Circulating tumour cells as a biomarker for diagnosis and staging in pancreatic cancer.

BACKGROUND: Current diagnosis and staging of pancreatic ductal adenocarcinoma (PDAC) has important limitations and better biomarkers are needed to guide initial therapy. We investigated the performance of circulating tumour cells (CTCs) as an adjunctive biomarker at the time of disease presentation. METHODS: Venous blood (VB) was collected prospectively from 100 consecutive, pre-treatment patients with PDAC. Utilising the microfluidic NanoVelcro CTC chip, samples were evaluated for the presence and number of CTCs. KRAS mutation analysis was used to compare the CTCs with primary tumour tissue. CTC enumeration data was then evaluated as a diagnostic and staging biomarker in the setting of PDAC. RESULTS: We found 100% concordance for KRAS mutation subtype between primary tumour and CTCs in all five patients tested. Evaluation of CTCs as a diagnostic revealed the presence of CTCs in 54/72 patients with confirmed PDAC (sensitivity=75.0%, specificity=96.4%, area under the curve (AUROC)=0.867, 95% CI=0.798-0.935, and P<0.001). Furthermore, a cut-off of ⩾3 CTCs in 4 ml VB was able to discriminate between local/regional and metastatic disease (AUROC=0.885; 95% CI=0.800-0.969; and P<0.001). CONCLUSION: CTCs appear to function well as a biomarker for diagnosis and staging in PDAC.

Risk of malignancy in resected cystic tumors of the pancreas < or =3 cm in size: is it safe to observe asymptomatic patients? A multi-institutional report.

Recent international consensus guidelines propose that cystic pancreatic tumors less than 3 cm in size in asymptomatic patients with no radiographic features concerning for malignancy are safe to observe; however, there is little published data to support this recommendation. The purpose of this study was to determine the prevalence of malignancy in this group of patients using pancreatic resection databases from five high-volume pancreatic centers to assess the appropriateness of these guidelines. All pancreatic resections performed for cystic neoplasms < or =3 cm in size were evaluated over the time period of 1998-2006. One hundred sixty-six cases were identified, and the clinical, radiographic, and pathological data were reviewed. The correlation with age, gender, and symptoms (abdominal pain, nausea and vomiting, jaundice, presence of pancreatitis, unexplained weight loss, and anorexia), radiographic features suggestive of malignancy by either computed tomography, magnetic resonance imaging, or endoscopic ultrasound (presence of solid component, lymphadenopathy, or dilated main pancreatic duct or common bile duct), and the presence of malignancy was assessed using univariate and multivariate analysis. Among the 166 pancreatic resections for cystic pancreatic tumors < or =3 cm, 135 cases were benign [38 serous cystadenomas, 35 mucinous cystic neoplasms, 60 intraductal papillary mucinous neoplasms (IPMN), 1 cystic papillary tumor, and 1 cystic islet cell tumor], whereas 31 cases were malignant (14 mucinous cystic adenocarcinomas and 13 invasive carcinomas and 4 in situ carcinomas arising in the setting of IPMN). A greater incidence of cystic neoplasms was seen in female patients (99/166, 60%). Gender was a predictor of malignant pathology, with male patients having a higher incidence of malignancy (19/67, 28%) compared to female patients (12/99, 12%; p < 0.02). Older age was associated with malignancy (mean age 67 years in patients with malignant disease vs 62 years in patients with benign lesions (p < 0.05). A majority of the patients with malignancy were symptomatic (28/31, 90%). Symptoms that correlated with malignancy included jaundice (p < 0.001), weight loss (p < 0.003), and anorexia (p < 0.05). Radiographic features that correlated with malignancy were presence of a solid component (p < 0.0001), main pancreatic duct dilation (p = 0.002), common bile duct dilation (p < 0.001), and lymphadenopathy (p < 0.002). Twenty-seven of 31(87%) patients with malignant lesions had at least one radiographic feature concerning for malignancy. Forty-five patients (27%) were identified as having asymptomatic cystic neoplasms. All but three (6.6%) of the patients in this group had benign disease. Of the patients that had no symptoms and no radiographic features, 1 out of 30 (3.3%) had malignancy (carcinoma in situ arising in a side branch IPMN). Malignancy in cystic neoplasms < or =3 cm in size was associated with older age, male gender, presence of symptoms (jaundice, weight loss, and anorexia), and presence of concerning radiographic features (solid component, main pancreatic duct dilation, common bile duct dilation, and lymphadenopathy). Among asymptomatic patients that displayed no discernable radiographic features suggestive of malignancy who underwent resection, the incidence of occult malignancy was 3.3%. This study suggests that a group of patients with small cystic pancreatic neoplasms who have low risk of malignancy can be identified, and selective resection of these lesions may be appropriate.

