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Introduction: Pancreatic cancer remains one of the deadliest cancers in the United States. Some types of pancreatic cysts, which are being detected more frequently and often incidentally on imaging, have the potential to develop into pancreatic cancer and thus provide a valuable window of opportunity for cancer interception. Although racial disparity in pancreatic cancer has been described, little is known regarding health disparities in pancreatic cancer prevention. In the present study, we investigate potential health disparities along the continuum of care for pancreatic cancer., Methods: The racial and ethnic composition of pancreatic patients at high-volume centers in Indiana were evaluated, representing patients undergoing surgery for pancreatic cancer (n=390), participating in biobanking (972 pancreatic cancer patients and 1984 patients with pancreatic disease), or being monitored for pancreatic cysts at an early detection center (n=1514). To assess racial disparities and potential differences in decision-making related to pancreatic cancer prevention and early detection, an exploratory online survey was administered through a volunteer registry (n=708).  Results: We show that despite comprising close to 10% or 30% of the Indiana or Indianapolis population, respectively, African Americans make up only about 4-5% of our study cohorts consisting of patients undergoing pancreatic surgery or participating in biobanking and early detection. Analysis of online survey results revealed that given the hypothetical situation of being diagnosed with a pancreatic cyst or pancreatic cancer, the vast majority of respondents (>90%) would agree to undergo surveillance or surgery, respectively, regardless of race. Only a minority (3-12%) acknowledged any significant transportation, financial, or emotional barriers that would impact a decision to undergo surveillance or surgery. This suggests that the observed racial disparities may be due in part to the existence of other barriers that lie upstream of this decision point., Conclusion: Racial disparities exist not only for pancreatic cancer but also at earlier points along the continuum of care such as prevention and early detection. To our knowledge, this is the first study to document racial disparity in the management of patients with pancreatic cysts who are at risk of developing pancreatic cancer. Our results suggest that improving access to information and care for such at-risk individuals may lead to more equitable outcomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Yip-Schneider et al.)

Background: Increasing evidence implicates microbiome involvement in the development and progression of pancreatic ductal adenocarcinoma (PDAC). Studies suggest that reflux of gut or oral microbiota can lead to colonization in the pancreas, resulting in dysbiosis that culminates in release of microbial toxins and metabolites that potentiate an inflammatory response and increase susceptibility to PDAC. Moreover, microbe-derived metabolites can exert direct effector functions on precursors and cancer cells, as well as other cell types, to either promote or attenuate tumor development and modulate treatment response., Content: The occurrence of microbial metabolites in biofluids thereby enables risk assessment and prognostication of PDAC, as well as having potential for design of interception strategies. In this review, we first highlight the relevance of the microbiome for progression of precancerous lesions in the pancreas and, using liquid chromatography-mass spectrometry, provide supporting evidence that microbe-derived metabolites manifest in pancreatic cystic fluid and are associated with malignant progression of intraductal papillary mucinous neoplasm(s). We secondly summarize the biomarker potential of microbe-derived metabolite signatures for (a) identifying individuals at high risk of developing or harboring PDAC and (b) predicting response to treatment and disease outcomes., Summary: The microbiome-derived metabolome holds considerable promise for risk assessment and prognostication of PDAC., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background/objectives: Pancreatic serous cystic neoplasms (SCN) present a diagnostic challenge given their increasing frequency of detection and benign nature yet relatively high rate of misdiagnosis. Here, imaging and analyses associated with EUS-guided fine-needle aspiration (EUS-FNA) are evaluated for their ability to provide a correct preoperative diagnosis of SCN., Methods: A surgical cohort with confirmed pathological diagnosis of SCN (n = 62) and a surveillance cohort with likely SCN (n = 31) were assessed for imaging (CT/MRI/EUS) and EUS-FNA-based analyses (cytology/DNA analysis for Von Hippel-Lindau [VHL] gene alterations/biomarkers)., Results: In the surgical cohort, CT/MRI and EUS respectively predicted SCN in 4 of 58(7%) and 19 of 62(31%). Cyst fluid cytology and VHL alterations predicted SCN in 1 of 51(2%) and 5 of 21(24%), respectively. High specificity cyst fluid biomarkers (vascular endothelial growth factor [VEGF]/glucose/carcinoembryonic antigen [CEA]/amylase) correctly identified SCN in 25 of 27(93%). In the surveillance cohort, cyst fluid biomarkers predicted SCN in 12 of 12(100%) while VHL alterations identified SCN 3 of 10(30%)., Conclusion: High specificity cyst fluid biomarkers provided the most sensitive means of diagnosing SCN preoperatively. To obtain a preoperative diagnosis of SCN at the highest level of certainty, a multidisciplinary approach should be taken to inform appropriate SCN management., (Copyright © 2023 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Introduction: The accurate identification of mucinous pancreatic cystic lesions (PCLs) is paramount for cancer risk stratification. Cyst fluid carcinoembryonic antigen (CEA), the only routinely used test, requires high volumes and has low sensitivity. We aimed to compare the performance of two investigational small-volume biomarkers, glucose and the protease gastricsin, to CEA for PCL classification., Methods: We obtained cyst fluid samples from 81 patients with pathologically confirmed PCLs from four institutions between 2003 and 2016. Gastricsin activity was measured using an internally quenched fluorescent substrate. Glucose levels were measured with a standard glucometer. CEA levels were obtained from the medical record. Models using Classification and Regression Trees were created to predict mucinous status. Model performance was evaluated using nested cross-validation., Results: Gastricsin activity, CEA, and glucose levels from patients with mucinous (n = 50) and nonmucinous (n = 31) PCLs were analyzed. Area under the curve (AUC) was similar for individual classifiers (gastricsin volume normalized [GVN] 0.88; gastricsin protein concentration normalized [GPN] 0.95; glucose 0.83; CEA 0.84). The combination of two classifiers did not significantly improve AUC, with CEA + GVN (0.88) performing similarly to CEA + GPN (0.95), GVN + glucose (0.87), GPN + glucose (0.95), and CEA + glucose (0.84). The three-analyte combination performed similarly to single and dual classifiers (GPN + glucose + CEA AUC 0.95; GVN + glucose + CEA AUC 0.87). After multiple comparison corrections, there were no significant differences between the individual, dual, and triple classifiers., Conclusions: Gastricsin and glucose performed similarly to CEA and required <5% of the volume required for CEA; these classifiers may be useful in patients with limited cyst fluid. Future multicenter prospective studies are needed to validate and compare these novel small-volume biomarkers., (Copyright © 2022. Published by Elsevier Inc.)

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that is often detected at an advanced stage. Earlier diagnosis of PDAC is key to reducing mortality. Circulating biomarkers such as microRNAs are gaining interest, but existing technologies require large sample volumes, amplification steps, extensive biofluid processing, lack sensitivity, and are low-throughput. Here, we present an advanced nanoplasmonic sensor for the highly sensitive, amplification-free detection and quantification of microRNAs (microRNA-10b, microRNA-let7a) from unprocessed plasma microsamples. The sensor construct utilizes uniquely designed -ssDNA receptors attached to gold triangular nanoprisms, which display unique localized surface plasmon resonance (LSPR) properties, in a multiwell plate format. The formation of -ssDNA/microRNA duplex controls the nanostructure-biomolecule interfacial electronic interactions to promote the charge transfer/exciton delocalization processes and enhance the LSPR responses to achieve attomolar (10 -18 M) limit of detection (LOD) in human plasma. This improve LOD allows the fabrication of a high-throughput assay in a 384-well plate format. The performance of nanoplasmonic sensors for microRNA detection was further assessed by comparing with the qRT-PCR assay of 15 PDAC patient plasma samples that shows a positive correlation between these two assays with the Pearson correlation coefficient value >0.86. Evaluation of >170 clinical samples reveals that oncogenic microRNA-10b and tumor suppressor microRNA-let7a levels can individually differentiate PDAC from chronic pancreatitis and normal controls with >94% sensitivity and >94% specificity at a 95% confidence interval (CI). Furthermore, combining both oncogenic and tumor suppressor microRNA levels significantly improves differentiation of PDAC stages I and II versus III and IV with >91% and 87% sensitivity and specificity, respectively, in comparison to the sensitivity and specificity values for individual microRNAs. Moreover, we show that the level of microRNAs varies substantially in pre- and post-surgery PDAC patients ( n = 75). Taken together, this ultrasensitive nanoplasmonic sensor with excellent sensitivity and specificity is capable of assaying multiple biomarkers simultaneously and may facilitate early detection of PDAC to improve patient care.

