Hiroyuki Matsubayashi

Peritumoral Fibroblast SPARC Expression and Patient Outcome With Resectable Pancreatic Adenocarcinoma

PURPOSE SPARC (secreted protein acidic and rich in cysteine) is a protein involved in cell matrix interactions, wound repair, and cell migration, and has been reported to inhibit cancer growth. SPARC undergoes epigenetic silencing in many pancreatic cancers, but stromal fibroblasts adjacent to infiltrating pancreatic adenocarcinomas frequently express SPARC. We evaluated the prognostic significance of tumor and peritumoral SPARC expression in patients with pancreatic adenocarcinoma. PATIENTS AND METHODS The expression patterns of SPARC were characterized by immunohistochemistry in 299 primary pancreatic ductal adenocarcinoma resection specimens from patients who underwent pancreaticoduodenectomy at Johns Hopkins Hospital (Baltimore, MD) between 1998 and 2003. Kaplan-Meier analysis and Cox proportional hazards regression modeling were used to assess the mortality risk associated with the presence or absence of tumor SPARC and peritumoral SPARC status. RESULTS By Kaplan-Meier analysis, patients whose pancreatic cancer stromal fibroblasts expressed SPARC (median survival, 15 months) had a significantly worse prognosis than patients whose tumor stroma did not express SPARC (median survival, 30 months; log-rank P < .001). In contrast, the expression of SPARC in pancreatic cancer cells was not associated with prognosis (log-rank P = .13). Controlling for other prognostic factors (tumor size, positive lymph nodes, margin status, tumor grade, and age), the relative hazard for patients whose stroma expressed SPARC compared with those whose stroma did not was 1.89 (95% CI, 1.31 to 2.74); the expression of SPARC in pancreatic cancer cells remained unrelated to prognosis (relative hazard, 1.02; 95% CI, 0.73 to 1.42). CONCLUSION The expression of SPARC by peritumoral fibroblasts portends a poorer prognosis for patients with pancreatic cancer.

SPARC/osteonectin is a frequent target for aberrant methylation in pancreatic adenocarcinoma and a mediator of tumor-stromal interactions.

Deregulated expression of SPARC/osteonectin, a secreted glycoprotein with multiple biological functions, has been associated with the progression of various cancers. Using microarrays, we previously identified SPARC as one of the genes induced by treatment with a DNA methylation inhibitor in pancreatic cancer cells. We therefore analysed the expression pattern and methylation status of the SPARC gene in pancreatic cancer. Gene expression profiling by oligonucleotide microarray and reverse transcription–PCR analyses demonstrated that SPARC mRNA was expressed in non-neoplastic pancreatic ductal epithelial cells, but was not expressed in a majority of pancreatic cancer cell lines. The loss of SPARC expression was associated with aberrant hypermethylation of its CpG island. Immunohistochemical labeling revealed that the SPARC protein was overexpressed in the stromal fibroblasts immediately adjacent to the neoplastic epithelium in primary pancreatic cancers, but rarely expressed in the cancers themselves. Primary fibroblasts derived from pancreatic cancer strongly expressed SPARC mRNA and secreted SPARC protein into the conditioned media, and treatment of pancreatic cancer cells with exogenous SPARC resulted in growth suppression. SPARC expression in fibroblasts from noncancerous pancreatic tissue was augmented by coculture with pancreatic cancer cells. These findings suggest that SPARC is a frequent target for aberrant methylation in pancreatic cancer and that SPARC expression in fibroblasts adjacent to pancreatic cancer cells is regulated through tumor–stromal interactions.

Identification of maspin and S100P as novel hypomethylation targets in pancreatic cancer using global gene expression profiling.