Unfavourable prognosis associated with K-ras gene mutation in pancreatic cancer surgical margins.

BACKGROUND: Despite intent to cure surgery with negative resection margins, locoregional recurrence is common in pancreatic cancer. AIMS: To determine whether detection of K-ras gene mutation in the histologically negative surgical margins of pancreatic cancer reflects unrecognised disease. PATIENTS: Seventy patients who underwent curative resection for pancreatic ductal adenocarcinoma were evaluated. METHODS: All patients had surgical resection margins (pancreatic transection and retroperitoneal) that were histologically free of invasive cancer. DNA was extracted from these paraffin embedded surgical margins and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation at codon 12. Detection of K-ras mutation was correlated with standard clinicopathological factors. RESULTS: K-ras mutation was detected in histologically negative surgical margins of 37 of 70 (53%) patients. A significant difference in overall survival was demonstrated between patients with margins that were K-ras mutation positive compared with negative (median 15 v 55 months, respectively; p = 0.0008). By univariate and multivariate analyses, detection of K-ras mutation in the margins was a significant prognostic factor for poor survival (hazard ratio (HR) 2.8 (95% confidence interval (CI) 1.5-5.3), p = 0.0009; and HR 2.8 (95% CI 1.4-5.5), p = 0.004, respectively). CONCLUSIONS: Detection of cells harbouring K-ras mutation in histologically negative surgical margins of pancreatic cancer may represent unrecognised disease and correlates with poor disease outcome. The study demonstrates that molecular-genetic evaluation of surgical resection margins can improve pathological staging and prognostic evaluation of patients with pancreatic ductal adenocarcinoma.

Identification of genes differentially induced by hypoxia in pancreatic cancer cells.

A hypoxic microenvironment is characteristic of many solid tumors, including pancreatic cancer, the fifth leading cause of cancer death in the United States. Hypoxia causes the stabilization of the HIF-1 (hypoxia-inducible factor-1) transcription factor and the induction of many genes that promote angiogenesis, tumor growth, and metastasis. We performed representational difference analysis (RDA) using mRNA extracted from hypoxic and normoxic Capan-2, a human pancreatic cancer cell line. cDNAs corresponding to hypoxia-inducible genes were cloned and sequenced. We identified GPI/NLK/AMF (glucose phosphate isomerase/neuroleukin/autocrine motility factor) as a hypoxic inducible gene. In addition, hexokinase II and DEC1/Stra13, genes known to be hypoxia inducible in other systems, were found to be hypoxia inducible in our pancreatic cancer system. We thus identified three genes that are induced by hypoxia in a human pancreatic cancer, including GPI/NLK/AMF, which was not previously known to be hypoxia inducible in any other system. These genes may provide new targets for diagnosis and treatment of pancreatic cancer.

Peroxisome proliferator-activated receptor gamma induces pancreatic cancer cell apoptosis.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) decreases the growth of certain cancer cells. In the present study, we found that six different human pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-2, HPAF-II, MIA PaCa-2, and PANC-1) expressed PPAR-gamma m-RNA and synthesized the protein. The endogenous and exogenous PPAR-gamma ligands 15-deoxy-d12,14-prostaglandin J(2) (15-PGJ(2)) and ciglitazone decreased cell number, cell viability, and increased floating/attached ratio, in a time- and dose-dependent fashion. 15-PGJ(2) increased intracellular nucleosome concentration after 6 h, but did not increase caspase-3 activity even after 96 h. Combined treatment with both 15-PGJ(2) and the caspase-3 inhibitor DEVD-CHO had no effect on cell viability, but the general caspase inhibitor ZVAD-FMK reduced 15-PGJ(2)-induced apoptosis. We concluded that the six human pancreatic cancer cells tested all expressed PPAR-gamma receptor, and treatment with PPAR-gamma agonists decreased cell viability and growth in a time- and dose-dependent manner. These effects were partially mediated by induction of caspase-3 independent apoptosis.