Background: The decision to routinely leave a nasogastric tube after pancreatoduodenectomy remains controversial. We sought to determine the impact of immediate nasogastric tube removal versus early nasogastric tube removal (<24 h) on postoperative outcomes., Methods: A retrospective review of our institution's prospective ACS-NSQIP database identified patients that underwent pancreatoduodenectomy from 2015 to 2018. Outcomes were compared among patients with immediate nasogastric tube removal versus early nasogastric tube removal., Results: A total of 365 patients were included in primary analysis (no nasogastric tube, n = 99; nasogastric tube removed <24 h, n = 266). Thirty-day mortality and infectious, renal, cardiovascular, and pulmonary morbidity were similar in comparing those with no nasogastric tube versus early nasogastric tube removal on univariable and multivariable analyses (P > 0.05). Incidence of delayed gastric emptying (11.1 versus 13.2%) was similar between groups. Patients with no nasogastric tube less frequently required nasogastric tube reinsertion (n = 4, 4%) compared to patients with NGT <24 h (n = 39, 15%) (OR = 3.83, 95% CI [1.39-10.58]; P = 0.009)., Conclusion: Routine gastric decompression can be safely avoided after uneventful pancreaticoduodenectomy., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Purpose: Assess the relationship between MRI-derived pancreatic fat fraction and risk of malignancy in intraductal papillary mucinous neoplasm (IPMN)., Methods: MRIs of patients with IPMN who underwent pancreaticoduodenectomy were analyzed. IPMN with low-grade dysplasia (n = 29) were categorized as low-risk while IPMN at high risk of malignancy consisted of those with high-grade dysplasia/invasive carcinoma (n = 33). Pancreatic fat-fraction (FF mean ) was measured using the 2-point Dixon-method. Images were evaluated for the high-risk stigmata and worrisome features according to the revised 2017 Fukuoka consensus criteria. Data on serum CA19-9, Diabetes Mellitus (DM) status, body mass index (BMI), and histological chronic pancreatitis were obtained., Results: A significant difference in FF mean was found between the high-risk IPMN (11.45%) and low-risk IPMN (9.95%) groups (p = 0.027). Serum CA19-9 level (p = 0.021), presence of cyst wall enhancement (p = 0.029), and solid mass (p = 0.008) were significantly associated with high-risk IPMN. There was a significant correlation between FF mean and mural nodule size (r = 0.36, p ˂ 0.01), type 2 DM (r = 0.34, p ˂ 0.01), age (r = 0.31, p ˂ 0.05), serum CA 19-9 (r = 0.30, p ˂ 0.05), cyst diameter (r = 0.30, p ˂ 0.05), and main pancreatic duct diameter (r = 0.26, p ˂ 0.05). Regression analysis revealed FF mean (OR 1.103, p = 0.035) as an independent predictive variable of high-risk IPMN., Conclusion: FF mean is significantly associated with high-risk IPMN and an independent predictor of IPMN malignant risk. FF mean may have clinical utility as a biomarker to complement the current IPMN treatment algorithm and improve clinical decision making regarding the need for surgical resection or surveillance., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Pancreatic cysts are incidentally detected in up to 13% of patients undergoing radiographic imaging. Of the most frequently encountered types, mucin-producing (mucinous) pancreatic cystic lesions may develop into pancreatic cancer, while nonmucinous ones have little or no malignant potential. Accurate preoperative diagnosis is critical for optimal management, but has been difficult to achieve, resulting in unnecessary major surgery. Here, we aim to develop an algorithm based on biomarker risk scores to improve risk stratification., Study Design: Patients undergoing surgery and/or surveillance for a pancreatic cystic lesion, with diagnostic imaging and banked pancreatic cyst fluid, were enrolled in the study after informed consent (n = 163 surgical, 67 surveillance). Cyst fluid biomarkers with high specificity for distinguishing nonmucinous from mucinous pancreatic cysts (vascular endothelial growth factor [VEGF], glucose, carcinoembryonic antigen [CEA], amylase, cytology, and DNA mutation) were selected. Biomarker risk scores were used to design an algorithm to predict preoperative diagnosis. Performance was tested using surgical (retrospective) and surveillance (prospective) cohorts., Results: In the surgical cohort, the biomarker algorithm outperformed the preoperative clinical diagnosis in correctly predicting the final pathologic diagnosis (91% vs 73%; p < 0.000001). Specifically, nonmucinous serous cystic neoplasms (SCN) and mucinous cystic neoplasms (MCN) were correctly classified more frequently by the algorithm than clinical diagnosis (96% vs 30%; p < 0.000008 and 92% vs 69%; p = 0.04, respectively). In the surveillance cohort, the algorithm predicted a preoperative diagnosis with high confidence based on a high biomarker score and/or consistency with imaging from ≥1 follow-up visits., Conclusions: A biomarker risk score-based algorithm was able to correctly classify pancreatic cysts preoperatively. Importantly, this tool may improve initial and dynamic risk stratification, reducing overdiagnosis and underdiagnosis., (Copyright © 2021 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: In patients undergoing pancreatoduodenectomy, non-home discharge is common and often results in an unnecessary delay in hospital discharge. This study aimed to develop and validate a preoperative prediction model to identify patients with a high likelihood of non-home discharge following pancreatoduodenectomy., Methods: Patients undergoing pancreatoduodenectomy from 2013 to 2018 were identified using an institutional database. Patients were categorized according to discharge location (home vs. non-home). Preoperative risk factors, including social determinants of health associated with non-home discharge, were identified using Pearson's chi-squared test and then included in a multiple logistic regression model. A training cohort composed of 80% of the sampled patients was used to create the prediction model, and validation carried out using the remaining 20%. Statistical significance was defined as P < 0.05., Results: Seven hundred sixty-six pancreatoduodenectomy patients met the study criteria for inclusion in the analysis (non-home, 126; home, 640). Independent predictors of non-home discharge on multivariable analysis were age, marital status, mental health diagnosis, functional health status, dyspnea, and chronic obstructive pulmonary disease. The prediction model was then used to generate a nomogram to predict likelihood of non-home discharge. The training and validation cohorts demonstrated comparable performances with an identical area under the curve (0.81) and an accuracy of 84%., Conclusion: A prediction model to reliably assess the likelihood of non-home discharge after pancreatoduodenectomy was developed and validated in the present study.

Objective: Intraductal papillary mucinous neoplasms (IPMNs) are non-invasive precursor lesions that can progress to invasive pancreatic cancer and are classified as low-grade or high-grade based on the morphology of the neoplastic epithelium. We aimed to compare genetic alterations in low-grade and high-grade regions of the same IPMN in order to identify molecular alterations underlying neoplastic progression., Design: We performed multiregion whole exome sequencing on tissue samples from 17 IPMNs with both low-grade and high-grade dysplasia (76 IPMN regions, including 49 from low-grade dysplasia and 27 from high-grade dysplasia). We reconstructed the phylogeny for each case, and we assessed mutations in a novel driver gene in an independent cohort of 63 IPMN cyst fluid samples., Results: Our multiregion whole exome sequencing identified KLF4 , a previously unreported genetic driver of IPMN tumorigenesis, with hotspot mutations in one of two codons identified in >50% of the analyzed IPMNs. Mutations in KLF4 were significantly more prevalent in low-grade regions in our sequenced cases. Phylogenetic analyses of whole exome sequencing data demonstrated diverse patterns of IPMN initiation and progression. Hotspot mutations in KLF4 were also identified in an independent cohort of IPMN cyst fluid samples, again with a significantly higher prevalence in low-grade IPMNs., Conclusion: Hotspot mutations in KLF4 occur at high prevalence in IPMNs. Unique among pancreatic driver genes, KLF4 mutations are enriched in low-grade IPMNs. These data highlight distinct molecular features of low-grade and high-grade dysplasia and suggest diverse pathways to high-grade dysplasia via the IPMN pathway., Competing Interests: Competing interests: LDW receives research support from Applied Materials. VBG is an employee of Personal Genome Diagnostics. The other authors declare no conflict of interest., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Background/aim: We created a novel, preoperative wellness program (WP) that promotes recovery. This study assessed its impact on patient outcomes after pancreatectomy., Patients and Methods: Pancreatoduodenectomies (PD) and distal pancreatectomies (DP) performed from 2015 to 2018 were reviewed using our institutional NSQIP database. Patients in the WP had their medical conditions optimized and were provided with the following: chlorhexidine, topical mupirocin, incentive spirometer, and immune-nutrition supplements., Results: Out of a total of 669 pancreatectomy patients (411 PD, 258 DP), 308 were enrolled in the WP (188 PD, 120 DP). In the PD subgroup, on multivariable analysis (MVA), the WP patients had shorter lengths of hospital stay (LOS) (12 vs. 10 days, p<0.001). On MVA, WP patients had less post-op transfusion (20 vs. 10%, p=0.027). For the combined groups on MVA, LOS continued to be significant (OR=0.89, 95%CI=0.82-0.97, p<0.007)., Conclusion: A preoperative patient centered WP may reduce the length of stay.

