Steven A. Ahrendt

Tissue factor expression, angiogenesis, and thrombosis in pancreatic cancer.

Purpose: Hemostatic activation is common in pancreatic cancer and may be linked to angiogenesis and venous thromboembolism. We investigated expression of tissue factor (TF), the prime initiator of coagulation, in noninvasive and invasive pancreatic neoplasia. We correlated TF expression with vascular endothelial growth factor (VEGF) expression, microvessel density, and venous thromboembolism in resected pancreatic cancer. Experimental Design: Tissue cores from a tri-institutional retrospective series of patients were used to build tissue microarrays. TF expression was graded semiquantitatively using immunohistochemistry in normal pancreas (n = 10), intraductal papillary mucinous neoplasms (n = 70), pancreatic intraepithelial neoplasia (n = 40), and resected or metastatic pancreatic adenocarcinomas (n = 130). Results: TF expression was observed in a majority of noninvasive and invasive pancreatic neoplasia, including 77% of pancreatic intraepithelial neoplasias, 91% of intraductal papillary mucinous neoplasms, and 89% of pancreatic cancers, but not in normal pancreas. Sixty-six of 122 resected pancreatic cancers (54%) were found to have high TF expression (defined as grade ≥2, the median score). Carcinomas with high TF expression were more likely to also express VEGF (80% versus 27% with low TF expression, P < 0.0001) and had a higher median MVD (8 versus 5 per tissue core with low TF expression, P = 0.01). Pancreatic cancer patients with high TF expression had a venous thromboembolism rate of 26.3% compared with 4.5% in patients with low TF expression (P = 0.04). Conclusions: TF expression occurs early in pancreatic neoplastic transformation and is associated with VEGF expression, increased microvessel density, and possibly clinical venous thromboembolism in pancreatic cancer. Prospective studies evaluating the role of TF in pancreatic cancer outcomes are warranted.

Serum microRNA Biomarkers for Detection of Non-Small Cell Lung Cancer

Non small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality world-wide and the majority of cases are diagnosed at late stages of disease. There is currently no cost-effective screening test for NSCLC, and the development of such a test is a public health imperative. Recent studies have suggested that chest computed tomography screening of patients at high risk of lung cancer can increase survival from disease, however, the cost effectiveness of such screening has not been established. In this Phase I/II biomarker study we examined the feasibility of using serum miRNA as biomarkers of NSCLC using RT-qPCR to examine the expression of 180 miRNAs in sera from 30 treatment naive NSCLC patients and 20 healthy controls. Receiver operating characteristic curves (ROC) and area under the curve were used to identify differentially expressed miRNA pairs that could distinguish NSCLC from healthy controls. Selected miRNA candidates were further validated in sera from an additional 55 NSCLC patients and 75 healthy controls. Examination of miRNA expression levels in serum from a multi-institutional cohort of 50 subjects (30 NSCLC patients and 20 healthy controls) identified differentially expressed miRNAs. A combination of two differentially expressed miRNAs miR-15b and miR-27b, was able to discriminate NSCLC from healthy controls with sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 100% in the training set. Upon further testing on additional 130 subjects (55 NSCLC and 75 healthy controls), this miRNA pair predicted NSCLC with a specificity of 84% (95% CI 0.73–0.91), sensitivity of 100% (95% CI; 0.93–1.0), NPV of 100%, and PPV of 82%. These data provide evidence that serum miRNAs have the potential to be sensitive, cost-effective biomarkers for the early detection of NSCLC. Further testing in a Phase III biomarker study in is necessary for validation of these results.

Surgical Therapy of Iatrogenic Lesions of Biliary Tract

Abstract. Iatrogenic injuries of the biliary tract have increased in incidence over the past decade with the introduction of laparoscopic cholecystectomy. Although a number of factors have been identified with a higher risk of injury (male gender, complicated gallstone disease, aberrant anatomy) and a number of technical steps have been emphasized to avoid these injuries, the incidence of bile duct injuries has reached a steady-state at least double the rate observed with open cholecystectomy. Most patients sustaining a bile duct injury are recognized in the weeks following laparoscopic cholecystectomy. Careful preoperative preparation should include control of sepsis by draining any bile collections or fistulas and complete cholangiography. Long-term results are best achieved in specialized hepatobiliary centers performing biliary reconstruction with a Roux-en-Y hepaticojejunostomy. Success rates over 90% have been reported from several centers to date with intermediate follow-up.

