Low-grade Intraductal Papillary Mucinous Neoplasms Research Articles

Abstract C030: Evolutionary and epistatic analyses reveal genic interactions with KRAS during malignant progression of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) patients face poor prognoses attributable to delayed disease detection, resulting in distant metastases and thereby reducing treatment efficacy. The challenge of detecting PDAC lesions during early stages emanates from an inauspicious trifecta of clinical factors: early-stage malignancies are often asymptomatic, existing biomarkers are insufficiently sensitive, and the physical location of the pancreas stymies reliable screening. A promising strategy towards improving outcomes is the resection of high-risk, pre-malignant lesions deemed likely to progress into carcinomas. Intraductal papillary mucinous neoplasms (IPMNs) are both large enough to be detected during abdominal imaging and can originate PDAC lesions. However, a patient may have many IPMN cysts, many of which will never progress. As pancreatic resection carries with it a high risk of morbidity, distinguishing the the evolutionary trajectories of IPMNs that will and will not progress could inform treatment strategies. Additionally, these trajectories may serve as disruptable targets to manage pre- neoplastic lesions, preventing their progression to PDAC. The vast majority PDAC cases contain mutations in KRAS and GNAS with secondary driver mutations in TP53, SMAD4, CDKN2A, and RNF43. Recent work has compared the frequency of these mutations in PDAC with low-grade and high-grade IPMN lesions, finding that they more common in high than low- grade lesions. Interestingly, unlike KRAS or GNAS, KLF4 mutations are prevalent in low-grade IPMNs, rare in high-grade IPMNs, and almost never observed in full-fledged carcinoma, suggesting distinct evolutionary trajectories possibly mediated by epistatic interactions between KLF4 and KRAS. However, the prevalence of a mutation is not dispositive as to its importance in tumorgenesis. Mutations in TTN, for instance, are frequently observed in PDAC and other cancers, but are widely considered to play a modest role, if any, in these cancers. To understand the contribution of a mutation to the evolution of cancer, we must instead deconvolve selection from baseline mutation rate across genomic sites and among patients to distinguish truly oncogenic mutations from mutations that occur frequently merely due to high underlying mutational rates. Here, we quantify the evolutionary and epistatic interplay between KRAS, KLF4, and other frequent mutations in IPMNs and PDAC during malignant transition. First, we report the cancer effect sizes of single-nucleotide variants in IPMNs at varying dysplastic stages. Then, we infer the synergistic and antagonistic interactions between driver mutations, providing insight into the order in which driver mutations occur. We then corroborate this epistasis-inferred ordering with the ordering of mutations recovered from ancestral state reconstruction derived from time-calibrated phylogenies. Lastly, we probe the mutational processes most responsible for conferring highly oncogenic mutations in IPMNs and PDACs to identify potentially actionable or preventable pathways of disease progression. Metabolism Citation Format: Nic Fisk, Derek Song, Margaret Moore, Cole G Jensen, Vincent L. Cannataro, Mofeed Nagib, Jeffrey D Mandell, Luisa Escobar-Hoyos, John W Kunstman, Jeffrey P Townsend. Evolutionary and epistatic analyses reveal genic interactions with KRAS during malignant progression of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C030.

Abstract 446: Integrated spatial transcriptomics and lipidomics analyses of intraductal papillary mucinous neoplasm identifies enrichment of long chain sulfatide as an early metabolic alteration