Background & Aims: There is substantial interest in liquid biopsy approaches for cancer early detection among subjects at risk, using multi-marker panels. CA19-9 is an established circulating biomarker for pancreatic cancer; however, its relevance for pancreatic cancer early detection or for monitoring subjects at risk has not been established., Methods: CA19-9 levels were assessed in blinded sera from 175 subjects collected up to 5 years before diagnosis of pancreatic cancer and from 875 matched controls from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial. For comparison of performance, CA19-9 was assayed in blinded independent sets of samples collected at diagnosis from 129 subjects with resectable pancreatic cancer and 275 controls (100 healthy subjects; 50 with chronic pancreatitis; and 125 with noncancerous pancreatic cysts). The complementary value of 2 additional protein markers, TIMP1 and LRG1, was determined., Results: In the PLCO cohort, levels of CA19-9 increased exponentially starting at 2 years before diagnosis with sensitivities reaching 60% at 99% specificity within 0 to 6 months before diagnosis for all cases and 50% at 99% specificity for cases diagnosed with early-stage disease. Performance was comparable for distinguishing newly diagnosed cases with resectable pancreatic cancer from healthy controls (64% sensitivity at 99% specificity). Comparison of resectable pancreatic cancer cases to subjects with chronic pancreatitis yielded 46% sensitivity at 99% specificity and for subjects with noncancerous cysts, 30% sensitivity at 99% specificity. For prediagnostic cases below cutoff value for CA19-9, the combination with LRG1 and TIMP1 yielded an increment of 13.2% in sensitivity at 99% specificity (P = .031) in identifying cases diagnosed within 1 year of blood collection., Conclusion: CA19-9 can serve as an anchor marker for pancreatic cancer early detection applications., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor (VEGF)-A is known to play key biological roles in angiogenesis and vascular permeability. We previously identified VEGF-A as an accurate biomarker of benign pancreatic cystic lesions known as serous cystic neoplasms (SCN). In the present study, we seek to further characterize the expression of VEGF-A and its splice isoforms in different pancreatic cysts including SCN. Patients undergoing surgery were consented for the collection of pancreatic cystic lesion tissue (SCN, pseudocysts, mucinous cysts) and normal adjacent pancreas as well as pancreatic cyst fluid. Following RNA isolation from the tissues, relative VEGF-A gene expression was quantitatively analyzed using real-time PCR (qPCR), and VEGF-A isoform expression was evaluated by reverse transcriptase (RT)-PCR. Relative VEGF-A gene expression was significantly increased in SCN, demonstrating transcriptional upregulation in SCN compared to other pancreatic cyst tissues (P < 0.0001). VEGF-189, -165, -145, and -121 splice variants were detected in both normal adjacent pancreas and pancreatic cystic lesions; the novel VEGF-111 isoform was variably expressed in normal and cyst tissues. Finally, VEGF isoform levels in pancreatic cyst fluid were measured by isoform-specific ELISAs. VEGF-165, -145, and -121 proteins were present in pancreatic cyst fluids; VEGF-165 levels were significantly higher in SCN cyst fluid. Thus, multiple VEGF isoforms were expressed in normal pancreas and pancreatic cysts. Of particular interest are VEGF-145 and -111, which have not previously been described in human pancreas where they may exhibit unique biological activities in health and/or disease., Competing Interests: Declaration of competing interest The authors have no conflict of interests to report., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Background: Recognition of the impact of social determinants on health care and surgical outcomes is imperative to improve patient care. This study aims to examine the impact social determinants have on hospital length of stay (LOS) after pancreatoduodenectomy (PD)., Methods: Retrospective review of a prospective American College of Surgeons-National Surgical Quality Improvement Program database identified patients who underwent PD from 2013 to 2018. Patients were categorized by insurance type (public/private/multiple), and electronic medical record review was performed to obtain distance from home, marital status, and race. Public insurance included Medicare and Medicaid; multiple types were defined as public insurance supplemented by a private insurance. Univariable analysis was used to identify potential confounders. Significant differences (P < 0.05) were controlled for using multivariable regression models to examine the effect of variables on LOS., Results: About 813 PDs were included (n = 341 public; n = 238 private; and n = 234 multiple). Patients with public insurance had significantly longer LOS than patients with private on univariate (P < 0.001) and multivariable analyses (P = 0.021) (8 versus 7 d). Patients with multiple insurance types showed significantly increased LOS compared with patients with private on univariable (P < 0.001) and multivariable analyses (P = 0.006) (8 versus 7 d). Single patients had significantly longer LOS compared with married patients on univariable (P = 0.012) and multivariable analyses (P = 0.005) (8 versus 7 d). Distance from home, race, gender, or age did not have a significant impact on LOS., Conclusions: Single patients and patients with public or multiple insurance types are more likely to have longer hospital LOS after PD. These findings will enable physicians to identify patients at risk and target them for enhanced recovery programming., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: This study evaluated closure techniques and incisional surgical site complications (SSCs) and incisional surgical site infections (SSIs) after pancreaticoduodenectomy (PD)., Methods: Retrospective review of open PDs from 2015 to 2018 was performed. Outcomes were compared among closure techniques (subcuticular + topical skin adhesive (TSA); staples; subcuticular only). SSCs were defined as abscess, cellulitis, seroma, or fat necrosis. SSIs were defined according to the National Surgical Quality Improvement Program (NSQIP)., Results: Patients with subcuticular + TSA (n = 205) were less likely to develop an incisional SSC (9.8%) compared to staples (n = 139) (20.1%) and subcuticular (n = 74) (16.2%) on univariable analysis (P = 0.024). Multivariable analysis revealed no statistically significant difference in incisional SSC between subcuticular + TSA and subcuticular (P = 0.528); a significant difference remained between subcuticular + TSA and staples (P = 0.014). Unadjusted median length of stay (LOS) (days) was significantly longer for staples (9) vs. subcuticular (8) vs. subcuticular + TSA (7); P < 0.001. Incisional SSIs were evaluated separately according to the NSQIP definition. When comparing rates, the subcuticular + TSA group experienced lower incisional SSIs compared to the other two techniques (4.9% vs. 10.1%, 10.8%). However, this difference was not statistically significant by either univariable or multivariable analysis., Conclusions: Subcuticular suture + TSA reduces the risk of incisional SSCs when compared to staples alone after pancreaticoduodenectomy., Competing Interests: Declaration of competing interest None declared., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: A proteomic discovery study was performed to determine if urine possesses a unique biosignature that could form the basis for a noninvasive test able to predict intraductal papillary mucinous neoplasm (IPMN) dysplasia., Methods: Urine was collected from patients undergoing surgery for IPMN (72 low/moderate, 27 high-grade/invasive). Quantitative mass spectrometry-based proteomics was performed. Proteins of interest were identified by differential expression analysis followed by principal component analysis., Results: Proteomics identified greater than 4800 urinary proteins. Low/moderate and high-grade/invasive IPMN were distinguished by 188 proteins (P < 0.05). Following principal component analysis and heatmap visualization, vitamin D binding protein (DBP), apolipoprotein A1 (APOA1), and alpha-1 antitrypsin (A1AT) were selected. The proteomic abundance of DBP (median [interquartile range]) was significantly higher for high-grade/invasive than for low/moderate IPMN (219,735 [128,882-269,943] vs. 112,295 [77,905-180,773] normalized reporter ion intensity units; P = 0.001). Similarly, APOA1 was more abundant in the high-grade/invasive than low/moderate groups (235,420 [144,933-371,247] vs 150,095 [103,419-236,591]; P = 0.0007) as was A1AT (567,514 [358,544-774,801] vs 358,393 [260,850-477,882]; P = 0.0006)., Conclusions: Urinary DBP, APOA1, and A1AT represent potential biomarker candidates that may provide a noninvasive means of predicting IPMN dysplastic grade.

