福嶋 敬宜

Recurrent respiratory papillomatosis (RRP), a chronic upper respiratory condition characterized by diffuse multiple recurring papillomas, is thought to result from human papillomavirus (HPV) type 6 or 11 infection. Although RRP is an intractable disease, malignant transformation of RRP is rare. The underlying mechanism, however, has not been elucidated. We describe the clinical course of a patient who underwent more than 130 operations for RRP associated with HPV type 6 infection and subsequently suffered spontaneous malignant transformation to squamous cell carcinoma. Immunohistochemical analysis revealed that malignant transformation might result from a genomic defect, such as p53 inactivation, leading to stimulation of uncontrolled cell proliferation by HPV type 6 for an extended period, but not directly because of HPV itself. Our results could help in the development of novel therapeutic strategies for severe RRP, although further studies are required before clinical application of molecular targeted therapies. © 2013 Kanazawa et al.

The 6th Diagnostic Pathology Summer Fest, held in Tokyo on August 25-26, 2012, opened its gates for everyone in the medical profession. Basic pathology training can contribute to the improvement of algorithms for diagnosis and treatment. The 6th Summer Fest with the theme 'Pathology and Clinical Treatment of Gastrointestinal Diseases' was held at the Ito International Research Center, The University of Tokyo. On August 25, Treatment of Early Gastrointestinal Cancer and New Guidelines' was discussed in the first session, followed by 'Biopsy Diagnosis of Digestive Tract: Key Points of Pathological Diagnosis for Inflammation and Their Clinical Significance' in the second session. On August 26, cases were discussed in the third session, and issues on pathological diagnosis and classification of neuroendorcrine tumor in the fourth session. The summaries of speeches and discussions are introduced along with the statements of each speaker. This meeting was not a formal evidence-based consensus conference, and 20 experts gave talks on their areas of specialty. Discussion was focused on how the management strategy should be standardized on the algorithm of patient care. (C) 2013 S. Karger AG, Basel

Intestinal strangulation caused by a mucocele of the appendix is extremely rare and difficult to diagnose. It is not usually suspected pre-operatively. This report presents a case of intestinal strangulation due to a mucous containing cystic lesion that was wrapped around the base of a loop of the small bowel. An 89-year-old female was transferred after an acute onset of abdominal pain. A physical examination revealed severe tenderness with guarding in the right upper quadrant. CT of the abdomen showed a loop of dilated small bowel with edema on the right side of the abdominal cavity suggesting a closed loop obstruction. In addition, a low-density thin-walled mass measuring 5 cm in diameter was also seen in the pelvis. Intestinal strangulation was suspected and emergency laparotomy was thus performed. A loop of terminal ileum 25 cm in length was strangulated by a dilated appendix, and ileocecal resection was performed. The resected appendix measured 9 x 3 cm in size and contained mucus. A histopathological examination showed a mucous containing cystic appendix without mucin-producing, neoplastic epithelial cells. A mucocele of the appendix can present in various ways and it is important to recognize this entity at the time of surgery.

We describe a case of an adult female who presented with nephrotic syndrome. She was diagnosed with systemic lupus erythematosus with serum antinuclear antibodies, leucopenia with lymphopenia, butterfly erythema, and nephrotic syndrome. Renal biopsy revealed normal glomeruli with diffuse effacement of the foot processes, consistent with lupus podocytopathy. Although human albumin replacement was performed initially, acute renal failure developed rapidly. Therefore, she was treated with double filtration plasmapheresis (DFPP) in addition to oral steroid. After steroid therapy combined with DFPP, the renal function and proteinuria improved rapidly. Although the impact of DFPP on the treatment of lupus nephritis remains to be delineated, our observations suggest that DFPP in lupus podocytopathy played a pivotal role in facilitating the early recovery from renal injuries. Because of the rapid improvement of renal function without any change in body weight by DFPP, acute renal failure in the setting of lupus podocytopathy might contribute to an alternative pathophysiological factor for the diminished glomerular filtration rate, similar to that observed in the setting of idiopathic minimal change glomerulopathy.

Purpose To retrospectively evaluate criteria for differentiating biliary tract changes in autoimmune pancreatitis (AIP-BTC) from extrahepatic cholangiocarcinoma (ECCA) based on CT findings and to determine predictors for differentiation between the two disorders. Materials and methods CT findings of 22 patients with AIP-BTC and 45 patients with ECCA, both with positive CT findings in the biliary system, were retrospectively assessed. The images were assessed for presence of biliary obstruction, diameter of the maximally dilated biliary duct, maximum thickness of the involved duct, presence of masses inside or around the involved ducts, lengths of the biliary lesions, concentricity of wall thickening, multifocality of the lesion, and degree of lesion enhancement. Results Compared with AIP-BTC, ECCA was significantly more frequently associated with biliary obstruction (p = 0.0037), shorter lengths of the biliary lesions (p = 0.0036), and masses (p < 0.001). No significant differences were found for other items. Conclusion Presence of obstructive dilatation of the bile ducts and intraluminal or peri-ductal masses and length of the thickened wall may help differentiate between AIP-BTC and ECCA.