Abstract Purpose: To define the metabolic alterations associated with malignant progression of intraductal papillary mucinous neoplasm (IPMN). Experimental Procedures: Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) was conducted for spatial characterization of lipid profiles on frozen tissue sections from resected human IPMN patients. The specimen set consisted of either low-grade dysplasia (LG) (n=15), high-grade (HG) dysplasia (n=4), or with an associated pancreatic ductal adenocarcinoma (PDAC) (n=4) as well as 15 PDAC tissues. Utilizing serial sections to the same human IPMN cases analyzed with MALDI-MS imaging, we used the Visium Spatial Gene Expression technology platform to characterize the spatiomolecular underpinnings of LG and HG IPMN. H&E images of the same tissue section used for the Visium workflow, were used to annotate the spots in the dataset covering the epithelial lining of the IPMN lesions (“epilesional” areas) and the spots covering the adjacent microenvironment surrounding the lesion (“perilesiopecific” area). Findings were compared with lipidomic analyses of cystic fluid from 89 IPMN patients. The anti-cancer efficacy of UGT8-IN-1, a selective small molecule inhibitor of ceramide galactosyltransferase (CGT, also known as UGT8), was assessed in vitro and in mutant Kras;Gnas allografts. Results: MALDI/MS-based spatial imaging of human resected IPMN tissues and pancreata from a mutant Kras;Gnas mouse model of IPMN revealed long-chain hydroxylated sulfatides, particularly the C24:0(OH) and C24:1(OH) species, to be selectively enriched in the IPMN and PDAC neoplastic epithelium. Visium spatial transcriptomics data confirmed that the cognate transcripts engaged in sulfatide biosynthesis, including UGT8, Gal3St1, and FA2H, were co-localized with areas of sulfatide enrichment. Lipidomic analyses of cystic fluid from 89 patients with IPMN identified several sulfatide species to be significantly elevated in patients with IPMN/PDAC compared to those with low-grade IPMN. Inhibition of sulfatide metabolism via UGT8-IN-1 reduced viability of Kras;Gnas murine cell lines in vitro and attenuated tumor growth in an allograft IPMN model. UGT8-IN-1 treatment resulted in reduced levels of several sulfatide species, accompanied by a concomitant increase in a subset of quantified sulfatides precursor ceramides, accumulation of mitochondrial mass and reactive oxygen species and time-dependent increases in autophagy/mitophagy markers p62/SQSTM1 and LC-3B, suggesting impaired mitophagy. Conclusion: Enhanced sulfatide metabolism is an early alteration in cystic pre-cancerous lesions of the pancreas that persists through invasive neoplasia. Targeting sulfatide biosynthesis might represent an actionable vulnerability for cancer interception. Citation Format: Yihui Chen, Marta Sans, Fredrik Thege, Rongzhang Dou, Jimin Min, Michele Yip-Schneider, Jianjun Zhang, Ranran Wu, Ehsan Irajizad, Yuki Makino, Kimal Rajapakshe, Mark Hurd, Ricardo León-Letelier, Jody Vykoukal, Jennifer Dennison, Kim-Anh Do, Robert Wolff, Paola Guerrero, Michael Kim, Max Schmidt, Anirban Maitra, Samir Hanash, Johannes Fahrmann. Integrated spatial transcriptomics and lipidomics analyses of intraductal papillary mucinous neoplasm identifies enrichment of long chain sulfatide as an early metabolic alteration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 446.

Immunohistochemical FAP Expression Reflects 68Ga-FAPI PET Imaging Properties of Low- and High-Grade Intraductal Papillary Mucinous Neoplasms and Pancreatic Ductal Adenocarcinoma.

Pancreatic intraductal papillary mucinous neoplasms (IPMNs) are grossly visible (typically > 5 mm) intraductal epithelial neoplasms of mucin-producing cells, arising in the main pancreatic duct or its branches. According to the current 2-tiered grading scheme, these lesions are categorized as having either low-grade (LG) dysplasia, which has a benign prognosis, or high-grade (HG) dysplasia, which formally represents a carcinoma in situ and thus can transform to pancreatic ductal adenocarcinoma (PDAC). Because both entities require different treatments according to their risk of becoming malignant, a precise pretherapeutic diagnostic differentiation is inevitable for adequate patient management. Recently, our group has demonstrated that 68Ga-fibroblast activation protein (FAP) inhibitor (FAPI) PET/CT shows great potential for the differentiation of LG IPMNs, HG IPMNs, and PDAC according to marked differences in signal intensity and tracer dynamics. The purpose of this study was to biologically validate FAP as a target for PET imaging by analyzing immunohistochemical FAP expression in LG IPMNs, HG IPMNs, and PDAC and comparing with SUV and time to peak (TTP) measured in our prior study. Methods: To evaluate the correlation of the expression level of FAP and α-smooth muscle actin (αSMA) in neoplasm-associated stroma depending on the degree of dysplasia in IPMNs, 98 patients with a diagnosis of LG IPMN, HG IPMN, PDAC with associated HG IPMN, or PDAC who underwent pancreatic surgery at the University Hospital Heidelberg between 2017 and 2023 were identified using the database of the Institute of Pathology, University Hospital Heidelberg. In a reevaluation of hematoxylin- and eosin-stained tissue sections of formalin-fixed and paraffin-embedded resection material from the archive, which was originally generated for histopathologic routine diagnostics, a regrading of IPMNs was performed by a pathologist according to the current 2-tiered grading scheme, consequently eliminating the former diagnosis of "IPMN with intermediate-grade dysplasia." For each case, semithin tissue sections of 3 paraffin blocks containing neoplasm were immunohistologically stained with antibodies directed against FAP and αSMA. In a masked approach, a semiquantitative analysis of the immunohistochemically stained slides was finally performed by a pathologist by adapting the immunoreactive score (IRS) and human epidermal growth factor receptor 2 (Her2)/neu score to determine the intensity and percentage of FAP- and αSMA-positive cells. Afterward, the IRS of 14 patients who underwent 68Ga-FAPI-74 PET/CT in our previous study was compared with their SUVmax, SUVmean, and TTP for result validation. Results: From 98 patients, 294 specimens (3 replicates per patient) were immunohistochemically stained for FAP and αSMA. Twenty-three patients had LG IPMNs, 11 had HG IPMNs, 10 had HG IPMNs plus PDAC, and 54 had PDAC. The tumor stroma was in all cases variably positive for FAP. The staining intensity, percentage of FAP-positive stroma, IRS, and Her2/neu score increased with higher malignancy. αSMA expression could be shown in normal pancreatic stroma as well as within peri- and intraneoplastic desmoplastic reaction. No homogeneous increase in intensity, percentage, IRS, and Her2/neu score with higher malignancy was observed for αSMA. The comparison of the mean IRS of FAP with the mean SUVmax, SUVmean, and TTP of 68Ga-GAPI-74 PET/CT showed a matching value increasing with higher malignancy in 68Ga-FAPI-74 PET imaging and immunohistochemical FAP expression. Conclusion: The immunohistochemical staining of IPMNs and PDAC validates FAP as a biology-based stromal target for invivo imaging. Increasing expression of FAP in lesions with a higher degree of malignancy matches the expectation of a stronger FAP expression in PDAC and HG IPMNs than in LG IPMNs and corroborates our previous findings of higher SUVs and a longer TTP in PDAC and HG IPMNs than in LG IPMNs.