Background: Intraductal papillary mucinous neoplasms (IPMN) are precursors of pancreatic cancer. Potential biomarkers of IPMN progression have not been identified in urine. A few urinary biomarkers were reported to be predictive of pancreatic ductal adenocarcinoma (PDAC). Here, we seek to assess their ability to detect high-risk IPMN., Methods: Urine was collected from patients undergoing pancreatic resection and healthy controls. TIMP-1(Tissue Inhibitor of Metalloproteinase-1), LYVE-1(Lymphatic Vessel Endothelial Receptor 1), and PGEM(Prostaglandin E Metabolite) levels were determined by ELISA and analyzed by Kruskal-Wallis., Results: Median urinary TIMP-1 levels were significantly lower in healthy controls (n = 9; 0.32 ng/mg creatinine) compared to PDAC (n = 13; 1.95) but not significantly different between low/moderate-grade (n = 20; 0.71) and high-grade/invasive IPMN (n = 20; 1.12). No significant difference in urinary LYVE-1 was detected between IPMN low/moderate (n = 16; 0.37 ng/mg creatinine) and high/invasive grades (n = 21; 0.09). Urinary PGEM levels were not significantly different between groups., Conclusions: Urinary TIMP-1, LYVE-1, and PGEM do not correlate with malignant potential of pancreatic cysts., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: We sought to determine if interleukin (IL)-1β and prostaglandin E2 (PGE2) (inflammatory mediators in pancreatic fluid) together with serum carbohydrate antigen (CA) 19-9 could better predict intraductal papillary mucinous neoplasm (IPMN) dysplasia than individual biomarkers alone., Methods: Pancreatic cyst fluid (n = 92) collected via endoscopy or surgery (2003-2016) was analyzed for PGE2 and IL-1β (enzyme-linked immunosorbent assay). Patients had surgical pathology-proven IPMN. Threshold values (PGE2 [>1100 pg/mL], IL-1β [>20 pg/mL], and serum CA 19-9 [>36 U/mL]) were determined., Results: Levels of IL-1β were higher in high-grade dysplasia (HGD)/invasive-IPMN (n = 42) compared with low/moderate IPMN (n = 37) (median [range], 54.6 [0-2671] vs 5.9 [0-797] pg/mL; P < 0.001; area under curve [AUC], 0.766). Similarly, PGE2 was higher in HGD/invasive IPMN (n = 45) compared with low/moderate IPMN (n = 47) (median [range], 1790 [20-15,180] vs. 140 [10-14,630] pg/mL; P < 0.001; AUC, 0.748). Presence of elevated PGE2 and IL-1β (AUC, 0.789) provided 89% specificity and 82% positive predictive value (PPV) for HGD/invasive IPMN. Elevated levels of all 3 provided 100% specificity and PPV for HGD/invasive IPMN., Conclusions: Cyst fluid PGE2, IL-1β, and serum CA 19-9 in combination optimize specificity and PPV for HGD/invasive IPMN and may help build a panel of markers to predict IPMN dysplasia.

Background: For pancreatic cysts with negative cytology, Integrated Molecular Pathology (IMP) is a malignancy risk score integrating clinical criteria with pancreatic cyst fluid DNA profiling. Aside from main pancreatic duct (MPD) diameter, integrated clinical criteria are not International Consensus Guidelines High-Risk Stigmata. We predicted exclusion of clinical criteria except MPD diameter could simplify the IMP and better distinguish invasive/malignant disease., Methods: Records of >1100 patients with IPMN were reviewed retrospectively. Sensitivity, specificity, and accuracy of conventional IMP for invasive/malignant disease was compared to DNA profile including only MPD ≥10mm (IMP-10.) Invasive outcomes were invasive-IPMN/adenocarcinoma on surgical pathology, pathologic or radiographic evidence of invasive/metastatic disease during surveillance. Malignant outcomes included high grade dysplastic IPMN (HGD-IPMN)., Results: 225 patients who met study criteria underwent 283 IMP evaluations: 98 followed by surgery, 185 followed by ≥ 23 months surveillance. IMP-10 had greater specificity (90.1% vs. 73.7%) and accuracy (89.8% vs. 74.2%) for invasive disease compared to IMP in surgery + surveillance patients, but lower sensitivity (77.8% vs. 88.9%). Trends were similar in surgery patients alone and malignant outcome analyses., Conclusion: IMP-10 excludes less-reliable clinical factors resulting in greater accuracy in predicting invasive/malignant disease and fewer patients with benign disease being recommended for surgery., (Copyright © 2018 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Background: The most common type of mucinous pancreatic cyst that may progress to pancreatic cancer is intraductal papillary mucinous neoplasm (IPMN). Low-risk IPMN with low-/moderate-grade dysplasia may be safely watched, whereas high-risk IPMN with high-grade dysplasia or invasive components should undergo resection. However, there is currently no reliable means of making this distinction. We hypothesize that blood concentrations of insulin resistance biomarkers may aid in the differentiation of low- and high-risk IPMN., Methods: Plasma/serum was collected from consented patients undergoing pancreatic resection. IPMN diagnosis and dysplastic grade were confirmed by surgical pathology. The study included 235 IPMN (166 low/moderate grade, 39 high grade, 30 invasive). Circulating levels of leptin, branched chain amino acids (BCAA), and retinol-binding protein-4 (RBP-4) were measured by enzyme-linked immunoassay and correlated with surgical pathology., Results: Circulating leptin levels (mean ± SE) were significantly higher in patients with low/moderate IPMN than in high-grade/invasive IPMN (15,803 ± 1686 vs. 10,275 ± 1228 pg/ml; p = 0.0086). Leptin levels were positively correlated with BMI (r = 0.65, p < 0.0001) and were higher in females (p < 0.0001). Stratified analysis showed that mean leptin levels were significantly different between low/moderate and high/invasive IPMNs only in females (24,383 ± 2748 vs. 16,295 ± 2040 pg/ml; p = 0.020). Conversely, circulating BCAA levels were lower in low/moderate IPMN than in high-grade/invasive IPMN (0.38 ± 0.007 vs. 0.42 ± 0.01 mM; p = 0.011). No significant differences in RBP-4 levels were observed., Conclusions: Circulating leptin in females and BCAA correlates with IPMN dysplastic grade and, if combined with clinical characteristics, have the potential to improve clinical decision-making.

Background: The risk of developing invasive cancer in the remnant pancreas after resection of multifocal high-grade pancreatic precursor lesions is not well known. We report three patients who were followed up after resection of multifocal high-grade pancreatic intraepithelial neoplasia (PanIN)-3 or intraductal papillary mucinous neoplasia (IPMN), two of whom eventually developed invasive carcinoma. Presentation: 1) 68-year-old woman who had a laparoscopic distal pancreatectomy for multifocal mixed-type IPMN, identified as high-grade on final pathology, with negative surgical margins. During semiannual monitoring, eight years from the first surgery, the patient developed suspicious features prompting surgical resection of the body with final pathology revealing invasive ductal adenocarcinoma in the setting of IPMN. 2) 48-year-old woman who had a distal pancreatectomy for severe acute/chronic symptomatic pancreatitis, with final pathology revealing multifocal high-grade PanIN-3, with negative surgical margins. Despite semiannual monitoring, two years from the first surgery, the patient developed pancreatic adenocarcinoma with liver metastasis. 3) 55-year-old woman who had a Whipple procedure for symptomatic chronic pancreatitis, with multifocal PanIN-3 on final pathology. The patient underwent completion pancreatectomy due to symptomatology and her high-risk profile, with final pathology confirming multifocal PanIN-3. Conclusion: Multifocal high-grade dysplastic lesions of the pancreas might benefit from surgical resection., Competing Interests: The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements).

Background: IPMNs are cystic pancreatic lesions with variable malignant potential. Thrombospondin-2 (THBS2)-an endogenous, anti-angiogenic matrix glycoprotein-may modulate tumor progression. We hypothesized that circulating levels of THBS2 could aid in preoperative prediction of malignant IPMN., Methods: Preoperative serum/plasma samples were procured from patients undergoing surgery. Circulating levels of THBS2 were measured (enzyme-linked immunosorbent assay) and compared to surgical pathology IPMN dysplastic grade., Results: 164 patients underwent THBS2 testing (100 Low/Moderate-IPMN; 64 High-Grade/Invasive-IPMN). Circulating THBS2 (mean ± SD) was greater in High-Grade/Invasive-IPMN than Low/Moderate-grade IPMN (26.6 ± 12.7 ng/mL vs. 20.4 ± 8.2 ng/mL; P < 0.001). THBS2 (AUC = 0.65) out-performed CA19-9 (n = 144; AUC = 0.59) in predicting IPMN grade. The combination of THBS2, CA19-9, radiographic main-duct involvement, main-duct diameter, age, sex, and BMI (AUC 0.82; n = 137) provided a good prediction model for IPMN grade., Conclusion: Circulating THBS2 is correlated with IPMN dysplasia grade. THBS2 alone did not strongly predict IPMN grade but rather strengthened prediction models for High-Grade/Invasive IPMN when combined with other clinical/biomarker data., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Predicting malignancy in intraductal papillary mucinous neoplasm remains challenging. Integrated molecular pathology combines pancreatic fluid DNA and clinical factors into a malignant potential score. We sought to determine the utility of DNA components alone in predicting high-grade dysplasia/invasive disease., Methods: We reviewed prospectively the records from 1,106 patients with intraductal papillary mucinous neoplasm. We excluded non-intraductal papillary mucinous neoplasm cases and cases with definitive malignant cytology. A total 225 patients had 283 DNA profiles (98 followed by surgery, 185 followed by ≥23-month surveillance). High-grade dysplasia/invasive outcomes were high-grade dysplasia, intraductal papillary mucinous neoplasm-invasive, and adenocarcinoma on surgical pathology or mesenteric or vascular invasion, metastases, or biopsy with high-grade dysplasia or adenocarcinoma during surveillance., Results: High-quantity DNA predicted (P = .004) high-grade dysplasia/invasive disease outcomes with sensitivity of 78.3%, but 52.7% specificity, indicating benign cases may exhibit high-quantity DNA. High clonality loss of heterozygosity of tumor suppressor genes was 98.0% specific, strongly predicted high-grade dysplasia/invasive disease but lacked sensitivity (20.0%). High-quantity DNA + high clonality loss of heterozygosity had 99.0% specificity for high-grade dysplasia/invasive disease. KRAS mutation alone did not predict high-grade dysplasia/invasive disease, but, when combined with high-quantity DNA (specificity 84.7%) and high clonality loss of heterozygosity (specificity 99.0%) strongly predicted high-grade dysplasia/invasive outcomes., Conclusion: Certain DNA components are highly specific for high-grade dysplasia/invasive disease and may indicate aggressive lesions, requiring resection when cytology fails., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The most widely accepted biochemical test for preoperative differentiation of mucinous from benign, nonmucinous pancreatic cysts is cyst fluid carcinoembryonic antigen. However, the diagnostic accuracy of carcinoembryonic antigen ranges from 70% to 86%. Based on previous work, we hypothesize that pancreatic cyst fluid glucose may be an attractive alternative to carcinoembryonic antigen., Methods: Pancreatic cyst fluid was collected during endoscopic or operative intervention. Diagnoses were pathologically confirmed. Glucose and carcinoembryonic antigen were measured using a patient glucometer and automated analyzer/enzyme-linked immunosorbent assay. Sensitivity, specificity, accuracy, and receiver operator characteristic analyses were performed., Results: Cyst fluid samples from 153 patients were evaluated (mucinous: 25 mucinous cystic neoplasms, 77 intraductal papillary mucinous neoplasms, 4 ductal adenocarcinomas; nonmucinous: 21 serous cystic neoplasms, 9 cystic neuroendocrine tumors, 14 pseudocysts, 3 solid pseudopapillary neoplasms). Median cyst fluid glucose was lower in mucinous versus nonmucinous cysts (19 vs 96 mg/dL; P < .0001). With a threshold of ≤ 50 mg/dL, cyst fluid glucose was 92% sensitive, 87% specific, and 90% accurate in diagnosing mucinous pancreatic cysts. In comparison, cyst fluid carcinoembryonic antigen with a threshold of >192 ng/mL was 58% sensitive, 96% specific, and 69% accurate. Area under the curve for glucose and CEA were similar at 0.91 and 0.92., Conclusion: Cyst fluid glucose has significant advantages over carcinoembryonic antigen and should be considered for use as a routine diagnostic test for pancreatic mucinous cysts., (Copyright © 2017. Published by Elsevier Inc.)