Primary sclerosing cholangitis: resect, dilate, or transplant?

OBJECTIVE The current study examines the results of extrahepatic biliary resection, nonoperative endoscopic biliary dilation with or without percutaneous stenting, and liver transplantation in the management of patients with primary sclerosing cholangitis (PSC). SUMMARY BACKGROUND DATA Primary sclerosing cholangitis is a progressive inflammatory disease leading to secondary biliary cirrhosis. The most effective management of sclerosing cholangitis before the onset of cirrhosis remains unclear. METHODS From 1980 to 1994, 146 patients with PSC were managed with either resection of the extrahepatic bile ducts and long-term transhepatic stenting (50 patients), nonoperative endoscopic biliary dilation with or without percutaneous stenting (54 patients), medical therapy (28 patients), and/or liver transplantation (21 patients). RESULTS Procedure-related morbidity and mortality rates were similar between surgically resected and nonoperatively managed patients. In noncirrhotic patients, the serum bilirubin level was significantly (p < 0.05) reduced from preoperative levels (8.3+/-1.5 mg/dL) 1 (1.7+/-0.4 mg/dL) and 3 (2.7+/-0.9 mg/ dL) years after resection, but not after endoscopic or percutaneous management. For noncirrhotic PSC patients, overall 5-year survival (85% vs. 59%) and survival until death or transplantation (82% vs. 46%) were significantly longer (p < 0.05) after resection than after nonoperative dilation with or without stenting. For cirrhotic patients, survival after liver transplantation was longer than after resection or nonoperative dilation with or without stenting. Five patients developed cholangiocarcinoma, including three (6%) of the nonoperatively managed patients but none of the resected patients. CONCLUSIONS In carefully selected noncirrhotic patients with PSC, resection and long-term stenting remains a good option. Patients with cirrhosis should undergo liver transplantation.

Cystic pancreatic neuroendocrine tumors: Is preoperative diagnosis possible?

Pancreatic neuroendocrine tumors rarely undergo cystic degeneration leading to a radiologic appearance, which is often interpreted as a pancreatic mucinous cystadenoma or pseudocyst. We reviewed our experience with 38 neuroendocrine tumors, four of which were cystic, and 24 other cystic pancreatic tumors (mucinous cystadenoma [n = 5], cystadenocarcinoma [n = 6], serous cystadenoma [n = 3], solid/cystic papillary neoplasm [n = 3], intraductal papillary mucinous tumor [n = 6], and mucinous adenocarcinoma [n = 1]) managed operatively between 1990 and 2000. This review was undertaken to identify clinical and pathologic features useful for preoperative diagnosis of cystic neuroendocrine tumors. Two of the four patients with cystic neuroendocrine tumors presented with abdominal pain, one patient was asymptomatic, and one patient had hypoglycemia. Three of the four cystic neuroendocrine tumors were identified by CT scan, and none were biopsied preoperatively. Preoperative diagnoses included mucinous cystadenoma in two patients (n = 2), pancreatic cystic neoplasm in one patient, (n = 1) and insulinoma in one patient (n = 1). All four cystic neuroendocrine tumors were benign and were completely resected (distal pancreatectomy [n = 2], enucleation [n = 2]). Cystic neuroendocrine tumors are difficult to diagnose preoperatively because the majority of these tumors are nonfunctional, and CT does not differentiate these tumors from other cystic neoplasms. Cystic neuroendocrine tumors represent a subgroup of pancreatic cystic and neuroendocrine tumors with malignant potential. Their high resectability rate further supports the role of surgical exploration and resection in the treatment of pancreatic cystic neoplasms.