Expression of PD-L1 and VISTA in Intraductal Papillary Mucinous Neoplasm With Associated Invasive Carcinoma of the Pancreas

Early detection and treatment of invasive carcinoma arising in association with intraductal papillary mucinous neoplasm (IPMN), which is biologically and (epi)genetically distinct from conventional pancreatic ductal adenocarcinoma, provide an opportunity to improve the prognosis of this lethal disease. Despite the successful application of programmed death (ligand) 1 (PD-[L]1)-blocking strategies in numerous cancers, the immune microenvironment of IPMN with associated invasive carcinoma remains elusive. Here, we investigated CD8+ T cells, CD68+ macrophages, PD-L1, and V-domain immunoglobulin suppressor of T-cell activation (VISTA) in 60 patients with IPMN with associated invasive carcinoma using immunohistochemistry, explored their correlations with clinicopathologic variables and prognosis, and compared them with those in 76 patients with IPMN without invasive carcinoma (60 low-grade and 16 high-grade lesions). Using antibodies against CD8, CD68, and VISTA, we evaluated tumor-infiltrating immune cells in 5 high-power fields (×400) and calculated the corresponding mean counts. PD-L1 with a combined positive score of ≥1 was regarded as positive, and VISTA expression on tumor cells (TCs) was deemed positive when ≥1% of TCs showed membranous/cytoplasmic staining. A reduction of CD8+ T cells and an increase of macrophages were observed during carcinogenesis. Positive PD-L1 combined positive score and VISTA expression on TCs were 13% and 11% in the intraductal component of IPMN with associated invasive carcinoma, 15% and 12% in the associated invasive carcinoma, and 6% and 4% in IPMN without an invasive carcinoma, respectively. Interestingly, the PD-L1 positivity rate was the highest in a subset of associated invasive carcinomas (predominantly gastric-type-derived) and was associated with higher counts of CD8+ T cells, macrophages, and VISTA+ immune cells. Accumulation of VISTA+ immune cells was observed in the intraductal component of IPMN with associated invasive carcinoma compared with that of low-grade IPMN, whereas in intestinal-type IPMN with associated invasive carcinoma, the number of these cells decreased during the transition from the intraductal component to the associated invasive carcinoma. Survival analysis revealed that a higher number of macrophages predicted poorer prognosis. In conclusion, our results might help in individualized immunotherapeutic strategies for these patients.

O144 The role of cytology in the investigation and management of cystic lesions of the pancreas with special reference to mucinous cystic neoplasms

Abstract Introduction Pancreatic cystic lesions encompass benign and malignant disease within the pancreas. Cytology has been increasingly used in the physician's toolbox to provide an accurate, non-invasive and cost-effective modality for the diagnosis of pancreatic lesions. The cytological appearance alone may be insufficient to establish the diagnosis and it's crucial for effective clinicopathological correlation in a multidisciplinary setting, highlighting the vital role of the pathologist to ensure effective and quality care. Methods We identified 178 cases of cystic lesions of the pancreas between 2017 and 2021. We used the C grading system and the Papanicolaou grading systems (Pap) to assess the potential risk of malignancy. Results We identified 20 cases with moderate and high grade intraductal papillary mucinous neoplasms (IPMN) and 12 cases of mucinous cystic neoplasms (MCN) by using the Pap grading system, 18% that may require surgical intervention. 39 cases (22%) are low grade IPMN. 72 cases are classified as high risk and potentially malignant (Pap grade 4b) in comparison with 25 cases, using the C grading (C4 and C5). The remaining cases 77 (43%) are benign to very low risk malignant potential that don't require surgical intervention. Conclusion The use of Pap grading allowed for a less invasive approach to tissue sampling which, when combined with routine staining and specialised immunohistochemistry can help establish the diagnosis and parameters associated with progressive malignancy. The study provides a powerful non-invasive diagnostic tool that helps in surgical decision making for patients with cystic lesions of the pancreas.