Background: With the increased frequency of diagnostic imaging, pancreatic cysts are now detected in >3% of American adults. Most of these are intraductal papillary mucinous neoplasms (IPMNs) with well-established but variable malignant potential. A biomarker that predicts malignant potential or dysplastic grade would help determine which IPMNs require removal and which can be observed safely. We previously reported that pancreatic fluid prostaglandin E 2 (PGE 2 ) levels might have promise as a predictor of IPMN dysplasia and we seek to validate those results in the current study., Study Design: Pancreatic cyst/duct fluid was prospectively collected from 100 patients with IPMN undergoing pancreatic resection. Surgical pathology revealed 47 low-/moderate-grade, 34 high-grade, and 20 invasive IPMNs. The PGE 2 levels were assessed by ELISA and correlated with IPMN dysplasia grade, demographics, clinical radiologic/pathologic variables, acute/chronic pancreatitis, and NSAID use., Results: Mean pancreatic cyst fluid PGE 2 levels in high-grade and invasive IPMNs were significantly higher than low-/moderate-grade IPMNs (3.5 and 4.4 pg/μL, respectively, vs 1.2 pg/μL; p < 0.0016). At a threshold of 1.1 pg/μL, PGE 2 was 63% sensitive, 79% specific, and 71% accurate for detection of high-grade/invasive IPMNs. When tested in the subset of IPMN patients with preoperative pancreatic cyst fluid CEA >192 ng/mL, PGE 2 at a threshold of 0.5 pg/μL demonstrated 78% sensitivity, 100% specificity, and 86% accuracy for detection of high-grade/invasive IPMN., Conclusions: Our results validate pancreatic cyst fluid PGE 2 as an indicator of IPMN dysplasia, especially in select patients with preoperative pancreatic cyst fluid CEA >192 ng/mL. The inclusion of PGE 2 /CEA in a diagnostic biomarker panel can facilitate more optimal treatment stratification of IPMN patients., (Copyright © 2017 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

The earlier diagnosis of cancer is one of the keys to reducing cancer deaths in the future. Here we describe our efforts to develop a noninvasive blood test for the detection of pancreatic ductal adenocarcinoma. We combined blood tests for KRAS gene mutations with carefully thresholded protein biomarkers to determine whether the combination of these markers was superior to any single marker. The cohort tested included 221 patients with resectable pancreatic ductal adenocarcinomas and 182 control patients without known cancer. KRAS mutations were detected in the plasma of 66 patients (30%), and every mutation found in the plasma was identical to that subsequently found in the patient's primary tumor (100% concordance). The use of KRAS in conjunction with four thresholded protein biomarkers increased the sensitivity to 64%. Only one of the 182 plasma samples from the control cohort was positive for any of the DNA or protein biomarkers (99.5% specificity). This combinatorial approach may prove useful for the earlier detection of many cancer types., Competing Interests: Conflict of interest statement: C.M.S. and M.T.Y.-S. are founders and coowners of B9, Inc. and have no conflicts of interest with respect to the new technology described in this article. N.P., K.W.K., and B.V. are founders of Personal Genome Diagnostics, Inc. and PapGene, Inc. K.W.K. and B.V. are members of the Scientific Advisory Board of Sysmex-Inostics and Morphotek. B.V. is also a member of the Scientific Advisory Board of Exelixis GP. These companies and others have licensed technologies from Johns Hopkins, including those related to early diagnostics. N.P., K.W.K., and B.V. are the inventors of some of these technologies and receive equity or royalties from their licenses. The terms of these arrangements are being managed by the university in accordance with its conflict of interest policies. N.P., K.W.K., and B.V. have no conflicts of interest with respect to the new technology described in this article, as defined by the Johns Hopkins University policy on conflict of interest.

Purpose: Pancreatic cysts are estimated to be present in 2%-3% of the adult population. Unfortunately, current diagnostics do not accurately distinguish benign cysts from those that can progress into invasive cancer. Misregulated pericellular proteolysis is a hallmark of malignancy, and therefore, we used a global approach to discover protease activities that differentiate benign nonmucinous cysts from premalignant mucinous cysts. Experimental Design: We employed an unbiased and global protease profiling approach to discover protease activities in 23 cyst fluid samples. The distinguishing activities of select proteases was confirmed in 110 samples using specific fluorogenic substrates and required less than 5 μL of cyst fluid. Results: We determined that the activities of the aspartyl proteases gastricsin and cathepsin E are highly increased in fluid from mucinous cysts. IHC analysis revealed that gastricsin expression was associated with regions of low-grade dysplasia, whereas cathepsin E expression was independent of dysplasia grade. Gastricsin activity differentiated mucinous from nonmucinous cysts with a specificity of 100% and a sensitivity of 93%, whereas cathepsin E activity was 92% specific and 70% sensitive. Gastricsin significantly outperformed the most widely used molecular biomarker, carcinoembryonic antigen (CEA), which demonstrated 94% specificity and 65% sensitivity. Combined analysis of gastricsin and CEA resulted in a near perfect classifier with 100% specificity and 98% sensitivity. Conclusions: Quantitation of gastricsin and cathepsin E activities accurately distinguished mucinous from nonmucinous pancreatic cysts and has the potential to replace current diagnostics for analysis of these highly prevalent lesions. Clin Cancer Res; 23(16); 4865-74. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background: Accurate differentiation of pancreatic cystic lesions is important for pancreatic cancer early detection and prevention as well as avoidance of unnecessary surgical intervention. Serous cystic neoplasms (SCN) have no malignant potential, but may mimic premalignant mucinous cystic lesions: mucinous cystic neoplasm (MCN) and intraductal papillary mucinous neoplasm (IPMN). We recently identified vascular endothelial growth factor (VEGF)-A as a novel pancreatic fluid biomarker for SCN. We hypothesize that combining cyst fluid carcinoembryonic antigen (CEA) with VEGF-A will improve the diagnostic accuracy of VEGF-A., Methods: Pancreatic cyst/duct fluid was collected from consenting patients undergoing surgical cyst resection with corresponding pathologic diagnoses. Pancreatic fluid VEGF-A and CEA levels were detected by ELISA., Results: One hundred forty-nine patients with pancreatic cystic lesions met inclusion criteria. Pathologic diagnoses included pseudocyst (n=14), SCN (n=26), MCN (n=40), low/moderate grade IPMN (n=34), high grade IPMN (n=20), invasive IPMN (n=10) and solid pseudopapillary neoplasm (n=5). VEGF-A was significantly elevated in SCN cyst fluid compared to all other diagnoses (p<0.001). With a threshold of >5,000 pg/ml, VEGF-A alone has 100% sensitivity and 83.7% specificity to distinguish SCN from other cystic lesions. With a threshold of ≤10ng/ml, CEA alone identifies SCN with 95.5% sensitivity and 81.5% specificity. Sensitivity and specificity of the VEGF-A/CEA combination are 95.5% and 100% respectively. The c-statistic increased from 0.98 to 0.99 when CEA was added to VEGF-A alone in the ROC analysis., Conclusions: Although VEGF-A alone is a highly accurate test for SCN, the combination of VEGF-A with CEA approaches the gold-standard of pathologic diagnosis, thus importantly avoiding false positives. Patients with a positive test indicating benign SCN can be spared a high risk surgical pancreatic resection., (Copyright © 2017. Published by Elsevier Inc.)