Platelet activating factor antagonism reduces the systemic inflammatory response in a murine model of acute pancreatitis.

BACKGROUND: The platelet activating factor (PAF) antagonist, Lexipafant, has been used in experimental models and clinical trials to treat severe acute pancreatitis (AP). The purpose of this study was to determine whether Lexipafant reduces the local and systemic components of AP in a murine model of mild, edematous AP. MATERIALS AND METHODS: Forty-eight female Swiss-Webster mice were divided into four groups. Group 1 received 50 microl of saline ip every hour for 6 h (sham). Group 2 received saline treatment, plus Lexipafant (25 mg/kg dose ip, every 3 h starting 1 h after the first saline injection) (sham/Lex). Group 3 received cerulein (50 microg/kg dose ip, every hour for 6 h) (AP). Group 4 received AP, plus therapeutic treatment with Lexipafant (AP/Lex). Animals were sacrificed 3 h after the last injection. Serum cytokine levels were determined by ELISA. Standard assays were performed for serum amylase activity and lung myeloperoxidase activity (MPO). Histology was scored by two blinded investigators. RESULTS: Serum cytokines (TNFalpha, IL-1beta), lung MPO, and serum amylase activity were reduced by PAF antagonism. Histology showed a trend toward improvement with Lexipafant, but did not reach statistical significance. CONCLUSION: The PAF antagonism reduces the severity of systemic inflammation when given after the induction of mild AP in mice. These results suggest that Lexipafant may be useful in the treatment of mild pancreatitis after its clinical onset.

Angiogenesis inhibitor TNP-470 reduces human pancreatic cancer growth.

In this study we investigated the effects of the angiogenesis inhibitor TNP-470 on human pancreatic cancer cells in vitro and in vivo. The action of TNP-470 on vascular endothelial growth factor (VEGF) was also assessed. In vitro human pancreatic cancer cells (MIAPaCa-2, AsPC-1, and Capan-1), and human umbilical vein endothelial cells (HUVEC) were exposed to increasing concentrations (1 pg/ml to 100 microg/ml) of TNP-470. Cell proliferation was assessed after 3 days by cell count and MTT assay. In vivo, 5 x 10(6) pancreatic cancer cells were injected subcutaneously into nude mice. Four weeks later, 1 mm3 fragments of the resulting tumors were implanted into the pancreas of other mice. Animals received either TNP-470 (30 mg/kg every other day) or vehicle subcutaneously for 14 weeks. The volume of the primary tumor and metastatic spread were determined at autopsy. Concentrations of VEGF were determined in serum (VEGF(S)) and ascites (VEGF(A)) by enzyme-linked immunosorbent assay. Microvessel density was analyzed by immunohistochemistry in CD31-stained tumor sections. In vitro, proliferation and viability of the human pancreatic cancer cell lines were significantly inhibited at high concentrations of TNP-470 (> 1 microg/ml). In contrast, TNP-470 effectively decreased the growth of HUVEC at 100 pg/ml. In vivo, tumor volume and dissemination scores were significantly lower in all three pancreatic cancer cell lines. VEGF(S) and VEGF(A) were not different between treated groups. Treatment with TNP-470 significantly reduced neoangiogenesis in tumors of all three human pancreatic cancer cell lines: MIAPaCa-2 = 74.8 +/- 7.8/0.74 mm2 vs. 24.8 +/- 3.7/0.74 mm2; AsPC-1 = 65.3 +/- 5.0/0.74 mm2 vs. 26.0 +/- 3.4/0.74 mm2; and Capan-1 = 82.2 +/- 5.8/0.74 mm2 vs. 26.9 +/- 2.5/0.74 mm2 (P < 0.001). However, survival was not statistically different between groups. TNP-470 reduced tumor growth and metastatic spread of pancreatic cancer in vivo. This was probably due to the antiproliferative effect of the agent on endothelial cells rather than to the direct inhibition of pancreatic cancer cell growth. TNP-470 activity was not associated with alteration of VEGF secretion.

