however, diagnosing PDAC at an early stage is challenging. In patients with localized high-grade pancreatic intraepithelial neoplasia (HG-PanIN), whose tumorous lesion is undetectable on cross-sectional images such as computed tomography or magnetic resonance image, long-term survival is expected. Pancreatic cystic lesions or main pancreatic duct (MPD) dilatation are important indirect findings for the initial diagnosis of HG-PanIN. Magnetic resonance cholangiopancreatography (MRCP) and endoscopic ultrasound (EUS) should play important roles in detecting abnormal image findings, such as local irregular MPD stenosis, caliber MPD changes, small cystic lesions, or branch duct dilatation. Additionally, EUS could detect hypoechoic areas around the MPD stenosis in some patients with HG-PanIN. Subsequently, endoscopic retrograde cholangiopancreatography (ERCP) and its associated pancreatic juice cytology, including serial pancreatic juice aspiration cytologic examination (SPACE) after placement of an endoscopic nasopancreatic drainage (ENPD) tube, may have high diagnostic accuracy for confirming the malignancy in HG-PanIN. Although ERCP and its associated pancreatic cytology, including SPACE, may be associated with post-ERCP pancreatitis (PEP), a recent randomized trial suggested that a 4-Fr ENPD tube may reduce the incidence of PEP. In the future, further prospective multicenter studies are required to establish a standard method of SPACE. Additionally, further studies for novel biomarkers could help to establish evolutionary methods with duodenal fluid and pancreatic juice for the early and accurate diagnosis of early-stage PDAC.

CRMP4 Up-regulates M2 Macrophages and Myeloid-derived Suppressor Cells to Promote Pancreatic Cancer in Mice.

We previously observed higher prevalence of high-grade pancreatic intraepithelial neoplasia (PanIN) in LSL-KrasG12D/+; Pdx1Cre/+ (KC-Crmp4wild) mice than LSL-KrasG12D/+; Pdx1Cre/+; Crmp4-/- (KC-Crmp4-/-) mice. This study investigated the relationship between collapsin response mediator protein 4 (CRMP4) and immune cell infiltration in pancreatic cancer. PanIN was induced by intraperitoneal injection of caerulein into KC-Crmp4wild and KC-Crmp4-/- mice, and immune cells in PanIN lesions were compared. Subcutaneous tumors were created by injecting Pan02 cells, and tumor diameter was compared between Crmp4wild and Crmp4-/- mice every 7 days. Peritumoral immune cells were examined immunohisto chemically. High-grade PanIN in KC mice showed statistically significantly high expression of CD163 (p=0.031) and CD11b (p=0.027). Following subcutaneous injection of Pan02 cells, tumor diameter was greater in Crmp4wild mice than Crmp4-/- mice. Crmp4wild mice exhibited higher CD163 and CD11b expression than Crmp4-/- mice in tumors (p<0.001). CRMP4 might promote pancreatic cancer by up-regulating M2 macrophages and myeloid-derived suppressor cells.

Downregulation of 5-hydroxymethylcytosine is an early event in pancreatic tumorigenesis.

Epigenetic alterations are increasingly recognized as important contributors to the development and progression of pancreatic ductal adenocarcinoma. 5-hydroxymethylcytosine (5hmC) is an epigenetic DNA mark generated through the ten-eleven translocation (TET) enzyme-mediated pathway and is closely linked to gene activation. However, the timing of alterations in epigenetic regulation in the progression of pancreatic neoplasia is not well understood. In this study, we hypothesized that aberrant expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and subsequent global 5hmC alteration are linked to early tumorigenesis in the pancreas. Therefore, we evaluated alterations of 5hmC and TET1 levels using immunohistochemistry in pancreatic neoplasms (n = 380) and normal ducts (n = 118). The study cohort included representation of the full spectrum of precancerous lesions from low- and high-grade pancreatic intraepithelial neoplasia (n = 95), intraductal papillary mucinous neoplasms (all subtypes, n = 129), intraductal oncocytic papillary neoplasms (n = 12), and mucinous cystic neoplasms (n = 144). 5hmC and TET1 were significantly downregulated in all types of precancerous lesion and associated invasive pancreatic ductal adenocarcinomas compared with normal ductal epithelium (all p < 0.001), and expression of 5hmC positively correlated with expression of TET1. Importantly, downregulation of both 5hmC and TET1 was observed in most low-grade precancerous lesions. There were no clear associations between 5hmC levels and clinicopathological factors, thereby suggesting a common epigenetic abnormality across precancerous lesions. We conclude that downregulation of 5hmC and TET1 is an early event in pancreatic tumorigenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pre-Operative Imaging and Pathological Diagnosis of Localized High-Grade Pancreatic Intra-Epithelial Neoplasia without Invasive Carcinoma