Intraductal papillary mucinous neoplasm originating from a jejunal heterotopic pancreas: A case report

Intraductal papillary mucinous neoplasm (IPMN) is a rare pancreatic tumor and has the potential to become malignant. Surgery is the most effective treatment at present, but there is no consensus on the site of resection. Heterotopic pancreas occurs in the gastrointestinal tract, especially the stomach and duodenum but is asymptomatic and rare. We report a case of ectopic pancreas with IPMN located in the jejunum. A 56-year-old male patient suffered from severe pain, nausea and vomiting due to a traffic accident and sought emergency treatment at our hospital. Contrast-enhanced computed tomography of the whole abdomen suggested splenic congestion, which was considered to be splenic rupture. Emergency laparotomy was performed, and the ruptured spleen was removed during the operation. Unexpectedly, a cauliflower-like mass of about 2.5 cm × 2.5 cm in size was incidentally found about 80 cm from the ligament of Treitz during the operation. A partial small bowel resection was performed, and postoperative pathology confirmed the small bowel mass as heterotopic pancreas with low-grade IPMN. Ectopic pancreas occurs in the jejunum and is pathologically confirmed as IPMN after surgical resection.

IPMN-LEARN: A linear support vector machine learning model for predicting low-grade intraductal papillary mucinous neoplasms.

We aimed to build a machine learning tool to help predict low-grade intraductal papillary mucinous neoplasms (IPMNs) in order to avoid unnecessary surgical resection. IPMNs are precursors to pancreatic cancer. Surgical resection remains the only recognized treatment for IPMNs yet carries some risks of morbidity and potential mortality. Existing clinical guidelines are imperfect in distinguishing low-risk cysts from high-risk cysts that warrant resection. We built a linear support vector machine (SVM) learning model using a prospectively maintained surgical database of patients with resected IPMNs. Input variables included 18 demographic, clinical, and imaging characteristics. The outcome variable was the presence of low-grade or high-grade IPMN based on post-operative pathology results. Data were divided into a training/validation set and a testing set at a ratio of 4:1. Receiver operating characteristics analysis was used to assess classification performance. A total of 575 patients with resected IPMNs were identified. Of them, 53.4% had low-grade disease on final pathology. After classifier training and testing, a linear SVM-based model (IPMN-LEARN) was applied on the validation set. It achieved an accuracy of 77.4%, with a positive predictive value of 83%, a specificity of 72%, and a sensitivity of 83% in predicting low-grade disease in patients with IPMN. The model predicted low-grade lesions with an area under the curve of 0.82. A linear SVM learning model can identify low-grade IPMNs with good sensitivity and specificity. It may be used as a complement to existing guidelines to identify patients who could avoid unnecessary surgical resection.

Accurate prediction of histological grading of intraductal papillary mucinous neoplasia using deep learning.

Risk stratification and recommendation for surgery for intraductal papillary mucinous neoplasm (IPMN) are currently based on consensus guidelines. Risk stratification from presurgery histology is only potentially decisive owing to the low sensitivity of fine-needle aspiration. In this study, we developed and validated a deep learning-based method to distinguish between IPMN with low grade dysplasia and IPMN with high grade dysplasia/invasive carcinoma using endoscopic ultrasound (EUS) images. For model training, we acquired a total of 3355 EUS images from 43 patients who underwent pancreatectomy from March 2015 to August 2021. All patients had histologically proven IPMN. We used transfer learning to fine-tune a convolutional neural network and to classify "low grade IPMN" from "high grade IPMN/invasive carcinoma." Our test set consisted of 1823 images from 27 patients, recruiting 11 patients retrospectively, 7 patients prospectively, and 9 patients externally. We compared our results with the prediction based on international consensus guidelines. Our approach could classify low grade from high grade/invasive carcinoma in the test set with an accuracy of 99.6 % (95 %CI 99.5 %-99.9 %). Our deep learning model achieved superior accuracy in prediction of the histological outcome compared with any individual guideline, which have accuracies between 51.8 % (95 %CI 31.9 %-71.3 %) and 70.4 % (95 %CI 49.8-86.2). This pilot study demonstrated that deep learning in IPMN-EUS images can predict the histological outcome with high accuracy.