A systematic study to identify the factors influencing the cytotoxicity of α-methylene-γ-hydroxy esters against three pancreatic cancer cell lines (Panc-1, MIA-PaCa-2, and BxPC-3) has established that, in addition to Michael acceptor abilities, the possibility to lactonize to α-methylene-γ-butyrolactones is as important. The substitution pattern and the number of carbons between the hydroxy and ester moieties also influence the bio-activity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Mucinous pancreatic cysts (intraductal papillary mucinous neoplasm and mucinous cystic neoplasm) have the potential to progress to invasive pancreatic adenocarcinoma, presenting an opportunity for early detection, prevention, and cure. Serous cystic neoplasms (SCN) have no malignant potential, but can mimic mucinous pancreatic cysts on imaging. Therefore, identification of biomarkers that can distinguish between cystic lesions is critically important. We hypothesize that vascular endothelial growth factor (VEGF)-A levels in pancreatic fluid correlate with pathologic diagnosis., Study Design: Pancreatic cyst/duct fluid samples were prospectively collected from patients undergoing pancreatic resection and correlated with surgical pathology. VEGF levels were detected by ELISA. VEGF-A and VEGF receptor 2 expression in pancreatic tissue was localized by immunohistochemistry. Genetic alterations of the von Hippel-Lindau gene were determined by targeted next-generation sequencing., Results: Eighty-seven patients met inclusion criteria for enrollment. Final pathologic diagnoses included pseudocyst (n = 9), SCN (n = 17), mucinous cystic neoplasm (n = 24), low/moderate grade intraductal papillary mucinous neoplasm (n = 16), high-grade/invasive intraductal papillary mucinous neoplasm (n = 10), and pancreatic ductal adenocarcinoma (n = 11). VEGF-A was significantly upregulated in SCN cyst fluid compared with all other diagnoses (p < 0.0001). With a cut-off of 8,500 pg/mL, VEGF-A has 100% sensitivity and 97% specificity as an SCN biomarker. VEGF-A and VEGF receptor 2 are overexpressed in SCN cyst tissue. VEGF-C was also significantly elevated in SCN cyst fluid (p < 0.0001). With a cut-off set at 200 pg/mL, VEGF-C identifies SCN with 100% sensitivity and 90% specificity. The presence of a von Hippel-Lindau mutation in SCN cyst tissue correlates with elevated cyst fluid VEGF levels., Conclusions: This is the first report of a cyst fluid protein biomarker that can positively identify SCN. The ability to distinguish SCN from premalignant/malignant pancreatic cysts can spare the cost and risk of surveillance and surgical intervention in select patients., (Copyright © 2014 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Alcohol consumption promotes hepatocellular carcinoma (HCC). The responsible mechanisms are not well understood. Hepatocarcinogenesis increases with age and is enhanced by factors that impose a demand for liver regeneration. Because alcohol is hepatotoxic, habitual alcohol ingestion evokes a recurrent demand for hepatic regeneration. The alcohol-preferring (P) rat model mimics the level of alcohol consumption by humans who habitually abuse alcohol. Previously, we showed that habitual heavy alcohol ingestion amplified age-related hepatocarcinogenesis in P rats, with over 80% of alcohol-consuming P rats developing HCCs after 18 months of alcohol exposure, compared with only 5% of water-drinking controls., Methods: Herein, we used quantitative real-time PCR and quantitative immunocytochemistry to compare liver tissues from alcohol-consuming P rats and water-fed P rat controls after 6, 12, or 18 months of drinking. We aimed to identify potential mechanisms that might underlie the differences in liver cancer formation and hypothesized that chronic alcohol ingestion would activate Hedgehog (HH), a regenerative signaling pathway that is overactivated in HCC., Results: Chronic alcohol ingestion amplified age-related degenerative changes in hepatocytes, but did not cause appreciable liver inflammation or fibrosis even after 18 months of heavy drinking. HH signaling was also enhanced by alcohol exposure, as evidenced by increased levels of mRNAs encoding HH ligands, HH-regulated transcription factors, and HH target genes. Immunocytochemistry confirmed increased alcohol-related accumulation of HH ligand-producing cells and HH-responsive target cells. HH-related regenerative responses were also induced in alcohol-exposed rats. Three of these processes (i.e., deregulated progenitor expansion, the reverse Warburg effect, and epithelial-to-mesenchymal transitions) are known to promote cancer growth in other tissues., Conclusions: Alcohol-related changes in Hedgehog signaling and resultant deregulation of liver cell replacement might promote hepatocarcinogenesis., (Copyright © 2013 by the Research Society on Alcoholism.)

Aminated α-methylene-γ-butyrolactones, which are readily synthesized with facile control of the diastereoisomerism, provide an economical and commercially-viable alternative to the use of aminated natural products. These aminoloactones, which exhibit excellent activity against three pancreatic cancer cell lines when measured at 10 μM-Panc-1, MIA PaCa-2, and BxPC-3-and are comparable to or better than parthenolide and dimethylaminoparthenolide (DMAPT, LC-1). It has also been shown that there is an effect on the biological activity depending on the identity of the amine., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Pancreatic cancer remains one of the deadliest cancers due to lack of early detection and absence of effective treatments. Gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer, has limited clinical benefit. Treatment of pancreatic cancer cells with gemcitabine has been shown to induce the activity of the transcription factor nuclear factor-kappaB (NF-κB) which regulates the expression of genes involved in the inflammatory response and tumorigenesis. It has therefore been proposed that gemcitabine-induced NF-κB activation may result in chemoresistance. We hypothesize that NF-κB suppression by the novel inhibitor dimethylaminoparthenolide (DMAPT) may enhance the effect of gemcitabine in pancreatic cancer., Methods: The efficacy of DMAPT and gemcitabine was evaluated in a chemoprevention trial using the mutant Kras and p53-expressing LSL-KrasG12D/+; LSL-Trp53R172H; Pdx-1-Cre mouse model of pancreatic cancer. Mice were randomized to treatment groups (placebo, DMAPT [40 mg/kg/day], gemcitabine [50 mg/kg twice weekly], and the combination DMAPT/gemcitabine). Treatment was continued until mice showed signs of ill health at which time they were sacrificed. Plasma cytokine levels were determined using a Bio-Plex immunoassay. Statistical tests used included log-rank test, ANOVA with Dunnett's post-test, Student's t-test, and Fisher exact test., Results: Gemcitabine or the combination DMAPT/gemcitabine significantly increased median survival and decreased the incidence and multiplicity of pancreatic adenocarcinomas. The DMAPT/gemcitabine combination also significantly decreased tumor size and the incidence of metastasis to the liver. No significant differences in the percentages of normal pancreatic ducts or premalignant pancreatic lesions were observed between the treatment groups. Pancreata in which no tumors formed were analyzed to determine the extent of pre-neoplasia; mostly normal ducts or low grade pancreatic lesions were observed, suggesting prevention of higher grade lesions in these animals. While gemcitabine treatment increased the levels of the inflammatory cytokines interleukin 1α (IL-1α), IL-1β, and IL-17 in mouse plasma, DMAPT and DMAPT/gemcitabine reduced the levels of the inflammatory cytokines IL-12p40, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 beta (MIP-1β), eotaxin, and tumor necrosis factor-alpha (TNF-α), all of which are NF-κB target genes., Conclusion: In summary, these findings provide preclinical evidence supporting further evaluation of agents such as DMAPT and gemcitabine for the prevention and treatment of pancreatic cancer.

Background: The Michael acceptor scaffolding is a source of rich biological activity for α-methylene-γ-butyrolactones and their derivatives. A wide variety of these structures are present in many natural products that are well-known for their useful medicinal properties., Results: The first example of a borylated α-methylene-γ-butyrolactone is presented herein, along with its antipancreatic cancer activities against Panc-1, MIA PaCa-2 and BXPC-3. The synthetic route chosen allows for a wide range of lactones to be synthesized through different cross-coupling reactions starting from arylic bromide precursors. The precursors were synthesized by way of a highly efficient, chemoselective and indium-promoted Barbier reaction. Specifically, the indium metal reacted with only one of two present bromide functionalities: an allylic bromide in the presence of an arylic one. The bromide precursors were also tested for activity in the same bioassay as the borylated lactone and parthenolide., Conclusion: Notably, these brominated compounds demonstrate a significantly higher level of activity than parthenolide.