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is typically associated with an extremely poor prognosis; however, small PDAC tumors show good prognosis. High-grade pancreatic intra-epithelial neoplasia (PanIN), which precedes invasive PDAC, is a primary target for improving the prognosis of PDAC. However, detection of high-grade PanIN without invasive carcinoma by existing imaging modalities is difficult because the lesions are only microscopically detectable. Recent studies have reported the characteristics of imaging findings associated with localized high-grade PanIN and the usefulness of serial pancreatic-juice aspiration cytologic examination as a method to confirm the pre-operative histopathology. In this review, we aimed to clarify recent clinical findings regarding detection of localized high-grade PanIN, which may contribute to improvement of the prognosis of patients with PDAC.Pancreatic ductal adenocarcinoma (PDAC) arises from precursor lesions, such as pancreatic intra-epithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN). The prognosis of high-grade precancerous lesions, including high-grade PanIN and high-grade IPMN, without invasive carcinoma is good, despite the overall poor prognosis of PDAC. High-grade PanIN, as a lesion preceding invasive PDAC, is therefore a primary target for intervention. However, detection of localized high-grade PanIN is difficult when using standard radiological approaches. Therefore, most studies of high-grade PanIN have been conducted using specimens that harbor invasive PDAC. Recently, imaging characteristics of high-grade PanIN have been revealed. Obstruction of the pancreatic duct due to high-grade PanIN may induce a loss of acinar cells replaced by fibrosis and lobular parenchymal atrophy. These changes and additional inflammation around the branch pancreatic ducts (BPDs) result in main pancreatic duct (MPD) stenosis, dilation, retention cysts (BPD dilation), focal pancreatic parenchymal atrophy, and/or hypoechoic changes around the MPD. These indirect imaging findings have become important clues for localized, high-grade PanIN detection. To obtain pre-operative histopathological confirmation of suspected cases, serial pancreatic-juice aspiration cytologic examination is effective. In this review, we outline current knowledge on imaging characteristics of high-grade PanIN.

MAb Das-1 identifies pancreatic ductal adenocarcinoma and high-grade pancreatic intraepithelial neoplasia with high accuracy

Pancreatic intraepithelial neoplasia (PanIN) is a microscopic precursor lesion to pancreatic ductal adenocarcinoma (PDAC); however, there are few biomarkers that segregate high-grade PanIN/PDAC from low-grade PanIN lesions. mAb Das-1 is a monoclonal antibody against a colonic epithelial antigen that is reactive to premalignant conditions of the upper gastrointestinal tract including Barrett's esophagus, incomplete-type gastric intestinal metaplasia, and intraductal papillary mucinous neoplasm of the pancreas at high risk of malignancy. We sought to examine a role for Das-1 expression in differentiating high-grade PanIN/PDAC from low-grade PanIN lesions. We examined surgical specimens from 86 patients and 2 autopsied pancreata (74 with and 14 without PDAC) with 107 distinct PanIN lesions, 74 PDAC cases, and 32 associated lymph node metastases, with internal controls of normal pancreatic ducts observed in 56 cases. All of the normal pancreatic duct controls (0/56) and low-grade PanIN (0/95) lesions were nonreactive to Das-1. Das-1 expression amonghigh-grade PanIN (7/12, 58%), PDAC (55/74, 74%), and lymph node metastasis (21/32, 66%) cases was significantly higher (p<0.0001). Clinicopathologically, Das-1 reactivity was significantly correlated with nodal metastasis (p=0.021). Overall, the sensitivity, specificity, and accuracy of Das-1 in segregating high-grade PanIN/PDAC from low-grade PanIN lesions and normal ducts were 72%, 100%, and 90%, respectively. Thus, mAb Das-1 reacts with high specificity with high-grade PanIN and PDAC and may help in preoperative diagnosis and/or clinical risk stratification.