Diagnosis and Management of Cholangiocarcinoma in Primary Sclerosing Cholangitis

Cholangiocarcinoma remains difficult to diagnose and is a major cause of death in patients with primary sclerosing cholangitis. Recently serum carcinoembryonic antigen and carbohydrate antigen 19-9 (CA 19-9) levels have been reported to improve diagnostic accuracy in patients with cholangiocarcinoma and primary sclerosing cholangitis. We reviewed our experience with cholangiocarcinoma complicating primary sclerosing cholangitis to identify clinical factors associated with cholangiocarcinoma in patients with primary sclerosing cholangitis and to determine the appropriate management of patients with confirmed or suspected cholangiocarcinoma. Between 1984 and 1997, 25 patients (18%) were diagnosed with cholangiocarcinoma among 139 patients with primary sclerosing cholangitis. The diagnosis of primary sclerosing cholangitis was made coincident with the diagnosis of cholangiocarcinoma in 12 patients and preceded it by a mean of 62 months in the remaining 13 patients. The incidence of inflammatory bowel disease was higher (P <0.05) in patients with cholangiocarcinoma (80% vs. 61%). Nine patients (36%) with cholangiocarcinoma were managed with either extrahepatic bile duct resection and/or partial hepatic resection (n = 5) or liver transplantation (n = 4), and the remaining 16 patients were unresectable at presentation. Serum CA 19-9 was elevated in all six patients with cholangiocarcinoma who were analyzed and in none of the eight patients without cholangiocarcinoma who were tested (P <0.01). Actuarial 1- and 3-year survival rates in the resected patients (56% and 28%, respectively) were significantly longer (P <0.02) than in the unresected patients (13% and 0%, respectively). The 10-year actuarial mortality rates for cholangiocarcinoma among all 139 patients was 25%. In summary, cholangiocarcinoma was the leading cause of death in patients with primary sclerosing cholangitis and was often diagnosed concurrently with or within months of its diagnosis. Early liver transplantation for patients with primary sclerosing cholangitis will not reduce the incidence of cholangiocarcinoma-related mortality in these patients.

Utility of magnetic resonance cholangiography in the evaluation of biliary obstruction.

BACKGROUND Evaluation of suspected biliary obstruction has traditionally involved a variety of imaging modalities including ultrasound, CT, and invasive cholangiography. These techniques have limitations because of poor visualization of intraductal stones (ultrasound and CT) and the need for an invasive procedure (ERCP and percutaneous transhepatic cholangiography). Magnetic resonance cholangiography (MRC) is a noninvasive imaging modality that provides good visualization of the hepatobiliary system. The aim of the present study was to determine the utility of MRC in evaluating patients with suspected biliary obstruction. STUDY DESIGN One hundred forty-three patients were identified with suspected acute biliary obstruction and underwent MRC. Patient selection was based on clinical criteria including an elevation in serum liver chemistries or evidence of biliary ductal dilatation on conventional imaging. MRC was performed using a half-Fourier acquisition single-shot turbo spin-echo sequence involving single breath-hold rapid image acquisition. A final diagnosis was determined in each patient based on invasive cholangiography, findings at surgery, and clinical course. RESULTS Of the 143 patients, 73 had an obstructing biliary lesion. A malignant process was identified in 25 patients with final diagnoses of pancreatic cancer (n = 15), ampullary cancer (n = 4), cholangiocarcinoma (n = 3), and hepatic or nodal metastases (n = 3). MRC correctly identified biliary obstruction in all these patients and accurately identified the level of biliary obstruction in 24 of 25 patients. Based on the MRC images alone, a malignant process was suspected in 21 of the 25 patients. Forty patients were found to have common bile duct stones and eight patients had a benign distal bile duct stricture. MRC correctly identified common bile duct stones in 37 patients with one false-positive exam (sensitivity = 92%; specificity = 99%). MRC also correctly identified distal biliary strictures in eight patients. In the remaining 70 patients, no definite biliary obstruction was identified by MRC, and in all patients the absence of mechanical obstruction was confirmed by invasive cholangiography or overall clinical course. CONCLUSIONS This study demonstrates that MRC is able to accurately identify the level and cause of biliary obstruction in both malignant and benign disease. MRC may prove to be an important noninvasive tool in preoperative evaluation of patients with suspected biliary obstruction and identification of patients most likely to benefit from an invasive radiologic or surgical procedure.