Objectives: Pancreatic cancer remains one of the deadliest diseases, with limited surgical and treatment options. Two targets of interest include the transcription factor nuclear factor-κB and cyclooxygenase-2, which are constitutively activated and overexpressed, respectively, in human pancreatic adenocarcinoma. We have previously shown that dimethylaminoparthenolide (DMAPT), a bioavailable nuclear factor-κB inhibitor, and the cyclooxygenase inhibitors sulindac and celecoxib have potential chemotherapeutic efficacy. The current study evaluates the efficacy of intervention with DMAPT and sulindac in the LSL-Kras(G12D);Pdx-1-Cre genetically engineered mouse model. Gemcitabine, traditionally a chemotherapeutic agent, has relatively low toxicity; thus, combinations with low-dose gemcitabine were also explored., Methods: LSL-Kras(G12D);Pdx-1-Cre mice at 7 months of age were randomized into placebo, DMAPT (40 mg/kg per day), sulindac (20 mg/kg per day), gemcitabine (50 mg/kg twice weekly), and combination treatment groups. After 3 months of treatment, the mice were killed., Results: The percentage of normal pancreatic ducts was significantly increased by the combinations of DMAPT/sulindac, DMAPT/gemcitabine, sulindac/gemcitabine, and DMAPT/sulindac/gemcitabine compared to placebo. Additionally, the percentage of mouse pancreatic intraepithelial neoplasia-2 lesions was significantly decreased by DMAPT/gemcitabine., Conclusions: Intervention with DMAPT and sulindac in combination with gemcitabine may delay or prevent progression of premalignant pancreatic lesions in the LSL-Kras(G12D);Pdx-1-Cre mouse model of pancreatic cancer.

Introduction: Gemcitabine is standard treatment for pancreatic cancer but has limited clinical benefit due to chemoresistance. Nuclear factor-kappaB (NF-κB) can promote chemoresistance and is therefore an attractive therapeutic target. We hypothesize that NF-κB suppression with the novel, orally bioavailable inhibitor dimethylamino parthenolide (DMAPT) will sensitize pancreatic cancer cells to gemcitabine., Methods: BxPC-3, PANC-1, and MIA PaCa-2 human pancreatic cancer cell lines were treated with gemcitabine and/or DMAPT. Effects on the NF-κB pathway were determined by electrophoretic mobility shift assay, ELISA, or Western blot. Proliferation and apoptosis were measured by cell counts and ELISA, respectively. The effect of gemcitabine in vivo was determined using a MIA PaCa-2 heterotopic xenograft model., Results: Gemcitabine induced NF-κB activity in BxPC-3, PANC-1, and MIA PaCa-2 cells and decreased the level of the NF-κB inhibitor IκBα in BxPC-3 and PANC-1 cells. DMAPT prevented the gemcitabine-induced activation of NF-κB. The combination of DMAPT/gemcitabine inhibited pancreatic cancer cell growth more than either agent alone. Gemcitabine also induced intratumoral NF-κB activity in vivo., Conclusions: DMAPT enhanced the anti-proliferative effects of gemcitabine in association with NF-κB suppression in pancreatic cancer cells in vitro. Furthermore, gemcitabine induced NF-κB activity in vivo, thus supporting the evaluation of NF-κB-targeted agents to complement gemcitabine-based therapies.

Objectives: The aims of this study were to characterize the proteome of normal pancreatic juice, to analyze the effect of secretin on the normal proteome, and to compare these results with published data from patients with pancreatic cancer., Methods: Paired pancreatic fluid specimens (before and after intravenous secretin stimulation) were obtained during endoscopic pancreatography from 3 patients without significant pancreatic pathology. Proteins were identified and quantified by mass spectrometry-based protein quantification technology. The human RefSeq (NCBI) database was used to compare the data in samples from patients without pancreatic disease with published data from 3 patients with pancreatic cancer., Results: A total of 285 proteins were identified in normal pancreatic juice. Ninety had sufficient amino acid sequences identified to characterize the protein with a high level of confidence. All 90 proteins were present before and after secretin administration but with altered relative concentrations, usually by 1 to 2 folds, after stimulation. Comparison with 170 published pancreatic cancer proteins yielded an overlap of only 42 proteins., Conclusions: Normal pancreatic juice contains multiple proteins related to many biological processes. Secretin alters the concentration but not the spectrum of these proteins. The pancreatic juice proteome of patients without pancreatic disease and that of patients with pancreatic cancer differ markedly.

Background: Alcohol is a significant risk factor for the development of hepatocellular carcinoma (HCC). To date, no rodent model has demonstrated the formation of hepatic neoplasia in the setting of chronic alcohol consumption alone., Methods: We investigated whether rats selectively bred for high alcohol preference (P rats), allowed free access to water, or water and 10% (v/v) alcohol, for 6, 12, or 18 months, develop hepatic neoplasia., Results: At necropsy, liver tumor incidence and multiplicity were significantly increased in 18-month alcohol-consuming versus water-consuming P rats. These data were confirmed histologically by glutathione-S-transferase pi-class (GSTp) staining. Phosphorylated mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK) staining was also increased in the sinusoidal lining cells within livers of alcohol-consuming versus water only P rats. In addition, cytochrome p450IIE1 (CYP2E1) mRNA, protein expression/activity, and intrahepatic oxidative stress were significantly increased in alcohol-consuming P rat livers versus water only. In contrast, acetaldehyde dehydrogenase expression decreased in alcohol-consuming versus water only P rats. No significant difference in alcohol dehydrogenase expression was detected., Conclusions: These data demonstrate that chronic alcohol consumption is associated with hepatic neoplasia, MAPK/ERK activation, increased CYP2E1 activity, and intrahepatic oxidative stress in P rats. As these rats are well characterized as a model of alcoholism, these findings identify a novel rodent model of alcohol or "alcoholism"-induced liver neoplasia., (Copyright © 2011 by the Research Society on Alcoholism.)

Unlabelled: To determine if the level of transforming growth factor α (TGF-α) in the pancreatic fluid (PF) can diagnose intraductal papillary mucinous neoplasm (IPMN) versus other cystic lesions of the pancreas in patients., Methods: Pancreatic fluid was prospectively obtained from patients during routine endoscopy and/or operation at Indiana University Hospital. Pancreatic fluid TGF-α levels were analyzed by enzyme-linked immunosorbent assay. Intraductal papillary mucinous neoplasm tissue was also analyzed by TGF-α immunohistochemistry., Results: Sixty-nine fluid samples from 58 patients with the following pathologically confirmed pancreatic disorders were analyzed: IPMN (26 patients), serous cystadenoma (6), mucinous cystic neoplasm (9), pseudocysts (5), non-IPMNY associated pancreatic ductal adenocarcinoma (6), and sphincter of Oddi dysfunction (6). There was no significant difference between the mean PF-TGF-α levels in each category or between different dysplastic grades of IPMN. However, of all the diagnoses examined, only IPMN demonstrated PF-TGF-α levels greater than 95 pg/mL. In low-grade IPMN specimens, TGF-α immunohistochemistry correlated with enzyme-linked immunosorbent assay levels., Conclusions: The mean PF-TGF-α levels are not significantly different in IPMN lesions compared with those in other cystic pancreatic lesions, pancreatic ductal adenocarcinoma, or sphincter of Oddi dysfunction. However, PF-TGF-α levels more than 95 pg/mL may be useful in diagnosing IPMN. This assertion requires prospective validation.

A selected series of racemic α-methylene-γ-butyrolactones (AMGBL) were synthesized via allylboration and screened against three human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3). This systematic study established a discernible relationship between the substitution pattern of AMGBL and their anti-proliferative activity. β,γ-diaryl-AMGBLs, particularly those with a trans-relationship exhibited higher potency than parthenolide and LC-1 against all three cell lines., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Hepatocellular carcinoma (HCC) is a common cause of death from solid organ malignancy worldwide. Extracellular signal-regulated/mitogen-activated protein kinase kinase (MEK) signaling is a critical growth regulatory pathway in HCC. Targeting MEK with a novel small molecule inhibitor, PD0325901, may inhibit HCC tumorigenesis. PD0325901 (0.01-100 nM) inhibited growth and MEK activity in vitro in immortalized murine transforming growth factor alpha (TGF-alpha) transgenic hepatocyte (TAMH) cells, derived from the livers of TGF-alpha transgenic mice. Treatment of athymic mice bearing TAMH flank tumors with vehicle or PD0325901 (20 mg/kg) revealed a significant reduction of MEK activity ex vivo 24 hours after a single PD0325901 dose. The growth rate of TAMH flank tumors over 16 days was reduced threefold in the treatment arm (1113 +/- 269% versus 3077 +/- 483%, P < 0.01). PD0325901 exhibited similar inhibitory effects in HepG2 and Hep3B human HCC cells in vitro and in Hep3B flank tumors in vivo. To confirm this in a developmental model, MT-42 (CD-1) TGF-alpha mice were treated with vehicle or PD0325901 (20 mg/kg) for 5 weeks. Gross HCC was detected in 47% and 13.3% of the control and treatment mice, respectively. Tumor growth suppression by PD0325901 relative to vehicle was also shown by magnetic resonance imaging. These studies provide compelling preclinical evidence that targeting MEK in human clinical trials may be promising for the treatment of HCC.