Objectives: There have been only a few reports on follow-up results of serous cystic neoplasm (SCN) of the pancreas. The frequency of malignancy and surgical indication of SCN are not determined yet. Methods: In this multi-institutional study of the Japan Pancreas Society, a total of 172 patients with SCN were enrolled. The mean follow-up period was 4.5 years. Surgical resection was performed in 90 patients, whereas the remaining 82 were simply observed. Results: Of all patients, 20% were symptomatic. The tumor was located in the pancreatic head (39%), body (35%), and tail (22%). The mean diameter of the tumor was 4.1 cm. None of the patients showed distant or lymph node metastasis except for liver metastasis found in 2 patients (1.2%). No patient died during the follow-up. The preoperative diagnosis did not correctly identify SCN in 57 (63%) of 90 resected cases. A honeycomb appearance, which is one of the most characteristic findings of SCN, could be diagnosed better by endoscopic ultrasonography than by other imaging diagnostic modalities. Conclusions: Surgical resection should be considered only when clear distinction from other surgical diseases is difficult, when symptoms or mass effects are present, and when the tumor size is large.

Glomerular crescents are most commonly associated with rapidly progressive crescentic glomerulonephritis; however, they also develop in response to a wide range of primary and secondary glomerular injuries. Since various kind of glomerulopathies occasionally overlay diabetic glomerular injuries, the presence of crescents in renal biopsy specimens of diabetics may have stimulated a search for etiologies other than diabetes. In this report, we describe an unusual case of diabetic glomerulosclerosis with peculiar extracapillary proliferation. Although such a relationship has so far been ignored in most of the literature, the etiological linkage between diabetic glomerulosclerosis and the development of crescents may not be exceptional. We have reviewed the previous literature and herein discuss the pathological implications of the development of crescents in patients with diabetic glomerulosclerosis. Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3950457896920255.

Intraductal papillary mucinous neoplasm (IPMN) is recognized as a precursor lesion to pancreatic cancer, a unique pathological entity. IPMN has subtypes with different clinical characteristics. However, the molecular mechanisms of cancer progression from IPMN remain largely unknown. In this study we examined the differences in genetic alteration(s) among the IPMN subtypes. Surgically resected IPMNs (n = 25) were classified into four subtypes by hematoxylin and eosin (H&E) and mucin immunostaining. Mutations in KRAS, BRAF, and PIK3CA genes and expression of CDKN2A, TP53, SMAD4, phospho-ERK, and phospho-SMAD1/5/8 proteins were examined. There were 11 gastric, 11 intestinal, one pancreatobiliary, and two oncocytic types in this study. We then compared the two major subtypes, gastric-type and intestinal-type IPMN. Gastric-type IPMN showed a significantly higher incidence of KRAS mutations (9/11, 81.8%) compared with intestinal type (3/11, 27.3%; p < 0.05), although the intestinal type showed a higher grade of dysplasia than gastric type (p < 0.01). All cases with KRAS mutations showed phospho-ERK immunostaining. In contrast, intestinal type (9/11, 81.8%) showed more frequent SMAD1/5/8 phosphorylation compared with gastric-type IPMN (3/11, 27.3%; p < 0.05%). There may be distinct mechanisms of pancreatic cancer progression in the different subtypes of IPMN. In particular, KRAS mutation and bone morphogenetic protein-SMAD signaling status may be crucial diverging steps for the two representative pathways to pancreatic cancer in IPMN patients.

Langerhans cell histiocytosis (LCH) is a clonal neoplasm that shows diverse clinical manifestations and courses of disease progression. The etiology and pathophysiology of LCH remain uncertain. We describe the clinical course of a 23-year-old Japanese woman with multi-system LCH, who showed rapid progression after steroid reduction and developed multi-organ failure. Liver biopsy showed LCH infiltration with fatty degeneration. She was treated with cytarabine, vincristine, and prednisolone according to the Japan LCH study group 02 protocol, without any clinical improvement. Low expression of Ki67 and bcl-2 failed to explain the rapid clinical course. Panhypopituitarism and hypothalamic dysfunction may have caused nonalcoholic fatty liver disease and liver failure. This case indicates that some multi-system LCH patients with hypopituitarism and hypothalamic dysfunction show very rapid progression and are difficult to treat. [J Clin Exp Hematopathol 52(2) : 121-126, 2012]

Forty-eight patients with gastrointestinal (GI) tract B-cell lymphoma (BCL) were analyzed retrospectively. The diagnosis was based on the histological examination of specimens obtained by endoscopic biopsy. Before the diagnosis was made, single-color flow cytometry was performed to analyze the expression of light chains and B-cell antigens including CD10 in the specimens. Restricted light chain (RLC) expression, a marker of B-cell clonality, was defined as κ and λ ratios of either more than 3.0 or less than 0.5. The specimens from 30 patients (62.5%) showed RLC expression. No RLC expression or RLC expression not examined was divided into two groups : those showing CD10 positivity in more than 20% of cells (4 patients, 8.3%) and those showing no positivity (14 patients, 29.2%). The cell number analyzed in the latter group was significantly smaller than that in the other two groups. Abnormal karyotypes were found in the specimens from 8 patients (16.7%). These results indicate that the flow cytometric analysis of endoscopic biopsy specimens is useful when BCL is suspected if an adequate number of cells are obtained. [J Clin Exp Hematopathol 52(2) : 127-131, 2012]