A hypoechoic, tumor-like lesion in the pancreatic head and neck on endoscopic ultrasonography may be due to a high-grade pancreatic intraepithelial neoplasia/carcinoma in situ

High-grade pancreatic intraepithelial neoplasia (HG PanIN)/carcinoma in situ (CIS) in the pancreatic body and tail can induce parenchymal atrophy through chronic inflammatory changes presenting as a Hypoechoic area on EUS (Hypocho) or focal pancreatic parenchymal atrophy (FPPA) on computed tomography (CT) and magnetic resonance imaging (MRI). We herein discussed two patients with a hypoechoic area in the pancreatic head and neck on EUS resembling pancreatic ductal adenocarcinoma (PDAC). The lesions consisted of dense fibrosis and fat infiltration with pancreatic parenchymal atrophy around the HG PanIN/CIS in the main pancreatic duct (MPD), which penetrated the lesion and showed mild stenosis and upstream dilation. CT and MRI were unable to visualize the lesions. A specimen was obtained from one lesion by fine-needle aspiration under EUS (EUS-FNA) guidance for histopathological and cytological analysis, but the tests returned negative for adenocarcinoma. However, serial pancreatic-juice aspiration cytologic examination (SPACE) revealed adenocarcinoma in both lesions, prompting surgical resection. Histopathological examination revealed non-invasive HG PanIN/CIS in the MPD surrounded by dense fibrosis and fat deposition in the area of parenchymal atrophy. The CIS was restricted to the area of parenchymal atrophy.These two cases are noteworthy in illustrating a hypoechoic area appearing on EUS as a tumor-like lesion resembling PDAC. EUS-FNA has recently been used histopathologically to diagnose a pancreatic lesion. However, in the present and similar cases, EUS-FNA can only reveal secondary changes due to CIS unless the pancreatic duct covered by the CIS is accidentally punctured. We should bear in mind that CIS can appear as a hypoechoic area resembling PDAC on EUS, and that SPACE is the best method for diagnosing CIS in such cases.

Left-posterior approach for artery-first en bloc resection in laparoscopic distal pancreatectomy for left-sided pancreatic cancer.

We describe a "left-posterior approach" in which the important steps in laparoscopic distal pancreatectomy (LDP) for left-sided pancreatic cancer are accomplished in the direction caudal and dorsal to the pancreas. The patients who underwent LDP with a left-posterior approach at our hospital from January 2016 to April 2020 were reviewed to evaluate the short-term postoperative outcomes. In LDP, we first dissected retroperitoneal tissues above the left renal vein and superior mesenteric artery, yielding the mobilization of the pancreatic body widely. Then, the splenic artery was divided behind the ventrally lifted pancreas as an artery-first approach. The regional lymphadenectomy was performed in an en bloc manner consecutively in the same operative field. The neck of the pancreas was transected with a linear stapler after mobilization of the spleen. In nine patients (five men and four women) aged 76 years (range: 64-82 years), the operative time was 398 min (276-482 min) with the estimated blood loss of 40 ml (0-80 ml). No patients developed grade B/C pancreatic fistula or delayed gastric emptying. Postoperative complications classified as grade III in the Clavien-Dindo classification occurred in one patient (abdominal abscess). The pathology confirmed R0 resection in all patients who had pancreatic cancer (n = 5), IPMNs (n = 3), and high-grade pancreatic intraepithelial neoplasia (PanIN) (n = 1). The number of retrieved lymph nodes was 35 (11-49). The procedure with a left-posterior approach is a rational surgical technique in LDP for left-sided pancreatic cancer.

Focal parenchymal atrophy of pancreas: An important sign of underlying high-grade pancreatic intraepithelial neoplasia without invasive carcinoma, i.e., carcinoma in situ

ObjectivesDiagnosing high-grade intraepithelial neoplasia without invasion, traditionally referred to as carcinoma in situ (CIS), is essential for improving prognosis. We examined the imaging findings of patients with and without CIS to identify significant aspects for the diagnosis of CIS. MethodsForty-six patients strongly suspected of early pancreatic cancer without nodule on imaging (CIS group, n = 27; non-malignant group, n = 19) were retrospectively evaluated according to ten factors of computed tomography/magnetic resonance imaging (CT/MRI), endoscopic ultrasonography (EUS), and endoscopic retrograde cholangiopancreatography (ERCP) using hierarchical cluster and univariate analyses. ResultsTwo clusters were formed by hierarchical cluster analysis. One cluster consisted of 83.3% CIS cases with similar image findings such as focal pancreatic parenchymal atrophy (FPPA) on CT/MRI, main pancreatic duct (MPD) stricture surrounded by hypoechoic areas on EUS, and MPD stricture with upstream MPD dilation on ERCP. On univariate analysis, the CIS and non-malignant groups had FPPA on CT/MRI in 15 (55.6%) and 3 (15.8%) cases (p = 0.013), and MPD stricture surrounded by hypoechoic areas on EUS in 20 (74.1%) and 4 (21.1%) cases (p = 0.001), respectively. MPD stricture surrounded by hypoechoic areas was observed in 80% (12/15) of CIS cases with FPPA on CT/MRI and correlated with FPPA. Moreover, FPPA and MPD stricture surrounded by hypoechoic areas had histopathologically observed fibrosis or fat replacement due to pancreatic parenchymal atrophy. ConclusionsFPPA and MPD stricture surrounded by hypoechoic areas are significant findings for the diagnosis of CIS.