DNA hypomethylation is one of the major epigenetic alterations in human cancers. We have previously shown that genes identified as hypomethylated in pancreatic cancer are expressed in pancreatic cancer cell lines, but not in normal pancreatic ductal epithelium and can be reexpressed in nonexpressing cells using ‘epigenetic modifying agents’ such as DNA methyltransferase inhibitors. To identify additional targets for aberrant hypomethylation in pancreatic cancer, we used oligonucleotide microarrays to screen for genes that displayed expression patterns associated with hypomethylation. This analysis identified a substantial number of candidates including previously reported hypomethylated genes. A subset of eight genes were selected for further methylation analysis, and two cancer-related genes, maspin and S100P, were found to be aberrantly hypomethylated in a large fraction of pancreatic cancer cell lines and primary pancreatic carcinomas. Combined treatment with 5-aza-2′-deoxycytidie and trichostatin A resulted in synergistic induction of maspin and S100P mRNA in MiaPaCa2 cells where both genes were methylated. Furthermore, there was an inverse correlation between methylation and mRNA expression level for maspin and S100P in a large panel of pancreatic cancer cell lines. We also found a significant difference in the methylation patterns of maspin and two previously identified hypomethylated genes (trefoil factor 2 and lipocalin 2) between pancreatic and breast cancer cell lines, suggesting cancer-type specificity for some hypomethylation patterns. Thus, our present results confirm that DNA hypomethylation is a frequent epigenetic event in pancreatic cancer, and suggest that gene expression profiling may help to identify potential targets affected by this epigenetic alteration.

Usefulness of a novel electrosurgical knife, the insulation-tipped diathermic knife-2, for endoscopic submucosal dissection of early gastric cancer

BackgroundAlthough endoscopic submucosal dissection (ESD) of early gastric cancer using an insulation-tipped diathermic (IT) knife enables the removal of large and ulcerative lesions en bloc, expert endoscopic skill is required. We developed an improved IT knife (IT-2) and compared its efficacy and safety with that of the original IT knife (IT-OM).MethodsWe performed ESD of 602 gastric cancers. Of these, 314 previously untreated single lesions of initial onset were analyzed. Operating time, rate of en-bloc resection, and incidence of complications were compared in the IT-2 group (161 patients) and IT-OM group (153 patients). Lesions were further analyzed as to whether they met the Japanese Gastric Cancer Association indications for ESD or extended indications.ResultsMean resection time was significantly shorter in the IT-2 than in the IT-OM group (48 vs 63 min). There were fewer surgeries lasting longer than 2 h in the IT-2 group than in the IT-OM group (3% vs 12%). En-bloc and margin-free resection rates in the IT-OM and IT-2 groups were 95% and 99%, respectively. Perforations occurred in 3.9% of patients in the IT-OM group and in 5% of patients in the IT-2 group (difference not significant [NS]). The incidence of postoperative hemorrhage was 7.8% in the IT-OM group and 8.7% in the IT-2 group (NS). In both groups, complications were treated endoscopically, and emergency surgery was unnecessary.ConclusionResectability and complication rates were similar in the two groups. However, operating time was shorter with IT-2, irrespective of the indications for the performance of ESD. This study suggests benefits of the IT-2 over the IT-OM.

Epigenetic Down-Regulation of CDKN1C/p57KIP2 in Pancreatic Ductal Neoplasms Identified by Gene Expression Profiling

Purpose: Intraductal papillary mucinous neoplasm (IPMN) of the pancreas is an increasingly identified precursor to infiltrating ductal adenocarcinoma. Although our knowledge of the clinical and pathologic features of IPMNs is increasing, the molecular mechanisms underlying these neoplasms remain poorly understood. Experimental Designs: To provide further insight into the molecular pathobiology of IPMNs, global expression profiling was done to determine genes that are inactivated/down-regulated in IPMNs using oligonucleotide microarrays (Affymetrix). Results: In total, 300 unique transcripts (217 known genes) were identified as highly underexpressed in 12 IPMNs (<10-fold lower and P < 0.05) compared with five normal pancreatic ductal epithelium samples obtained by laser capture microdissection. The differential expression of a selection of genes was confirmed using reverse-transcription PCR. One of the genes underexpressed at both the transcriptional and protein level in a significant proportion of IPMNs was the cyclin-dependent kinase inhibitor, CDKN1C/p57KIP2. CDKN1C expression was also decreased in many pancreatic cancer cell lines and was restored following treatment with a DNA methylation inhibitor (5-aza-2′-deoxycitidine) or, more potently, with a histone deacetylase inhibitor (trichostatin A). Partial methylation of the CDKN1C promoter CpG island was found in most, but not all, pancreatic cancer cell lines with reduced CDKN1C expression, and was also detectable in IPMNs. Furthermore, a subset of pancreatic cancers showed complete hypomethylation of LIT1, an imprinting control region important for the regulation of CDKN1C expression. Complete hypomethylation in these cancers was the result of deletion of the methylated LIT1 allele at 11p15.5 rather than loss of imprinting. Conclusions: These findings suggest that CDKN1C is commonly down-regulated in pancreatic ductal neoplasms through a combination of promoter hypermethylation, histone deacetylation, and loss of the maternal allele expressing CDKN1C.