Static and Dynamic 68Ga-FAPI PET/CT for the Detection of Malignant Transformation of Intraductal Papillary Mucinous Neoplasia of the Pancreas.

Pancreatic ductal adenocarcinoma (PDAC) may arise from intraductal papillary mucinous neoplasms (IPMN) with malignant transformation, but a significant portion of IPMN remains to show benign behavior. Therefore, it is important to differentiate between benign IPMN and IPMN lesions undergoing malignant transformation. However, nonoperative differentiation by ultrasound, CT, MRI, and carbohydrate antigen 19-9 (CA19-9) is still unsatisfactory. Here, we assessed the clinical feasibility of additional assessment of malignancy by PET using 68Ga-labeled fibroblast activation protein inhibitors (68Ga-FAPI PET) in 25 patients with MRI- or CT-proven cystic pancreatic lesions. Methods: Twenty-five patients with cystic pancreatic lesions who were followed up in the European Pancreas Center of Heidelberg University hospital and who were led to surgical resection or fine-needle aspiration due to suspicious clinical, laboratory chemistry, or radiologic findings were examined by static (all patients) and dynamic (20 patients) 68Ga-FAPI PET. Cystic pancreatic lesions were delineated and SUVmax and SUVmean were determined. Time-activity curves and dynamic parameters (time to peak, K 1, k 2, K3, k 4) were extracted from dynamic PET data. Receiver-operating curves of static and dynamic PET parameters were calculated. Results: Eleven of the patients had menacing IPMN (high-grade IPMN with [6 cases] or without [5 cases] progression into PDAC) and 11 low-grade IPMN; 3 patients had other benign entities. Menacing IMPN showed significantly elevated 68Ga-FAPI uptake compared with low-grade IPMN and other benign cystic lesions. In dynamic imaging, menacing IPMN showed increasing time-activity curves followed by slow decrease afterward; time-activity curves of low-grade IPMN showed an immediate peak followed by rapid decrease for about 10 min and slower decrease for the rest of the time. Receiver-operating curves showed high sensitivity and specificity (area under the curve greater than 80%) of static and dynamic PET parameters for the differentiation of IPMN subtypes. Conclusion: 68Ga-FAPI PET is a helpful new tool for the differentiation of menacing and low-grade IPMN and shows the potential to avoid unnecessary surgery for nonmalignant pancreatic IPMN.

Use of Autoreactive Antibodies in Blood of Patients with Pancreatic Intraductal Papillary Mucinous Neoplasms (IPMN) for Grade Distinction and Detection of Malignancy.

Simple SummaryRisk stratification via biomarkers used with imaging could support predicting high-risk pancreatic intraductal papillary mucinous neoplasms (IPMN) with malignant potential. We explored the presence of autoreactive antibodies in the blood of patients with IPMN of different grades of dysplasia, including IPMN with associated invasive carcinoma and early-stage pancreatic ductal adenocarcinoma (PDAC), to identify signatures of early malignancy. Multivariate predictive models retained 14 proteins as potential biomarkers for discrimination between all disease classes. The integration of the autoreactive-antibody panel with clinical variables may aid in risk stratification of high-risk IPMN patients, which would subsequently improve clinical management.(1) Background: A reliable non-invasive distinction between low- and high-risk pancreatic intraductal papillary mucinous neoplasms (IPMN) is needed to effectively detect IPMN with malignant potential. This would improve preventative care and reduce the risk of developing pancreatic cancer and overtreatment. The present study aimed at exploring the presence of autoreactive antibodies in the blood of patients with IPMN of various grades of dysplasia. (2) Methods: A single-center cohort was studied composed of 378 serum samples from patients with low-grade IPMN (n = 91), high-grade IPMN (n = 66), IPMN with associated invasive cancer (n = 30), pancreatic ductal adenocarcinoma (PDAC) stages T1 (n = 24) and T2 (n = 113), and healthy controls (n = 54). A 249 full-length recombinant human protein microarray was used for profiling the serum samples. (3) Results: 14 proteins were identified as potential biomarkers for grade distinction in IPMN, yielding high specificity but mediocre sensitivity. (4) Conclusions: The identified autoantibodies are potential biomarkers that may assist in the detection of malignancy in IPMN patients.