Introduction. Primary involvement of the salivary glands in small cell carcinoma is rare, and has one of the worst prognoses of salivary gland neoplasms. However, it has been reported that some cases have a favorable outcome, although the prognostic factors are still under consideration. Multidisciplinary therapy was usually required to achieve long-term survival. Recently, a resemblance of some small cell carcinomas of the salivary gland to cutaneous Merkel cell carcinoma was suggested the latter have the potential for spontaneous regression, which is related to a favorable clinical outcome. Case presentation. We present a locoregional advanced parotid small cell carcinoma with multiple lymph node metastases in an 87-year-old Asian woman. The tumor was controlled by surgery alone, and nine-year disease-free survival was achieved without any adjunctive therapy. To the best of our knowledge, this is the longest reported follow-up of head and neck small cell carcinoma. Conclusion: We believe this to be the first case of small cell carcinoma with involvement of the salivary glands reported in the literature with a good outcome after surgery alone without any adjunctive therapy. © 2012 Kanazawa et al. licensee BioMed Central Ltd.

Pancreatic ductal neoplasms exhibit gastric epithelium-like characteristics. In this study, we evaluated the expression of claudin-18 (CLDN18), a gastric epithelium-associated claudin, in pancreatic intraepithelial neoplasias (PanINs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and pancreatic ductal adenocarcinomas (PDACs) using immunohistochemistry. We observed a high level of expression of CLDN18 in PanINs (31/32, 97%), IPMNs (61/65, 95%), and MCNs (4/5, 80%) using ordinary tissue section analysis. Furthermore, we observed a high level of CLDN18 expression in PDACs (109/156, 70%) using tissue microarray analysis. However, the normal pancreatic duct or the ductal metaplasia of the acinar cells was not immunoreactive. Comparative analysis of CLDN18 and phenotypic markers in IPMNs revealed that simultaneous expression of CLDN18 and intestinal markers frequently occurred, even in intestinal-type IPMNs. CLDN18 variant 2 mRNA was expressed and was similarly upregulated by phorbol 12-myristate 13-acetate (PMA) treatment in pancreatic cancer cell lines and in a gastric cancer cell line. An inhibitor of pan-PKC (GF109203X) completely suppressed this upregulation in pancreatic cancer cells. These results indicate that CLDN18, a marker for the early carcinogenetic process, is commonly expressed in precursor lesions of PDAC. Activation of the PKC pathway might be involved in CLDN18 expression associated with pancreatic carcinogenesis. (J Histochem Cytochem 59:942-952, 2011)

Objective: The aim of this study was to elucidate the clinicopathological features and prognosis of mucinous cystic neoplasms (MCNs).Materials and Methods: We performed a multi-institutional, retrospective study on a collected series of patients with MCN pathologically defined by ovarian-type stroma. Clinicopathological features and prognosis were investigated.Result: Mucinous cystic neoplasm was confirmed in 156 cases, including 129 adenomas (82.7%) and 21 noninvasive (13.4%) and 6 invasive carcinomas (3.9%). Patients with MCN were exclusively women (98.1%) with the mean age of 48.1 years. All but 1 MCN were in the pancreatic body/tail region with a mean size of 65.3 mm. Communication between the cyst and the pancreatic duct was found in 18.1%. The 3-, 5-, and 10-year survival rates were 97.6%, 96.6%, and 96.6%, respectively. A significant difference in the survival rates was observed between adenomas and carcinomas and between minimally invasive carcinomas and invasive carcinomas. Cyst diameter and presence of mural nodule were predictive of malignant MCN.Conclusions: Mucinous cystic neoplasm is a rare but distinctive pancreatic cystic neoplasm with a favorable overall prognosis. All MCNs should be resected to prevent malignant changes but can be observed an appropriate time when the lesion is small without the presence of mural nodules.

Most patients with thymic cysts complain of a slowly enlarging, asymptomatic cervical mass. Only 6-10% suffer dysphagia, dyspnoea, stridor, cervical pain or vocal paralysis. In some rare cases sudden onset of severe dyspnoea or asphyxia is the first symptom, especially in neonates and small infants. We report a unique case of a 20-month-old child, who required emergency tracheal intubation due to asphyxia. Cervicomediastinal thymic cyst might need to be included in causes of life-threatening airway obstruction in young children.

Intraductal oncocytic papillary neoplasm is known as a distinct subtype of intraductal papillary mucinous neoplaSm of the pancreas. Similar neoplasms of the bile duct are rarely reported, and their disease characteristics are not well established. In this study, we examined 6 cases of biliary neoplasms consisting of oncocytic cells with almost exclusively intraductal growth. The patients were 5 women and I mail of 51 to 68 years. Grossly, 4 appeared to be cystic neoplasms with papillary projections located in the liver and the other two were papillary neoplasms of the dilated hilar bile duct that ranged from 1.5 to 16 cm in size. The most prominent neoplastic cells were cuboidal epithelial cells that showed abundant eosinophilic granular cytoplasm with strongly positive staining for antimitochondrial antibody. Four neoplasms were mixed with minor components of nononcocytic cells. All neoplasms showed arborized papillary and/or cribriform formations except one, which showed a villous architecture. All neoplasms were adenocarcinomas accompanied by a microscopic minimally invasive carcinoma. The oncocytic neoplastic cells, as well as the nononcocytic cells, produced gastric-type mucin (MUC5AC and MUC6) and showed claudin18 and HepPar-1 positivity. Five patients lived disease-free for 10 to 112 months after resection, and I died of tumor recurrence at 26 months postoperatively. The present series of biliary tumors are intraductal papillary neoplasms with oncocytic features and call be clinicopathologically regarded as counterparts of pancreatic intraductal oncocytic papillary neoplasm. Our results also Suggest that oncocytic changes occur in epithelial cells of biliary tracts that show a predominant gastric phenotype. (C) 2009 Elsevier Inc. All rights reserved.