Usefulness of positron emission tomography in the evaluation of distribution and activity of systemic lesions associated with autoimmune pancreatitis.

Background/Aims: Autoimmune pancreatitis (AIP) is an IgG4-related systemic disease often accompanied with a variety of lesions outside of the pancreas and is treated with steroid therapy. The aim of this study is to analyze the usefulness of positron emission tomography with 18F-fluorodeoxyglucose (FDG-PET) in the evaluation of distribution and activity of systemic lesions of AIP during steroid therapy. Methods: Eleven cases of AIP had their FDG-PET images evaluated before and 3 months after steroid therapy and another 2 cases only before therapy. AIP activity was determined by the level of serum markers, IgG and IgG4, and compared with findings of PET. Results: In all 13 cases of AIP, a moderate to intense level of FDG accumulation was recognized in the pancreatic lesion before steroid therapy. Of 13 patients, 11 (84.6%) showed FDG accumulation in the multiple organs, such as mediastinal and other lymph nodes, salivary gland, biliary tract, prostate, and aortic wall. In 11 patients who underwent PET before and after steroid therapy, FDG accumulation was diminished in almost all systemic lesions, with a mean of maximum standardized uptake value (SUVmax) in the pancreatic lesion from 5.12 to 2.69. Similar to the SUV level, serum IgG and IgG4 were decreased in most of the cases after steroid therapy. Conclusions: FDG-PET is an effective modality to evaluate the response of steroid therapy and the distribution and activity of various systemic lesions of AIP.

Increased prevalence of the BRCA2 polymorphic stop codon K3326X among individuals with familial pancreatic cancer

Germline BRCA2 mutations predispose to the development of pancreatic cancer. A polymorphic stop codon in the coding region of BRCA2 (K3326X) has been described, and although an initial epidemiological study suggested it was not disease causing, subsequent studies have been inconclusive. To investigate the biological significance of the K3326X polymorphism, we determined its prevalence in patients with sporadic and familial pancreatic cancer. Using a case–control design, we studied 250 patients with resected sporadic pancreatic adenocarcinomas, 144 patients with familial pancreatic adenocarcinoma, 115 spouses of patients with pancreatic cancer, and a disease control group of 135 patients without a personal history of cancer who had undergone cholecystectomy for non-neoplastic disease. The K3326X polymorphism was detected using heteroduplex analysis and DNA sequencing. The BRCA2 K3326X polymorphism was significantly more prevalent in individuals with familial pancreatic cancer: 8/144 (5.6%) vs 3/250 controls (1.2%) (odds ratio, 4.84; 95% CI, 1.27–18.55, P<0.01). One K3326X carrier with familial pancreatic cancer carried an alteration (IVS 16-2A>G) suspected to be deleterious. Excluding this case did not alter the significance of the association (OR: 4.24, P<0.01). In contrast, there was no difference in prevalence among individuals with sporadic pancreatic cancer – 7/250 (OR: 2.37, 95% CI: 0.61–9.27). The increased prevalence of the BRCA2 K3326X polymorphism in patients with familial pancreatic cancer suggests that this polymorphism is deleterious and contributes to pancreatic cancer risk.