Pancreatic resections for benign intraductal papillary mucinous neoplasms: Collateral damages from friendly fire

BackgroundSurgical resection of intraductal papillary mucinous neoplasms is based on preoperative high-risk stigmata/worrisome features, but the risk of overtreatment remains high. The aim of this study was to evaluate surgical indications and perioperative and long-term complications in patients with low-grade intraductal papillary mucinous neoplasms. MethodsPatients who underwent surgical resection between 2009 and 2018 with a final histology of low-grade intraductal papillary mucinous neoplasms were included. Surgical indications, type of surgery, and short- and long-term outcomes were evaluated. ResultsA significant decrease in the rate of patients resected for low-grade intraductal papillary mucinous neoplasms was observed (43.6% in 2009–2012 vs 27.8% in 2013–2018; P = .003), and 133 patients were finally included (62 women, median age: 68 years). Of these, 24.1% had 1 worrisome feature, 39.8% had ≥2 worrisome features, 18.8% had ≥1 high-risk stigmata, and 15.8% had ≥1 worrisome features + 1 high-risk stigmata. Overall surgical morbidity was 55.6%, 15.8% had Clavien-Dindo ≥3 complications, reoperation rate was 3.8%, and 90-day postoperative mortality was 1.5%. After a median follow-up of 60 months, 13 patients (11.5%) had a recurrence of benign intraductal papillary mucinous neoplasm in the pancreatic remnant, and 2 patients (1.8%) developed pancreatic ductal adenocarcinoma. After partial pancreatectomy, 51.3% of patients were taking pancreatic enzyme replacement therapy. Among nondiabetics, 26% developed diabetes after partial pancreatectomy, of which 38% were insulin-dependent. Eighteen patients (13.7%) developed incisional hernia. ConclusionGiven the rates of morbidity and long-term complications after pancreatic resections, surgeons should attentively balance the true risks of intraductal papillary mucinous neoplasm degeneration with the risks of surgical resection in each patient.

Diminished Immune Surveillance during Histologic Progression of Intraductal Papillary Mucinous Neoplasms Offers a Therapeutic Opportunity for Cancer Interception.

Intraductal papillary mucinous neoplasms (IPMN) are bona fide precursors to pancreatic ductal adenocarcinoma (PDAC). While genomic alterations during multistep IPMN progression have been well cataloged, the accompanying changes within the tumor immune microenvironment (TIME) have not been comprehensively studied. Herein, we investigated TIME-related alterations during IPMN progression, using multiplex immunofluorescence (mIF) coupled with high-resolution image analyses. Two sets of formalin-fixed, paraffin-embedded tissue samples from surgically resected IPMNs were analyzed. The training set of 30 samples consisted of 11 low-grade IPMN (LG-IPMN), 17 high-grade IPMN (HG-IPMN), and 2 IPMN with PDAC, while a validation set of 93 samples comprised of 55 LG-IPMN and 38 HG-IPMN. The training set was analyzed with two panels of immuno-oncology-related biomarkers, while the validation set was analyzed with a subset of markers found significantly altered in the training set. Cell types indicative of enhanced immune surveillance, including cytotoxic and memory T cells, and antigen-experienced T cells and B cells, were all found at higher densities within isolated LG-IPMNs compared with HG-IPMNs. Notably, the TIME of LG-IPMNs that had progressed at the time of surgical resection (progressor LGD) resembled that of the synchronous HG-IPMNs, underscoring that attenuated immune surveillance occurs even in LG-IPMNs destined for progression. Our findings provide a basis for interception of cystic neoplasia to PDAC, through maintenance of sustained immune surveillance using vaccines and other prevention approaches.

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.

Neutrophil-to-lymphocyte Ratio as a Predictor of Malignancy of Intraductal Papillary Mucinous Neoplasms.

Intraductal papillary mucinous neoplasm (IPMN) can lead, via the adenoma-carcinoma sequence, to invasive adenocarcinoma, which has a poor prognosis. Most IPMNs do not meet the indications for surgery and instead are monitored regularly, but no biomarkers of malignant transformation during surveillance have been established. A total of 50 patients with IPMN who underwent pancreatectomy were evaluated retrospectively. Clinicopathological parameters during the surveillance period before surgery were assessed to explore biomarkers for predicting malignancy. The serum level of carbohydrate antigen 19-9 was significantly higher in IPMN-derived invasive adenocarcinoma. The neutrophil-to-lymphocyte ratio was significantly lower in low-grade IPMN than high-grade and Inv-IPMN cases by univariate and multivariate analysis. Disease-free survival was significantly shorter in patients with high-grade and invasive IPMN compared with those with low-grade IPMN. In patients whose preoperative surveillance was performed for more than 12 months, the neutrophil-to-lymphocyte ratio was significantly higher in those with high-grade and invasive IPMNs compared with patients with low-grade IPMN in the year before surgery. The NLR is a useful biomarker for distinguishing between low-grade and high-grade IPMN and thus should be monitored during surveillance.