Background and aims Infiltration of IgG4-positive plasma cells in the pancreas and other organs is characteristic of autoimmune pancreatitis (AIP). However, it is undetermined whether needle or forceps biopsy of pancreas or other organs is indeed useful for the diagnosis of AIP. We aimed to clarify this point. Methods Among 39 AIP patients, tissue sampling without laparotomy was performed in 27. Biopsy of pancreas, gastric mucosa, liver, bile duct, and duodenal papilla was performed in 15, 17, 11, 5 and 7, respectively. The obtained specimens were examined for IgG4-positive plasma cells. We also examined gastric mucosa of 18 patients with pancreatic cancer as controls. When the number of IgG4-positive plasma cells was more than 10 per high-power field, we regarded it as diagnostic. Results Diagnostic sensitivity in pancreas, gastric mucosa, liver, bile duct, and duodenal papilla was 47% (7/15), 47% (8/17), 36% (4/11), 0% (0/5), and 57% (4/7), respectively. Conclusions Sensitivity of IgG4 immunostaining was unsatisfactory when tissue sampling was performed by needle or forceps biopsy. Biopsy of gastric mucosa might be a good subsidiary diagnostic tool.

BACKGROUND: We have often encountered difficulties in identifying small liver cancers during surgery. Fluorescent imaging using indocyanine green (ICG) has the potential to detect liver cancers through the visualization of the disordered biliary excretion of ICG in cancer tissues and noncancerous liver tissues compressed by the tumor. METHODS: ICG had been intravenously injected for a routine liver function test in 37 patients with hepatocellular carcinoma (HCC) and 12 patients with metastasis of colorectal carcinoma (CRC) before liver resection. Surgical specimens were investigated using a near-infrared light camera system. Among the 49 subjects, the 26 patients examined during the latter period of the study (20 with HCC and 6 with metastasis) underwent ICG-fluorescent imaging of the liver surfaces before resection. RESULTS: ICG-fluorescent imaging identified all of the microscopically confirmed HCCs (n=63) and CRC metastases (n=28) in surgical specimens. Among the 63 HCCs, 8 tumors (13%, including 5 early HCCs) were not evident grossly unless observed by ICG-fluorescent imaging. Five false-positive nodules (4 large regenerative nodules and 1 bile duct proliferation) were identified among the fluorescent lesions. Well-differentiated HCCs appeared as uniformly fluorescing lesions with higher lesion-to-liver contrast than that of moderately or poorly differentiated HCCs (162.6 [71.1-218.2] per pixel vs 67.7 [-6.3-211.2] per pixel, P<.001), while CRC metastases were delineated as rim-fluorescing lesions. Fluorescent microscopy confirmed that fluorescence originated in the cytoplasm and pseudoglands of HCC cells and in the noncancerous liver parenchyma surrounding metastases. ICG-fluorescent imaging before resection identified 21 of the 41 HCCs (51%) and all of the 16 metastases that were examined. CONCLUSIONS: ICG-fluorescent imaging enables the highly sensitive identification of small and grossly unidentifiable liver cancers in real time, enhancing the accuracy of liver resection and operative staging. Cancer 2009;115:2491-504. (C) 2009 American Cancer Society.

Desmoplasia is a common feature of infiltrating ductal adenocarcinoma of the pancreas. This process is intricately interacted between the host and neoplastic cells. Recently, by transcriptome analysis, periostin was identified as a significantly highly expressed gene in pancreatic stellate cells. To investigate the characteristics of periostin immunodeposition in pancreatic ductal neoplasms, we performed immunohistochemistry and in situ hybridization, focusing on tumor -stromal cells interactions. Eighty-one surgically resected pancreatic lesions, including 35 pancreatic ductal adenocarcinoma, 26 intraductal papillary-mucinous neoplasms, 11 mucinous cystic neoplasms and 9 chronic pancreatitis, were studied. In all ductal adenocarcinomas, periostin deposition was observed in the stroma around the infiltrating cancer on immunohistochemistry. Cellular stroma of mucinous cystic neoplasm, called 'ovarian-type' stroma, did not show periostin deposition. In chronic pancreatitis, most of the staining patterns of periostin were perilobular and meshwork-like. Periostin gene expression was detected solely in the stromal cells on in situ hybridization. Intraductal papillary-mucinous neoplasms were classified into four groups on the basis of the histological grade, namely, adenoma, non-invasive adenocarcinoma, adenocarcinoma with microscopical invasion and with macroscopically evident invasion. In intraductal papillary-mucinous neoplasm, periostin deposition in the periductal stroma increased in frequency and intensity in adenocarcinoma compared with adenomas (P = 0.014). Furthermore, our results showed that a higher frequency of periostin deposition was correlated with a higher frequency of ` intestinal phenotype' of proliferating epithelium (P = 0.036) and laminin-5 gamma 2 chain expression (P<0.001) in intraductal papillary-mucinous neoplasm, the latter of which is frequently expressed in invasive carcinoma. This is the first report to describe the periostin immunohistochemistry in intraductal papillary mucinous neoplasm of the pancreas.