Diagnosing Pancreatic Cancer Using Methylation Specific PCR Analysis

The aim of this study was to determine the utility of detecting methylated ppENK and p16 in pancreatic juice by methylation specific PCR as a marker of pancreatic adenocarcinoma. Pancreatic juice samples were collected either intraoperatively, from 92 patients undergoing pancreaticoduodenectomy for benign (n = 20) and malignant periampullary disease (n = 72) or endoscopically (by duodenal aspiration after secretin infusion), from 13 patients undergoing investigation for pancreatic disease. Methylated ppENK was detected in the pancreatic juice of 30 (66.7%) of 45 patients with pancreatic ductal adenocarcinoma, in 4 (44.4%) of 9 patients with intraductal papillary-mucinous adenocarcinoma, and in 7 (41.2%) of 17 patients with other periampullary carcinomas, using methylation specific PCR. Methylated p16 was detected in a lower percentage of these patients (11.1%, 11.1% and 23.5%, respectively). In contrast, methylated ppENK and p16 were not detected in 21 patients with non-malignant disease including 12 patients with chronic pancreatitis. Methylated ppENK was detected in 30 of 33 (90.9%) primary pancreatic adenocarcinoma and methylated p16 was in 6/33 (18.2%). Despite the absence of ppENK and p16 methylation in normal pancreas, methylated ppENK and p16 was present in the duodenum of 90.5% and 28.6%, respectively of patients without cancer. Further, methylated ppENK and p16 was seen in 88.9% and 11.1%, respectively of pancreatic juice samples obtained by duodenal aspiration from patients without cancer. We conclude that since ppENK and p16 are not normally methylated in pancreatic secretions, detection of methylated ppENK and p16 in pure pancreatic juice obtained by direct cannulation of the pancreatic duct to avoid duodenal secretions may suggest the presence of pancreatic adenocarcinoma Key Words: Pancreatic juice, Methylation, ppENK, Pancreatic adenocarcinoma

Tumor COX-2 expression and prognosis of patients with resectable pancreatic cancer.

Background: COX-2 is over-expressed in many cancers and precursor neoplasms including pancreatic cancer and in experimental settings its overexpression has multiple tumorigenic effects including increasing proliferation and angiogenesis, and inhibition of apoptotic and immunologic responses. We evaluated the prognostic significance of COX-2 expression in pancreatic adenocarcinomas. Patients and Methods: We analyzed COX-2 expression by immunohistochemistry in a prospective cohort of 299 patients with resectable infiltrating adenocarcinoma of the pancreas that had undergone a pancreaticoduodenectomy at Johns Hopkins Hospital between January of 1998 and July of 2003. The survival associated with COX-2 expression was assessed by Kaplan-Meier survival analysis and multivariate Cox proportional hazards regression models that controlled for other known prognostic factors associated with pancreas cancer mortality. Results: By Kaplan-Meier analysis, patients whose pancreatic cancer cells expressed COX-2 (median survival, 15 months) had a significantly worse prognosis than patients whose tumor cells did not express COX-2 (median survival, 20 months; log rank, P=0.002). In the multivariate Cox regression model (which included tumor size, node status, margin status, histologic grade, and age), COX-2 expression remained independently prognostic of a worse survival with a hazard ratio (HR) of 1.41 (95% CI 1.08-1.84, P=0.01). However, the adverse prognosis associated with COX-2 expression appeared greater in larger tumors: For tumors ≥ 3cm in diameter, the HR was HR of 1.52 (95% CI 1.04-2.22) versus 1.11 (95% CI 0.75-1.67) in cancers < 3cm. Conclusions: Tumor COX-2 expression portends a poor prognosis for patients with resected adenocarcinoma of the pancreas, particularly in tumors ≥ 3 cm.

Characteristics of autoimmune pancreatitis based on serum IgG4 level.

BACKGROUND Autoimmune pancreatitis is categorized as an IgG4-related autoimmune disease, mostly associated with serological alterations, however characteristics of autoimmune pancreatitis based on serum markers have not been fully evaluated. METHODS We evaluated demographics, symptoms, imaging and therapeutic outcome in 27 cases of autoimmune pancreatitis stratified by serum IgG4 level. RESULTS Twenty patients (74%) had elevated serum IgG4 and 7 (26%) had normal IgG4 levels. Compared to patients with normal serum IgG4 levels, patients with elevated IgG4 had higher incidence of jaundice at onset (14.3% vs. 80%, respectively; P=0.002), more frequent diffuse pancreatic enlargement at imaging (14.3% vs. 60%, respectively; P=0.04), significantly higher 18F-2-fluoro-2-deoxy-d-glucose uptake of pancreatic lesions (SUV max: 4.0 vs. 5.7, respectively; P=0.02), more frequent extrapancreatic lesions (42.9% vs. 85%, respectively; P=0.03). Response to steroids was recognized regardless of serum IgG4 level, however maintenance therapy was required more frequently amongst patients with elevated compared to normal IgG4 (85.7% vs. 33.3%, respectively; P=0.04). CONCLUSIONS Clinical features of autoimmune pancreatitis are different based on level of serum IgG4. Further studies are needed to clarify if normal serum IgG4 cases are a precursor of active type 1 or type 2 autoimmune pancreatitis.