Therapy for pancreatic cancer with a recombinant humanized anti-HER2 antibody (herceptin).

The HER2/neu oncogene is overexpressed in human pancreatic cancer, but the clinical significance of that overexpression is uncertain. In the present study we investigated the antitumor efficacy of Herceptin, a new recombinant humanized anti-HER2/neu antibody, which exhibits cytostatic activity on breast and prostate cancer cells that overexpress the HER2 oncogene. That antibody may retard tumor growth in certain patients with those diseases. We quantified HER2 expression in various human pancreatic cancer cell lines and studied the bioactivity of this antibody both in vitro and in vivo. Growth inhibition by Herceptin was observed in vitro in cell lines with high levels of HER2/neu expression. Cell lines with low levels of this protein did not respond significantly to the antibody. In vivo we studied two different pancreatic cancer cell lines in an orthotopic mouse model of the disease. Herceptin treatment suppressed tumor growth in the MIA PaCa-2 tumor cell line, which expressed high levels of HER2/neu. These data suggest that Herceptin treatment of patients with pancreatic cancer who express high levels of the HER2/neu oncogene may be reasonable.

Real-time quantitative PCR of telomerase mRNA is useful for the differentiation of benign and malignant pancreatic disorders.

The presence of telomerase activity has been proposed as a specific and sensitive marker for malignant tissue, and positivity rates of up to 95% have been reported in pancreatic cancer. In the present study telomerase activity analysis was reevaluated in 29 pancreatic cancer tissues compared with 36 chronic pancreatitis tissues and 21 normal controls, and a study was made of whether malignant and benign pancreatic disorders can be better differentiated using a novel technique real-time quantitative PCR analysis-analyzing telomerase mRNA expression. Telomerase activity was present in 35% (10 of 29) of pancreatic cancer samples, 3% (one of 36) of chronic pancreatitis samples, and none of the normal pancreatic tissue samples in the TRAP assay. Real-time quantitative PCR analysis revealed the presence of telomerase mRNA expression in 50% (10 of 20) of normal, 86% (31 of 36) of chronic pancreatitis, and 90% (26 of 29) of pancreatic cancer samples. However, quantification of the expression data revealed that the relative increase above normal was 5.5 (range, 3.5-8.6) for chronic pancreatitis and 23.9 (range, 18.6-30.7) for pancreatic cancer samples (p < 0.01). No relationship was found between telomerase activity and the fold increase of telomerase mRNA above normal and gender, patient age, tumor stage, or tumor grade. These data indicate that detection of telomerase activity using the TRAP assay has limitations in differentiating benign and malignant pancreatic disorders. However, telomerase mRNA analysis by real-time quantitative PCR analysis allows a highly sensitive detection and differentiation of pancreatic cancer from normal pancreas and chronic pancreatitis and thereby may serve as a new reliable, easy, and effective diagnostic tool for cancer diagnosis.

An improved clinical model of orthotopic pancreatic cancer in immunocompetent Lewis rats.

The study of pancreatic cancer (PaCa) requires orthotopic, clinically relevant animal models. The aims of this study were to establish an orthotopic model of ductal pancreatic adenocarcinoma in immunocompetent Lewis rats and to develop a scoring system to quantify local tumor infiltration and distant metastasis. Cells (10(7)) of the rat ductal PaCa cell line DSL-6A/C1 were injected s.c. into donor rats. After 8 weeks, either three (IPL-3) or five (IPL-5) fragments (1 mm3) of the resulting s.c. tumors were microsurgically implanted into the pancreas of recipient rats. In another series of animals, 10(7) DSL-6A/C1 cells were directly injected (INJ) into the pancreas. All animals were monitored daily until death or for 16 weeks. At autopsy, volume of primary tumors and ascites, local and systemic tumor spread, and histologic phenotype were assessed. IPL-5 resulted in significantly larger tumors (12,224 +/- 1,933 mm3), more local infiltration and systemic spread (score: 18.3 +/- 2.0 points), severe clinical tumor disease, and lethality (50%) in comparison to the other induction techniques (IPL-3: 283 +/- 115 mm3/3.5 +/- 0.8 points/0; INJ: 752 +/- 207 mm3/4.3 +/- 0.8 points/8%). Histologic examination revealed moderately to well-differentiated ductal tumors, surrounded by dense stroma. Intraperitoneal tumor dissemination in the INJ group occurred simultaneous with primary tumor growth, indicating PaCa cell spread during injection. Orthotopic implantation of five DSL-6A/C1 tumor fragments into the rat pancreas provides a valid clinical model of ductal pancreatic adenocarcinoma in immunocompetent rodents for preclinical treatment studies. The dissemination score we used permitted quantification of local and systemic tumor spread.