Reported herein is a case of endometrial adenocarcinoma without myometrial invasion that metastasized to the pancreas in a 69-year-old Japanese woman who had a history of hysterectomy. Although systemic radiography could not detect any metastasis in the whole body before hysterectomy, imaging performed 2 months after the hysterectomy consisting of CT, magnetic resonance imaging, gallium scintigraphy and positron emission tomography identified a solitary pancreatic tumor. Imaging demonstrated an intracystic papillary growth in the pancreas, suggesting intraductal papillary mucinous neoplasm or mucinous cystic neoplasm. Excised in distal pancreatectomy, the tumor was diagnosed as a pancreatic primary, an invasive papillary adenocarcinoma at first, but both the endometrial tumor and the pancreatic tumor demonstrated similar morphology and immunohistochemistry. Furthermore, the identical nucleotide mutation of TP53 gene was observed from both the endometrial and pancreatic tumors. The pancreatic tumor was therefore confirmed to be a metastasis from the primary endometrial adenocarcinoma. Metastasis to the pancreas from endometrial carcinoma is extremely rare but must be considered even if the previous cancer was treated at an early stage. Histopathological comparison study and genetic analysis are important for the correct diagnosis of metastasis.

In contrast to the relatively uniform pathology and the unyielding dismal outcome associated with infiltrating ductal adenocarcinoma of the pancreas, cystic lesions have a broad spectrum of gross and microscopic pathologies, and a range of clinical outcomes. The common cystic lesions of the pancreas are reviewed with emphasis on practical tips for distinguishing between the main entities. © 2008 Elsevier Ltd. All rights reserved.

KRAS2 gene mutations are found in 75-90% of infiltrating pancreatic ductal adenocarcinomas but can also be present with other nonneoplastic pancreatic diseases. We recently developed a novel sensitive assay for point mutation detection, called "LigAmp", which can detect one mutant molecule in the presence of 10,000 wild-type molecules and can quantify mutant DNA over a wide dynamic range. We analyzed KRAS2 mutations in surgically-collected pancreatic duct juice samples from patients with pancreatic adenocarcinoma ( n = 27) and chronic pancreatitis ( n = 9). DNA sequencing demonstrated that 17 of the 27 pancreatic cancers harbored KRAS2 mutations at codon 12, including G12D ( GGT -> GAT), G12V ( GTT), and G12R ( CGT). We determined the relative amounts of each KRAS2 mutant by simultaneously quantifying wild-type and mutant KRAS2 DNA. For all pancreatic adenocarcinoma patients, the dominant KRAS2 mutation detected in the pancreatic juice corresponded to that found in the primary cancer. Mutation levels were substantially higher in patients with pancreatic cancer ( 0.05 to 82% of total KRAS2 molecules) compared to those with chronic pancreatitis ( 0 to 0.7%). Among patients with mutant KRAS2 positive cancers, all but one ( 94%) had mutant KRAS2 DNA concentrations of more than 0.5% in their pancreatic juice samples, whereas only 1 of 9 ( 11%) pancreatic juice samples from patients with chronic pancreatitis had more than 0.5% mutant KRAS2 DNA, corresponding to a sensitivity of 94% and a specificity of 89%. LigAmp quantification of mutant KRAS2 in pancreatic juice differentiates pancreatic adenocarcinoma from chronic pancreatitis, and may be a useful early detection tool for pancreatic cancer.

Infiltrating adenocarcinoma of the pancreas is thought to develop through well-defined precursor lesions called pancreatic intraductal neoplasia (PanIN). Despite the exponential growth in our understanding of genetic events that characterize the progression of PanINs to invasive carcinoma, little is known about the role of epigenetic alterations in these precursor lesions. To define the timing and prevalence of methylation abnormalities during early pancreatic carcinogenesis, we investigated the CpG island methylation profile in the various grades of PanINs. Using methylation-specific PCR, we analyzed DNA samples from 65 PanIN lesions for methylation status of eight genes recently identified by microarray approach as aberrantly hypermethylated in invasive pancreatic cancer. Aberrant methylation at any of the eight genes was identified in 68% of all the PanIN lesions examined, and, notably, aberrant methylation was identified in more than 70% of the earliest lesions (PanIN-1A). The average number of methylated loci was 1.1 in PanIN-1A, 0.8 in PanIN-1B, 1.1 in PanIN-2, and 2.9 in PanIN-3 lesions (P = 0.01 for PanIN -3 vs earlier PanINs). Among the genes analyzed, NPTX2 demonstrated an increase in methylation prevalence from PanIN-1 to PanIN-2 (P = 0.0008), and from PanIN-2 to PanIN-3 for SARP2 (P = 0.001), Reprimo (P = 0.01), and LHX1 (P = 0.03). These results suggest that aberrant CpG island hypermethylation begins in early stages of PanINs, and its prevalence progressively increases during neoplastic progression.