Molecular characterization of organoids derived from pancreatic intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are commonly identified non-invasive cyst-forming pancreatic neoplasms with the potential to progress into invasive pancreatic adenocarcinoma. There are few in vitro models with which to study the biology of IPMNs and their progression to invasive carcinoma. Therefore, we generated a living biobank of organoids from seven normal pancreatic ducts and ten IPMNs. We characterized eight IPMN organoid samples using whole genome sequencing and characterized five IPMN organoids and seven normal pancreatic duct organoids using transcriptome sequencing. We identified an average of 11,344 somatic mutations in the genomes of organoids derived from IPMNs, with one sample harboring 61,537 somatic mutations enriched for T→C transitions and T→A transversions. Recurrent coding somatic mutations were identified in 15 genes, including KRAS, GNAS, RNF43, PHF3, and RBM10. The most frequently mutated genes were KRAS, GNAS, and RNF43, with somatic mutations identified in six (75%), four (50%), and three (37.5%) IPMN organoid samples, respectively. On average, we identified 36 structural variants in IPMN derived organoids, and none had an unstable phenotype (> 200 structural variants). Transcriptome sequencing identified 28 genes differentially expressed between normal pancreatic duct organoid and IPMN organoid samples. The most significantly upregulated and downregulated genes were CLDN18 and FOXA1. Immunohistochemical analysis of FOXA1 expression in 112 IPMNs, 113 mucinous cystic neoplasms, and 145 pancreatic ductal adenocarcinomas demonstrated statistically significant loss of expression in low-grade IPMNs (p < 0.0016), mucinous cystic neoplasms (p < 0.0001), and pancreatic ductal adenocarcinoma of any histologic grade (p < 0.0001) compared to normal pancreatic ducts. These data indicate that FOXA1 loss of expression occurs early in pancreatic tumorigenesis. Our study highlights the utility of organoid culture to study the genetics and biology of normal pancreatic duct and IPMNs. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Noninvasive Discrimination of Low and High-risk Pancreatic Intraductal Papillary Mucinous Neoplasms.

To propose a noninvasive diagnostic approach, which allows reliable distinction between low- and high-risk pancreatic intraductal papillary mucinous neoplasms (IPMNs). IPMNs are identifiable precursor lesions of pancreatic cancer, of which surgical resection is warranted prior to the development of invasive carcinoma, but low-grade IPMNs should not be unnecessarily resected. However, diagnostic tools that preoperatively enable accurate risk stratification of IPMNs are missing. This single-center, retrospective cohort study included 56 patients who underwent surgical resection for IPMN including 18 low-risk (low-grade) and 38 high-risk (high-grade/invasive carcinoma) IPMNs, from whom clinical features and serum samples were prospectively obtained. An antibody microarray platform was used to analyze the serum proteome. Based on serum markers and selected clinical characteristics support vector machine models were constructed to predict the risk of IPMN malignancy. A serum protein signature discriminating low- and high-risk IPMN patients was identified. Combinations of established clinical features and the newly identified serum biomarkers correctly distinguished low- and high-risk IPMNs in 93% on 1000-fold cross-validation. This study highlights the synergistic predictive value of combining a novel serum protein signature with conventional clinical characteristics to risk-stratify IPMN patients. If these findings are supported by larger validation studies, they might enable more rational decision-making in clinical management of IPMN patients in conjunction with clinical guidelines.

1050 – Linear Support Vector Machine Learning Model is Highly Specific for Predicting Low-Grade Intraductal Papillary Mucinous Neoplasm Lesions

1050 – Linear Support Vector Machine Learning Model is Highly Specific for Predicting Low-Grade Intraductal Papillary Mucinous Neoplasm Lesions

Single-Cell Transcriptomics of Pancreatic Cancer Precursors Demonstrates Epithelial and Microenvironmental Heterogeneity as an Early Event in Neoplastic Progression.

Early detection of pancreatic ductal adenocarcinoma (PDAC) remains elusive. Precursor lesions of PDAC, specifically intraductal papillary mucinous neoplasms (IPMNs), represent a bona fide pathway to invasive neoplasia, although the molecular correlates of progression remain to be fully elucidated. Single-cell transcriptomics provides a unique avenue for dissecting both the epithelial and microenvironmental heterogeneities that accompany multistep progression from noninvasive IPMNs to PDAC. Single-cell RNA sequencing was performed through droplet-based sequencing on 5,403 cells from 2 low-grade IPMNs (LGD-IPMNs), 2 high-grade IPMNs (HGD-IPMN), and 2 PDACs (all surgically resected). Analysis of single-cell transcriptomes revealed heterogeneous alterations within the epithelium and the tumor microenvironment during the progression of noninvasive dysplasia to invasive cancer. Although HGD-IPMNs expressed many core signaling pathways described in PDAC, LGD-IPMNs harbored subsets of single cells with a transcriptomic profile that overlapped with invasive cancer. Notably, a proinflammatory immune component was readily seen in low-grade IPMNs, composed of cytotoxic T cells, activated T-helper cells, and dendritic cells, which was progressively depleted during neoplastic progression, accompanied by infiltration of myeloid-derived suppressor cells. Finally, stromal myofibroblast populations were heterogeneous and acquired a previously described tumor-promoting and immune-evading phenotype during invasive carcinogenesis. This study demonstrates the ability to perform high-resolution profiling of the transcriptomic changes that occur during multistep progression of cystic PDAC precursors to cancer. Notably, single-cell analysis provides an unparalleled insight into both the epithelial and microenvironmental heterogeneities that accompany early cancer pathogenesis and might be a useful substrate to identify targets for cancer interception.See related commentary by Hernandez-Barco et al., p. 2027.