Gene Expression Network Analysis of Precursor Lesions in Familial Pancreatic Cancer.

Purpose: High-grade pancreatic intraepithelial neoplasia (PanIN) are aggressive premalignant lesions, associated with risk of progression to pancreatic ductal adenocarcinoma (PDAC). A depiction of co-dysregulated gene activity in high-grade familial pancreatic cancer (FPC)-related PanIN lesions may characterize the molecular events during the progression from familial PanIN to PDAC.Materials and Methods: We performed weighted gene coexpression network analysis (WGCNA) to identify clusters of coexpressed genes associated with FPC-related PanIN lesions in 13 samples with PanIN-2/3 from FPC predisposed individuals, 6 samples with PDAC from sporadic pancreatic cancer (SPC) patients, and 4 samples of normal donor pancreatic tissue.Results: WGCNA identified seven differentially expressed gene (DEG) modules and two commonly expressed gene (CEG) modules with significant enrichment for Gene Ontology (GO) terms in FPC and SPC, including three upregulated (p < 5e-05) and four downregulated (p < 6e-04) gene modules in FPC compared to SPC. Among the DEG modules, the upregulated modules include 14 significant genes (p < 1e-06): ALOX12-AS1, BCL2L11, EHD4, C4B, BTN3A3, NDUFA11, RBM4B, MYOC, ZBTB47, TTTY15, NAPRT, LOC102606465, LOC100505711, and PTK2. The downregulated modules include 170 genes (p < 1e-06), among them 13 highly significant genes (p < 1e-10): COL10A1, SAMD9, PLPP4, COMP, POSTN, IGHV4–31, THBS2, MMP9, FNDC1, HOPX, TMEM200A, INHBA, and SULF1. The DEG modules are enriched for GO terms related to mitochondrial structure and adenosine triphosphate metabolic processes, extracellular structure and binding properties, humoral and complement mediated immune response, ligand-gated ion channel activity, and transmembrane receptor activity. Among the CEG modules, IL22RA1, DPEP1, and BCAT1 were found as highly connective hub genes associated with both FPC and SPC.Conclusion: FPC-related PanIN lesions exhibit a common molecular basis with SPC as shown by gene network activities and commonly expressed high-connectivity hub genes. The differential molecular pathology of FPC and SPC involves multiple coexpressed gene clusters enriched for GO terms including extracellular activities and mitochondrion function.

Low Incidence of High-Grade Pancreatic Intraepithelial Neoplasia Lesions in a Crmp4 Gene–Deficient Mouse Model of Pancreatic Cancer

Pancreatic intraepithelial neoplasia (PanIN), the most common premalignant lesion of the pancreas, is a histologically well-defined precursor to invasive pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms underlying the progression of PanINs have not been fully elucidated. Previously, we demonstrated that the expression of collapsin response mediator protein 4 (CRMP4) in PDAC was associated with poor prognosis. The expression of CRMP4 was also augmented in a pancreatitis mouse model. However, the role of CRMP4 in the progression of PanIN lesions remains uncertain. In the present study, we examined the relationship between CRMP4 expression and progression of PanIN lesions using genetically engineered mouse models. PanIN lesions were induced by peritoneal injection of the cholecystokinin analog caerulein in LSL-KRASG12D; Pdx1-Cre (KC-Crmp4 wild-type, WT) mice and LSL-KRASG12D; Pdx1-Cre; Crmp4−/− (KC-Crmp4 knockout, KO) mice. We analyzed pancreatic tissue sections from these mice and evaluated PanIN grade by hematoxylin and eosin staining. CRMP4 expression was examined and the cellular components assessed by immunohistochemistry using antibodies against CRMP4, CD3, and α-smooth muscle actin (SMA). The incidence of high-grade PanIN in KC-Crmp4 WT mice was higher than that in KC-Crmp4 KO animals. CRMP4 was expressed not only in epithelial cells but also in αSMA-positive cells in stromal areas of PanIN lesions. The CRMP4 expression in stromal areas correlated with PanIN grade in WT mice. These results suggested that the expression of CRMP4 in stromal cells may underlie the incidence or progression of PanIN.