Background: Intrahepatic cholangiocarcinoma (ICC) incidence and mortality are increasing in the United States and worldwide. ICC etiologies involve chronic inflammation. We hypothesize that the nonsteroidal anti-inflammatory agent sulindac may prevent ICC by targeting cyclooxygenase-1 and -2 (COX-1, -2) as well as COX-independent pathways., Materials and Methods: ICC was induced with the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) in Syrian golden hamsters. Cholangiocarcinogenesis was accelerated by a choline-deficient diet and administration of DL-ethionine and L-methionine. Hamsters were gavaged twice daily for 10 wk with vehicle or sulindac 25, 50, or 75 mg/kg/dose. Harvested livers underwent gross and histopathological examinations. Tissues were analyzed by immunostaining, Western blot, and enzyme-linked immunosorbent assay (ELISA)., Results: ICC incidence and multiplicity were decreased in sulindac treatment groups versus control (P < 0.05). In addition, ICC and nontumor lesion sizes decreased in treatment versus control animals. Proliferative indices (Ki-67 immunostaining) decreased and apoptosis (ApopTag immunostaining) increased in treatment versus control (P < 0.05). No changes in COX-1 and -2 protein levels were detected by Western blot. Furthermore, prostaglandin E(2) (PGE(2)) levels were unchanged in treatment and control serum and liver tissues (P > 0.05), suggesting that the antitumor effects of sulindac are mediated by COX-independent mechanisms. Nuclear p65 (activated NF-kappaB) immunostaining decreased (P < 0.05), and protein levels of the NF-kB inhibitor IkappaB-alpha increased in treatment versus control groups. p65 ELISA of liver extracts confirmed decreased NF-kappaB binding activity in sulindac-treated versus control animals (P < 0.05)., Conclusion: Sulindac effectively prevents experimental cholangiocarcinogenesis, in part by inhibiting the NF-kappaB pathway.

Background: Chronic ethanol intake is a significant risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). The effects of ethanol on extracellular signal-regulated kinase (ERK) activation, transforming growth factor alpha (TGF-alpha), and HCC growth were examined in this study., Methods: HepG2, SKHep, Hep3B human HCC cells, or normal human hepatocytes were treated with ethanol (0-100 mM), exogenous TGF-alpha, TGF-alpha neutralization antibody or the MEK inhibitor U0126. TGF-alpha levels were quantified by ELISA. Growth was determined by trypan blue-excluded cell counts. Cell cycle phase distribution was determined by flow cytometry. Protein expression was determined by Western blot., Results: Ethanol treatment (10-40 mM) increased ERK activation in HepG2 and SKHep HCC cells but not in Hep3B or human hepatocyte cells. Growth increased in HepG2 (174 +/- 29%, P < 0.05) and SKHep (149 +/- 12%, P < 0.05) cells in response to ethanol treatment. Correspondingly, ethanol increased S phase distribution in these cells. U0126 suppressed ethanol-induced growth increases. Ethanol treatment for 24 h also raised TGF-alpha levels in HepG2 cells (118%-198%) and SKHep cells (112%-177%). Exogenous administration of recombinant TGF-alpha mimicked the ethanol-induced growth in HepG2 and SKHep cells; TGF-alpha neutralization antibody effectively abrogated this effect. The TGF-a neutralization antibody also prevented ERK activation by ethanol in HepG2 cells., Conclusions: These data demonstrate that clinically relevant doses of ethanol stimulate ERK-dependent proliferation of HCC cells. Ethanol up-regulates TGF-alpha levels in HCC cells and enhances growth through cell cycles changes, which appear to be mediated through TGF-alpha-MEK-ERK signaling. Ethanol-MEK signaling in normal hepatocytes is absent, suggesting that ethanol promotion of HCC growth may in part depend upon the acquisition of cancer-specific signaling by hepatocytes.

The extracellular signal-regulated (ERK), mitogen-activated protein kinase (p42/p44 MAPK) pathway is up-regulated in hepatocellular carcinoma (HCC). Molecular targeting of this critical mitogenic pathway may have therapeutic potential for the treatment of HCC; however, chemoresistance to long-term therapy may develop. In the present study, we employed small-molecule MAPK kinase (MEK) inhibitors, including U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene] and PD184161 (Neoplasia 8:1-8, 2006), in HepG2 and Hep3B human HCC cell lines to identify potential mechanism(s) of resistance. U0126 dose-dependently suppressed ERK phosphorylation at both 1- and 24-h time points in HepG2 cells, previously shown to be sensitive to growth inhibition by U0126. In contrast, ERK phosphorylation was only decreased at the 1-h time point but not at 24 h in the more resistant Hep3B cells. It is interesting that the lack of prolonged phospho-ERK suppression was associated with MEK hyperphosphorylation in Hep3B cells. Several MEK/ERK pathway intermediates were up-regulated in Hep3B cells; furthermore, transfection of Raf-1 small interfering RNA to suppress MEK/ERK pathway activation sensitized Hep3B cells to U0126. MEK inhibitor resistance was independent of p53 or hepatitis Bx protein status. Finally, we showed that combining two chemically distinct MEK inhibitors enhanced growth inhibition and apoptosis compared with the single agents. Taken together, these results suggest that up-regulated expression or activity of the MEK/ERK pathway contributes to MEK inhibitor resistance in HCC cells. Our findings also provide preclinical evidence suggesting that the status of the MEK/ERK pathway in patients may predict response to MEK/ERK-targeted therapeutics.

Objectives: Cancer of the exocrine pancreas is the fourth leading cause of cancer-related deaths in the United States. The efficacy of a novel bioavailable anticancer agent, dimethylamino-parthenolide (DMAPT), and the cyclooxygenase 2 inhibitor, celecoxib, was evaluated in a carcinogen-induced developmental model of pancreatic cancer., Methods: Syrian golden hamsters were injected with N-nitrosobis(2-oxopropyl)amine, once weekly for 6 weeks. Upon the first injection, hamsters were randomized as follows: placebo, low-/high-dose DMAPT (20 and 40 mg/kg per day), low-/high-dose celecoxib (10and 50 mg/kg per day), or combination DMAPT/celecoxib (low/low, high/high)., Results: The 32-week trial showed that 40 mg/kg DMAPT alone significantly decreased the size of gross pancreatic cancers relative to placebo. No significant difference in gross tumor number was observed between the treatment groups and placebo with the exception of 50 mg/kg celecoxib with a higher tumor incidence; this group also exhibited lower lymphotactin levels suggestive of decreased immune surveillance. Tumor invasion into adjacent organs and metastasis were not observed in the DMAPT/celecoxib treatment groups. Drug targets including prostaglandin E2, prostaglandin E2 metabolite and activated nuclear factor kappaB were significantly decreased., Conclusions: Dimethylamino-parthenolide and celecoxib have the potential to be novel chemotherapeutic agents for pancreatic cancer; however, further optimization or the use of other modalities may be required for chemoprevention.

Current management of intraductal papillary mucinous neoplasm (IPMN) according to recently published International Consensus Guidelines depends upon distinguishing it from mucinous cystic neoplasms (MCNs). We have previously shown that prostaglandin E(2) (PGE(2)) is increased in pancreatic cancer tissue over normal controls. Thus, we hypothesized that PGE(2) level in pancreatic fluid differentiates IPMN and MCN and is a biomarker of IPMN dysplasia. Pancreatic fluid was collected in 65 patients at the time of endoscopy (EUS or ERCP) or operation (OR) and analyzed by PGE(2) enzyme-linked immunosorbent assay (ELISA). PGE(2) level was correlated with surgical pathologic diagnosis and dysplastic stage. Mean PGE(2) level (pg/microl) in IPMNs (2.2 +/- 0.6) was greater than in MCNs (0.2 +/- 0.1) (p < 0.05). Mean PGE(2) level of IPMN by dysplastic stage was 0.1 +/- 0.01 (low grade), 1.2 +/- 0.6 (medium grade), 4.4 +/- 0.9 (high grade), and 5.0 +/- 2.3 (invasive). Among invasive IPMN, PGE(2) level dropped in advanced cases with pancreatic ductal obstruction by tumor (0.3 +/- 0) vs non-obstructed (8.6 +/- 2.9). PGE(2) level may help in distinguishing IPMN from MCN in patients with known mucinous lesions. PGE(2) level may also be an indicator of malignant progression of IPMN before ductal obstruction by tumor. Prospective evaluation will be necessary to evaluate the clinical role of PGE(2) level in pancreatic fluid.