Purpose: Most familial cancer susceptibility genes are tumor suppressor genes that are biallelically inactivated in familial neoplasms through somatic deletion of the wild-type allele. Identifying the genomic losses that occur in pancreatic neoplasms, particularly those that occur in familial and precursor neoplasms, may help localize the genes responsible for pancreatic cancer susceptibility. Experimental Design: Normal and neoplastic tissue DNA was isolated from fresh-frozen surgically resected tissues from 20 patients with primary familial pancreatic adenocarcinoma (defined as having at least one first-degree relative with pancreatic cancer), 31 with sporadic intraductal papillary mucinous neoplasms (IPMN), and 7 with familial IPMNs using laser capture microdissection. Microdissected DNA was whole genome amplified using multiple strand displacement. Genome-wide allelotypes were determined using 391 microsatellite markers. The accuracy of microdissection and fidelity of the whole genome amplification were determined by comparing the genotypes of microdissected primary pancreatic cancers to the genotypes of xenografts derived from these cancers and by comparing the results of amplified to nonamplified specimens. Results: The concordance of genotypes between LCM whole genome amplified primary pancreatic cancers and their corresponding pancreatic cancer xenograft DNAs was 98%. Among the 20 primary familial pancreatic adenocarcinomas, we found a high prevalence of loss of heterozygosity (LOH) with an average fractional allelic loss (FAL) of 49.9% of an aggregate of 2,378 informative markers. The level of FAL in the IPMNs (10%) was significantly lower than in the pancreatic adenocarcinomas. The most common locus of LOH in the IPMNs was at 19p (LOH at 24% of markers). The regions of frequent allelic loss observed in the familial pancreatic cancers were similar to those found in sporadic pancreatic cancers. Conclusions: The allelic loss patterns of familial and sporadic pancreatic cancers and IPMNs provide clues as to the genomic locations of tumor suppressor genes inactivated in these neoplasms.

Mucinous cystic neoplasm (MCN) of the pancreas is a distinct clinicopathological entity characterized by mucin-producing epithelial and cyst-forming neoplasm with "ovarian-type" stroma beneath the epithelial component. It is clearly distinguished from ductal adenocarcinoma and intraductal papillary mucinous neoplasm (IPMN). However, MCN can progress to infiltrating carcinoma, and frequently shows a similar histological pattern to ductal adenocarcinoma. Several genetic alterations such as K-ras oncogene mutation, and epigenetic alterations such as hypermethylation of p16 in the invasive component of MCN are also common with ductal adenocarcinoma. Furthermore, recent technologies, including a laser-assisted microdissection system for histological slides and global gene expression profilings using DNA microarrays, made possible to identify more information about molecular abnormalities of MCNs. It is important to diagnose the lesions before they progress to an invasive carcinoma. MCN is one of the precursors of invasive pancreatic carcinoma.

Background. Gemcitabine is an efficacious cytotoxic agent used in the treatment of unresectable pancreatic carcinoma (PC). Recently, gemcitabine resistance has been associated with the ribonucleotide reductase subunit M2 (RRM2). In this prospective study, we hypothesized that RRM2 expression in PC biopsy specimens would be a significant predictor of outcome. Methods. RRM2 mRNA expression in 35 endoscopic ultrasonography-guided fine needle aspiration biopsy (EUS-FNAB) samples was quantified using real-time quantitative reverse transcription -polymerase chain reaction. Results. Thirty-one of 35 biopsy specimens could be assessed for RRM2 expression levels. The mean RRM2 expression relative to glyceraldehyde-3-phcsphate dehydrogenase was 0.248 (range, 0.00739 to 0.858). Eighteen patients (64.5%) had low RRM2 levels, and 13 patients (35.5%) had high RRM2 levels with a cutoff of 0.1. The median survival was 8.8 months for patients with low RRM2 levels and 5.0 months for patients with high levels (P < 0.05). In the low RRM2 expression group, a complete response (CR) was observed in one patient, and a partial response (PR) was observed in eight patients. In contrast, in the high RRM2 expression group, PR was observed in one patient, and CR was not observed. The overall response rate between the high and low expression groups was significantly different (50.0% vs. 7.7%, P < 0.05). Conclusions. RRM2 mRNA expression of EUS-FNAB specimens is a key predictive marker of survival in gemcitabine-treated patients with PC.

Objectives: Histological subclassification of intraductal papillary mucinous neoplasms (IPMNs) is important because the malignant potential of each subtype is different. We investigated whether pancreatic juice cytology can be used to define the subtypes of IPMNs preoperatively. Methods: The cytological findings and pathological parameters in 19 cases of IPMN were analyzed for correlations. Pancreatic juice cytology specimens were reviewed and classified into 4 types according to the criteria previously described for histological diagnosis: intestinal (Int), gastric foveolar (GF), oncocytic (Onc), and pancreatobiliary (PB), and the resected IPMNs were classified histologically using the same criteria for comparison. Immunochemical testing for MUC proteins was also performed. Results: fit 15 cases (79%), the cytological and histological subclassifications were in agreement. The cytology specimens displayed different features corresponded to their histological subtypes. The sensitivities of the cytological diagnosis of each subtype were 80.0% (Int), 72.7% (GF), and 100% (Onc/PB); and the specificities were 85.7% (Int), 87.5% (GF), and 93.8% (Onc/PB). The cytoplasm of the Int-type cells in the pancreatic juice cytology specimens was positive for MUC2. Conclusions: Most of the cytological findings of IPMNs corresponded to the histological findings. The use of MUC2 immunocytochemistry in IPMN subtyping was also demonstrated. Subtyping of IPMNs is useful for preoperative evaluation in addition to cytomorphological grading.