Molecular detection approaches for smoking associated tumors.

Smoking is directly responsible for approximately 90% of lung cancers and is also strongly associated with cancers of the head and neck, esophagus and urinary bladder. Our growing understanding of the molecular changes that underlie cancer progression has contributed to the development of novel molecular approaches for the detection of cancer. In this study, we review a number of recent studies that have used molecular techniques to detect neoplastic DNA from lung, head and neck, esophagus and bladder cancer. The majority of these approaches are based on polymerase chain reaction (PCR) based assays. These PCR-based techniques can detect a few clonal cancer cells containing a specific DNA mutation, microsatellite alteration, or CpG island methylation among an excess background of normal cells. The ability to accurately detect a small number of malignant cells in a wide range of clinical specimens including sputum, saliva, bronchoalveolar lavage fluid, urine, serum, plasma, or tissue has significant implications for screening high-risk individuals (such as cigarette smokers) for cancer.

Integrative Discovery of Epigenetically Derepressed Cancer Testis Antigens in NSCLC

Background Cancer/testis antigens (CTAs) were first discovered as immunogenic targets normally expressed in germline cells, but differentially expressed in a variety of human cancers. In this study, we used an integrative epigenetic screening approach to identify coordinately expressed genes in human non-small cell lung cancer (NSCLC) whose transcription is driven by promoter demethylation. Methodology/Principal Findings Our screening approach found 290 significant genes from the over 47,000 transcripts incorporated in the Affymetrix Human Genome U133 Plus 2.0 expression array. Of the top 55 candidates, 10 showed both differential overexpression and promoter region hypomethylation in NSCLC. Surprisingly, 6 of the 10 genes discovered by this approach were CTAs. Using a separate cohort of primary tumor and normal tissue, we validated NSCLC promoter hypomethylation and increased expression by quantitative RT-PCR for all 10 genes. We noted significant, coordinated coexpression of multiple target genes, as well as coordinated promoter demethylation, in a large set of individual tumors that was associated with the SCC subtype of NSCLC. In addition, we identified 2 novel target genes that exhibited growth-promoting effects in multiple cell lines. Conclusions/Significance Coordinated promoter demethylation in NSCLC is associated with aberrant expression of CTAs and potential, novel candidate protooncogenes that can be identified using integrative discovery techniques. These findings have significant implications for discovery of novel CTAs and CT antigen directed immunotherapy.

The p53 Codon 72 Proline Allele Is Associated with p53 Gene Mutations in Non–Small Cell Lung Cancer

The p53 gene plays a critical role in cell cycle control, the initiation of apoptosis, and in DNA repair. An Arg/Pro polymorphism at codon 72 of the p53 gene alters the ability of the p53 protein to induce apoptosis, influences the behavior of mutant p53, decreases DNA repair capacity, and may be linked with an increased risk of lung cancer. To further define the role of the p53 codon 72 polymorphism on DNA repair, lung cancer risk, and mutant p53 function, we examined the effect of this polymorphism on mutation of the p53 gene and patient survival in non–small cell lung cancer (NSCLC). Tumor and nonneoplastic (lung or lymphocyte) samples were collected from 182 patients with NSCLC. p53 mutations were detected by direct sequencing and/or the Gene Chip p53 assay in 93 of 182 (51%) tumors. p53 codon 72 polymorphisms were identified by PCR/RFLP analysis. p53 mutations were significantly (P = 0.01) associated with the number of codon 72 Pro alleles: Pro/Pro homozygotes, 17 of 26 (65%); Arg/Pro heterozygotes, 45 of 79 (57%); and Arg/Arg homozygotes, 31 of 77 (40%). The number of codon 72 Pro alleles was independently associated with p53 mutations (odds ratio, 1.97; 95% confidence interval, 1.14-3.40; P = 0.01) in a multiple logistic regression model. The codon 72 polymorphism did not influence patient survival in either the entire patient group or among patients with p53 mutant tumors. In summary, the p53 Pro allele is associated with an increased frequency of p53 mutations in NSCLC.