Amplification-Free, High-Throughput Nanoplasmonic Quantification of Circulating MicroRNAs in Unprocessed Plasma Microsamples for Earlier Pancreatic Cancer Detection.

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.

Biomarker Risk Score Algorithm and Preoperative Stratification of Patients with Pancreatic Cystic Lesions.

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.

Novel expression of vascular endothelial growth factor isoforms in the pancreas and pancreatic cystic lesions.

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.

A Quantitative Global Proteomics Approach Identifies Candidate Urinary Biomarkers That Correlate With Intraductal Papillary Mucinous Neoplasm Dysplasia.

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.

Performance of candidate urinary biomarkers for pancreatic cancer - Correlation with pancreatic cyst malignant progression?

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.

Pancreatic Fluid Interleukin-1ß Complements Prostaglandin E2 and Serum Carbohydrate Antigen 19-9 in Prediction of Intraductal Papillary Mucinous Neoplasm Dysplasia.

OBJECTIVES: We sought to determine if interleukin (IL)-1ß 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-1ß (enzyme-linked immunosorbent assay). Patients had surgical pathology-proven IPMN. Threshold values (PGE2 [>1100 pg/mL], IL-1ß [>20 pg/mL], and serum CA 19-9 [>36 U/mL]) were determined. RESULTS: Levels of IL-1ß 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-1ß (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-1ß, 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.

Circulating Leptin and Branched Chain Amino Acids-Correlation with Intraductal Papillary Mucinous Neoplasm Dysplastic Grade.

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.

Circulating Thrombospondin-2 enhances prediction of malignant intraductal papillary mucinous neoplasm.

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 ±â€¯SD) was greater in High-Grade/Invasive-IPMN than Low/Moderate-grade IPMN (26.6 ±â€¯12.7 ng/mL vs. 20.4 ±â€¯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.

Pancreatic cyst fluid glucose: rapid, inexpensive, and accurate diagnosis of mucinous pancreatic cysts.

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.

Prostaglandin E2: A Pancreatic Fluid Biomarker of Intraductal Papillary Mucinous Neoplasm Dysplasia.

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 E2 (PGE2) 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 PGE2 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 PGE2 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, PGE2 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, PGE2 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 PGE2 as an indicator of IPMN dysplasia, especially in select patients with preoperative pancreatic cyst fluid CEA >192 ng/mL. The inclusion of PGE2/CEA in a diagnostic biomarker panel can facilitate more optimal treatment stratification of IPMN patients.

Pancreatic Cyst Fluid Vascular Endothelial Growth Factor A and Carcinoembryonic Antigen: A Highly Accurate Test for the Diagnosis of Serous Cystic Neoplasm.

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.

Vascular endothelial growth factor, a novel and highly accurate pancreatic fluid biomarker for serous pancreatic cysts.

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.

Dimethylaminoparthenolide and gemcitabine: a survival study using a genetically engineered mouse model of pancreatic cancer.

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-1ß, 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-1ß), 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.

Efficacy of dimethylaminoparthenolide and sulindac in combination with gemcitabine in a genetically engineered mouse model of pancreatic cancer.

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.

Alcohol induces liver neoplasia in a novel alcohol-preferring rat model.

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.

Tailored α-methylene-γ-butyrolactones and their effects on growth suppression in pancreatic carcinoma cells.

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. ß,γ-diaryl-AMGBLs, particularly those with a trans-relationship exhibited higher potency than parthenolide and LC-1 against all three cell lines.

Targeting mitogen-activated protein kinase kinase with the inhibitor PD0325901 decreases hepatocellular carcinoma growth in vitro and in mouse model systems.

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.

Ethanol-TGFalpha-MEK signaling promotes growth of human hepatocellular carcinoma.

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.

Effect of celecoxib and the novel anti-cancer agent, dimethylamino-parthenolide, in a developmental model of pancreatic cancer.

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.

Targeting MEK is effective chemoprevention of hepatocellular carcinoma in TGF-alpha-transgenic mice.

Hepatocellular carcinoma (HCC) causes 600,000 mortalities per year worldwide. Previous studies from our lab provide evidence for altered mitogen-activated protein kinase and extracellular signal-regulated kinase kinase (MEK) signaling in HCC pathogenesis. We hypothesized that pharmacologic targeting of MEK may prevent HCC. Transforming growth factor-alpha-transgenic mice (CD1-MT42) exposed to diethylnitrosamine were randomized to 20 (trial I) or 35 (trial II) weeks of MEK inhibitor PD0325901 (1, 10 mg/kg) or control via orogastric gavage. Ten HCC (44%) formed in trial I controls versus 0 in treatment arms (p<0.05). Fourteen HCC (50%) formed in trial II controls versus 1 (9%) in treatment arms (p<0.05). Mean HCC volume was 578 mm3 in control versus 46 mm3 in the single tumor formed in trial II. In trial I, foci of altered hepatocytes (FAH) formed in 78% of control versus 40% and 0% (1 and 10 mg/kg PD0325901) in treatment arms (p<0.05). In trial II, incidence of FAH was 80% in control versus 20% and 50% (1 and 10 mg/kg PD0325901) in treatment arms (p<0.05). Hepatocyte expression of phosphorylated extracellular signal-regulated kinase dose-dependently decreased in trial I but remained the same in trial II. Control and treated HCC demonstrated similar proliferation rates, but apoptosis appeared increased with treatment. MEK targeting is effective HCC chemoprevention, perhaps by lowering the apoptotic threshold.