Pancreatic cancer is the fourth leading cause of cancer death in the US. Most pancreatic cancers are infiltrating ductal adenocarcinomas. The careful application of well-defined morphologic criteria can be used to differentiate between infiltrating ductal adenocarcinoma and reactive glands. While most pancreatic cancers are ductal adenocarcinomas, a number of histologically defined variants have been described. These are important to recognize because they have distinct clinical pathologic features. Pancreatic intraepithelial neoplasia (PanIN) is the presumed precursor lesion to infiltrating ductal adenocarcinoma, and PanIN lesions can mimic infiltrating cancer.

Background: The gene expression profile of pancreatic cancer is significantly different from that of normal pancreas. Differences in gene expression are detectable using microarrays, but microarrays have traditionally been applied to pancreatic cancer tissue obtained from surgical resection. We hypothesized that gene expression alterations indicative of pancreatic cancer can be detected by profiling the RNA of pancreatic juice. Methods: We performed oligonucleotide microarray analysis on RNA isolated from pancreatic juice obtained endoscopically after secretin stimulation from six patients with pancreatic cancer and ten patients with nonneoplastic diseases of the pancreas or upper gastrointestinal tract. Extracted RNA was subjected to two rounds of linear RNA amplification, and then hybridized with U133A or X3P gene chips (Affymetrix). Results: Using the U133A or X3P chips, 37 and 133 gene fragments respectively, were identified as being at least 3-fold more abundant in the pancreatic juice of patients with pancreatic cancer compared to the noncancer controls ( p < 0.05, Mann-Whitney test). For example, pancreatic juice from patients with pancreatic cancer contained increased levels of IL8, IFITM1, fibrinogen, osteopontin, CXCR4, DAF and NNMT RNA, genes that have been previously reported as overexpressed in primary pancreatic cancers or pancreatic cancer cell lines relative to control tissues. Conclusions: These results demonstrate that RNA analysis of pancreatic juice can reveal some of the same RNA alterations found in invasive pancreatic cancers. RNA analysis of pancreatic juice deserves further investigation to determine its utility as a tool for the evaluation of pancreatic lesions.

BACKGROUND. The p53-dependent G2/M checkpoint plays a key role in the maintenance of genomic integrity, thereby protecting cells from neoplastic progression. Reprimo, a gene involved in the p53-induced G2 cell cycle arrest, has been recently identified as a novel target for aberrant methylation in pancreatic and other cancers. The biological and clinical relevance of Reprimo methylation in pancreatic cancer was investigated.METHODS. The methylation status of Reprimo CpG island was analyzed by methylation-specific polymerase chain reaction in a large series of pancreatic cancers and was correlated with p53 mutation status, genetic instability (as measured by the fractional allelic loss), and clinicopathologic features.RESULTS. Aberrant methylation of Reprimo was identified in 60% (75 of 125) of pancreatic cancer xenografts and primary pancreatic adenocarcinomas. Reprimo methylation was also detectable in 30% (19 of 63) of pancreatic intraepithelial neoplasias (PanIN), known precursors to infiltrating carcinoma. Reprimo methylation was unrelated to the p53 mutation status and associated with the increased degree of genetic instability (P=.04). Furthermore, we found that patients with Reprimo methylation in their primary pancreatic cancers have significantly worse prognosis than those without Reprimo methylation (P=.007). In contrast, other methylation targets in pancreatic cancers (SPARC and CXCR4) did not correlate with prognosis.CONCLUSIONS. These results suggest that aberrant methylation of Reprimo is a common event in pancreatic carcinogenesis and is associated with genetic instability and unfavorable outcome after surgical resection.

Background & Aims: Recently described genome-wide approaches robustly detect many candidate genes that are regulated by DNA methylation, but many of these genes do not represent important targets for functional inactivation. Here we used a microarray-based strategy to identify biologically relevant genes associated with epigenetic silencing in pancreatic cancer. Methods: We compared information from differential gene expression analysis with the transcriptional responses to epigenetic modifiers. Results: Using this approach, we identified 7 novel targets for aberrant methylation in pancreatic cancer. One of the genes identified, RELN (Reelin), a key regulator of neuronal migration, is frequently silenced in pancreatic cancers, as are several of its downstream mediators. Importantly, small interfering RNA-mediated knockdown of RELN in pancreatic cancer cells that retain RELN expression resulted in greatly enhanced cell motility, invasiveness, and colony-forming ability. Increased cell motility was also induced by knockdown of downstream components of the RELN pathway, including ApoER2, VLDLR, and DAB1. Treatment of pancreatic cancer cells with histone deacetylase inhibitors, valproic acid and suberoylanilide hydroxamic acid, restored the expression of RELN and DAB1 and markedly inhibited their migration. Conclusions: The high prevalence of the silencing of RELN pathway components and its reversal by histone deacetylase inhibitors suggest the importance of this pathway as a diagnostic and therapeutic target for pancreatic cancer.