Increased SOX9 Expression in Premalignant and Malignant Pancreatic Neoplasms.

SOX9, a progenitor cell marker, is important for pancreatic ductal development. Our goal was to examine SOX9 expression differences in intraductal papillary mucinous neoplasms (IPMNs) and ductal adenocarcinoma (PDAC) compared with benign pancreatic duct (BP). SOX9 expression was evaluated by immunohistochemistry performed on 93 specimens: 37 BP, 24 low grade (LG) IPMN, 12 high grade (HG) IPMN, and 20 PDAC. A linear mixed-effects model was used to compare the percentage of cells expressing SOX9 by specimen type. A separate linear mixed-effects model evaluated differences in SOX9 expression by staining intensity in pancreatic epithelial cells. Nuclear SOX9 expression was detected in the epithelial cells of 98% HG IPMN, 93% LG IPMN, 83% PDAC, and 60% BP. Compared with BP, SOX9 was expressed from a significantly greater percentage of cells in LG IMPN, HG IMPN, and PDAC (p < 0.001 for each). BP and PDAC showed greater variability in SOX9 expression in epithelial cells compared with IPMNs which showed strong, homogenous SOX9 expression in almost all cells. Compared with BP, both LG and HG IPMN showed significantly greater SOX9 expression (p < 0.001 for each), but there was no significant difference in SOX9 expression between LG and HG IPMN (p > 0.05). PDAC had significantly higher expression of SOX9 compared with BP but significantly lower SOX9 expression compared with LG or HG IPMN (p < 0.001 for each). IPMNs demonstrated the highest expression levels of SOX9. SOX9 expression in BP and PDAC demonstrated much more heterogeneity compared with the strong, uniform expression in IPMN.

Surgical overtreatment of pancreatic intraductal papillary mucinous neoplasms: Do the 2017 International Consensus Guidelines improve clinical decision making?

BackgroundSignificant overtreatment of intraductal papillary mucinous neoplasms can be attributed to low specificity of the current International Consensus Guidelines as well as nonconformity with the guidelines. We compare the ability of the 2012 and revised 2017 intraductal papillary mucinous neoplasms International Consensus Guidelines to predict high-grade dysplasia/invasive cancer and to determine the preoperative variables that predict resection of benign or low-grade dysplasia in tertiary care centers. MethodsClinical, radiographic, and pathologic data for resected intraductal papillary mucinous neoplasms at 3 high-volume National Cancer Institute Cancer Centers were reviewed and the 2012 and 2017 consensus criteria were retrospectively applied. When International Consensus Guidelines were not met, clinical decision analysis was used to determine the primary indication for resection. Logistic regression identified variables associated with pathologic grade. ResultsRecords for a total of 251 patients were reviewed, 129 of whom (52%) had low-grade dysplasia. The revised 2017 International Consensus Guidelines had high sensitivity (98.4%) and negative predicted value (96.1%), and all high-risk stigmata predicted high-grade dysplasia/invasive cancer; however, specificity remained low (14.8%). Nonconformity with International Consensus Guidelines was the most powerful predictor of low-grade dysplasia on final pathologic examination (9.5; 2.12–40.78). Independent predictors of low-grade dysplasia included age younger than 50 (2.46; 1.08–5.62), fine-needle aspiration without epithelial cells (2.6; 1.43–4.72), and normal duct diameter (3.07; 1.99–4.75). Diabetes developed in 30% of patients after resection. ConclusionManagement of intraductal papillary mucinous neoplasms remains clinically challenging. Low specificity of the International Consensus Guidelines and nonconformity with the guidelines continue to contribute to unnecessary pancreatic resections. Improved tools for disease classification as well as a better understanding of the natural history, biology, and rates of progression of intraductal papillary mucinous neoplasms are needed to avoid surgical overtreatment of low-grade intraductal papillary mucinous neoplasms.