G protein-coupled receptor signaling in human ductal pancreatic cancer cells: neurotensin responsiveness and mitogenic stimulation.

Neuropeptides and their corresponding G protein-coupled receptors (GPCRs) are increasingly implicated in the autocrine/paracrine stimulation of growth of human cancers. We report that neurotensin induced rapid Ca2+ mobilization from intracellular stores followed by Ca2+ influx in five human ductal pancreatic cancer cell lines: HPAF-II, Capan-1, Capan-2, PANC-1, and MIA PaCa-2. In addition, most cell lines exhibited Ca2+ responses to multiple neuropeptides including bombesin, bradykinin, cholecystokinin, and vasopressin and to bioactive lipids, including lysophosphatidic acid (LPA), that also act via GPCRs. The well-differentiated line HPAF-II responded to at least seven independent GPCR agonists. The concentrations of neurotensin required to induce half-maximal effects (EC50) in HPAF-II and PANC-1 cells were 5 and 8nM, respectively. Digital fluorescence image analysis to measure Ca2+ responses in single cells revealed that 90% or more of HPAF-II and PANC-1 cells responded to 10nM neurotensin. Addition of neurotensin to PANC-1 cells also induced rapid and dose-dependent extracellular-regulated protein kinase (ERK-1 and ERK-2) activation and subsequently, stimulated DNA synthesis. The signaling complexity of GPCRs uncovered by these studies reveals a new aspect in the biology of human pancreatic cancer and could offer the basis for new approaches to the treatment of this disease.

Solid and papillary epithelial neoplasms of the pancreas: aggressive resection for cure.

Solid and papillary epithelial neoplasms of the pancreas (SPENP) are extremely rare and usually affect young women. We retrospectively reviewed our experience with pancreatic neoplasms from 1986 to the present and identified nine patients with SPENP. All nine patients were female with a mean age of 32 years (range 16-66). All patients presented with gastrointestinal complaints including pain, mass, dyspepsia, or bloating and were subsequently diagnosed with a tumor of the pancreas by CT scan. All patients underwent surgical resection. Two patients had tumors located in the head of the pancreas and underwent a pancreaticoduodenectomy. The remainder had tumors located in the tail of the pancreas and underwent distal pancreatectomy. Pathology demonstrated solid and papillary or solid and cystic pseudopapillary neoplasm of the pancreas. Three tumors were positive for both vimentin and alpha-1 antitrypsin on immunohistochemical studies, and three were positive for neuron-specific enolase. All nine patients underwent curative resection and are alive without any evidence of recurrence with a mean follow-up of 5.4 years. SPENP is considered to be a low-grade malignancy with an excellent prognosis. Prompt diagnosis and surgical resection can result in cure.

Pancreatic surgery.

Recent advances have been made in both laparoscopic and endoscopic techniques for diagnosing and treating pancreatic neoplasms. These advances are reviewed. In addition to the traditional methods of pancreatic resection (eg, standard Whipple and pylorus-preserving Whipple), several other techniques have been described and are discussed (eg, median pancreatectomy and extended pancreatectomy). The morbidity and mortality rates of pancreatic operations have diminished, but significant complications still occur (eg, delayed gastric emptying, pancreatic fistula, and biliary strictures). These are discussed. In managing acute and chronic pancreatitis, less invasive and more conservative approaches are being advocated.

Kupffer cell blockade reduces hepatic and systemic cytokine levels and lung injury in hemorrhagic pancreatitis in rats.

Severe acute pancreatitis (AP) is associated with both the local (pancreatic) release of cytokines and an elevation in their systemic plasma concentrations. This may lead to organ dysfunction and death of the patient. The aims of this study were to investigate the source(s) of systemic cytokine production during experimental AP. Forty-two rats were allocated to five groups (control, sham operation and saline injection, sham operation and gadolinium chloride injection, intraductal sodium-taurocholate infusion and saline injection, or intraductal sodium-taurocholate infusion and gadolinium chloride injection). Blood from the iliac artery, portal vein, and hepatic vein, along with tissue from the pancreas, liver, and lung, were collected. Serum levels of TNFalpha, IL-1beta, IL-6, and IL-10 were determined by enzyme-linked immunosorbent assay. Tissue mRNA for IL-1beta and IL-10 was assessed by reverse-transcription polymerase chain reaction. In untreated animals with AP, the lowest serum cytokine levels were found in the portal vein. In the hepatic vein, the levels of TNFalpha, IL-1beta, and IL-6 were higher. The highest serum levels were detected in the systemic circulation. In the gadolinium chloride-treated group, there was no increase in hepatic or systemic cytokine levels and less lung injury was observed. Extrapancreatic cytokine production from both the liver and the lung contributed significantly to systemic levels of TNFalpha, IL-1beta, IL-6, and IL-10 in this experimental model of AP.

The spectrum of mucin-producing adenocarcinoma of the pancreas.

The clinical distinction between cystic and mucinous carcinomas of the pancreas has been poorly defined. Therefore we sought to stratify the entity known as pancreatic mucinous adenocarcinoma based on pathologic and clinical criteria. Clinical data and pathology specimens were reviewed for patients (n = 40) who had been diagnosed as having mucin-producing pancreatic adenocarcinoma and had undergone either resection or intraoperative biopsy of their pancreatic tumor during a 40-year period at the UCLA Medical Center. Based on histologic criteria, three distinct classes of pancreatic adenocarcinoma were identified: mucinous noncystic (colloid) adenocarcinoma (group I), mucinous cystadenocarcinoma (group II), and ductal adenocarcinoma (group III). Based on clinical behavior, groups I and III were indistinguishable. Compared to patients from groups I and III, those from group II were younger, more likely to be female, and had a better prognosis. Among mucin-producing adenocarcinomas of the pancreas, mucinous noncystic adenocarcinoma and ductal adenocarcinoma share similar clinical features, whereas true cystic lesions represent a distinct clinical entity.

Primary duodenal adenocarcinoma: a 40-year experience.

HYPOTHESIS: In patients with duodenal adenocarcinoma, certain pathologic features of the tumor will have prognostic significance. DESIGN: Retrospective case series. PATIENTS: Forty-nine patients diagnosed with duodenal adenocarcinoma between 1957 and 1998. RESULTS: The tumors of 31 (63%) of the 49 patients underwent resection, 18 (37%) had surgical palliation or underwent biopsy. Mean (+/- SEM) survival for all patients was 49 +/- 9 months. The patients whose tumors were resected had longer survival than those who underwent palliation (mean +/- SEM, 66 +/- 13 months vs 18 +/- 6 months, P =.02). Multivariate analysis revealed large tumor size (P =.01), transmural invasion (P =.004), and moderate to poor tumor grade (P =.03) were negatively correlated with survival. Lymph node status did not influence survival. CONCLUSIONS: Our 40-year experience with duodenal adenocarcinoma demonstrates that large tumor size, advanced histological grade, and transmural invasion are associated with decreased survival. These results underscore the importance of early diagnosis, and suggest the presence of nodal spread is not a contraindication to resection.

Animal models of exocrine pancreatic cancer.

Clinically and biologically relevant animal models are mandatory to further evaluate both the pathophysiology and novel strategies for diagnosis and treatment of exocrine pancreatic cancer. This review briefly summarizes the features of human pancreatic cancer in order to define requirements for animal models of the disease. The described model systems in rodents include pancreatic cancer induced by chemicals, pancreatic cancer in transgenic, and immunodeficient animals.