Now that more than two decades have passed since the first reports of intraductal papillary-mucinous neoplasms (IPMNs), it has become clear that IPMN consists of a spectrum of neoplasms with both morphological and immunohistochemical variations. At a meeting of international experts on pancreatic precursor lesions held in 2003, it was agreed that a consensus classification of IPMN subtypes should be established to enable a more detailed analysis of the clinicopathological significance of the variations. Based on our experience and on information from the literature, we selected representative histological examples of IPMNs and defined a consensus nomenclature and criteria for classifying variants as distinctive IPMN subtypes including gastric type, intestinal type, pancreatobiliary type, and oncocytic type. These definitions can be used for further analyses of the clinicopathological significance of the variations of IPMN.

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/p57K1P2. 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 hypornethylation of LIT1, an imprinting control region important for the regulation of CDKN1C expression. Complete hypornethylation in these cancers was the result of deletion of the methylated LIT1 allele at 11p15.5 rather than loss of imprinting. dConclusions: 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.

The non-neoplastic pancreatic parenchyma adjacent to infiltrating ductal adenocarcinoma demonstrates inflammation, fibrosis, acinar cell loss and small duct-like metaplasia of acinar cells. Similar morphologic changes are also observed in the setting of chronic pancreatitis. In addition, peritumoral acini have been shown to have alterations in gene expression even in the absence of morphological changes. To better understand the pancreatic acinar responses to infiltrating pancreatic ductal adenocarcinoma, we characterized gene expression patterns of pancreatic acinar tissue adjacent to infiltrating pancreatic ductal adenocarcinomas and compared them to gene expression patterns of acinar tissue affected by chronic pancreatitis as well as to those of normal pancreatic acini. Fresh-frozen pancreatic acinar tissue was microdissected from nine patients ( three with pancreatic cancer, three with chronic pancreatitis, three with normal pancreata) using laser capture microdissection, and extracted RNA from each microdissection was subjected to two rounds of linear amplification and hybridized to oligonucleotide microarrays. Gene expression patterns were confirmed using quantitative RT-PCR and/or immunohistochemistry. A total of 20 genes was found to be overexpressed in peritumoral acinar tissue compared to normal acinar tissue and to acini affected by chronic pancreatitis. These 20 genes included pancreatitis-associated protein (HIP/PAP), a gene known to be overexpressed in acini adjacent to infiltrating pancreatic cancer, and the gene cartilage glycoprotein-39 (HC gp-39 or TKL-40). Serum HC gp-39 protein levels were significantly higher in patients with pancreatic cancer and in those with chronic pancreatitis than in controls without pancreatic disease. There was no significant difference in the levels of serum HC gp-39 in patients with pancreatic cancer and those with chronic pancreatitis. Our results demonstrate some of the molecular alterations in acinar cells that occur in response to adjacent infiltrating pancreatic ductal adenocarcinoma and reveal that such alterations can provide a rich source of markers of pancreatic cancer.

Aberrant gene expression in pancreatic ductal adenocarcinomas contributes to the dismal outcome of patients who develop this disease. The 5' region of 14-3-3 sigma (stratifin) is hypomethylated in pancreatic adenocarcinomas and is associated with gene overexpression. In multiple experimental systems, ezrin (ERM, Radixin, Moesin) has been identified as being important in the metastatic behavior of pancreatic and other cancers. We investigated the prognostic significance of aberrant expression of 14-3-3 sigma and the ERM proteins (Ezrin, radixin, Moesin) in a series of invasive periampullary adenocarcinomas including 300 infiltrating pancreatic adenocarcinomas, 54 ampullary adenocarcinomas, and 33 noninvasive intraductal papillary mucinous neoplasms from patients who underwent pancreaticoduodenal resection at The Johns Hopkins Hospital, Baltimore, MD, between 1991 and 2003. Two-hundred fourty-four (82%) primary infiltrating adenocarcinomas of the pancreas demonstrated positive expression of the 14-3-3 sigma, 45 (15%) showed weak immunolabelling, and 9 (3%) were negative. 201 (68%) showed positive immunolabeling of the ERM proteins, 75 (25%) demonstrated weak expression and 20 (7%) no expression. A similar proportion of ampullary cancers showed 14-3-3 sigma and ERM protein expression. Expression of 14-3-3 sigma and ERM protein was more likely in poorly differentiated cancers (p = 0.00005), and their expression was associated with poor survival in univariate analysis (p = 0.09). By multivariate analysis, patients whose cancers expressed 14-3-3 sigma, but not ERM tended to have a poorer prognosis (Hazard ratio, 1.4; 0.9-2.2, p = 0.14). Aberrant expression of 14-3-3 sigma may contribute to the outcome of patients with pancreatic ductal adenocarcinoma.

Pancreatic cancer, once invasive, is almost uniformly fatal. In order to alleviate the dismal prognosis associated with this disease, it is imperative that pancreatic cancer be recognized and treated prior to invasion. Understanding the morphology and biology of precursor lesions of invasive pancreatic cancer has therefore become an issue of paramount importance. In the last decade, significant progress has been in the recognition and appropriate classification of these precursor lesions, and the current review will focus on our state-of-the-art knowledge on this topic. Mucinous cystic neoplasms (MCNs), intraductal papillary mucinous neoplasms (IPMNs), and pancreatic intraepithelial neoplasia (PanIN) encompass the three known morphologically distinct precursors to invasive pancreatic cancer. In addition to discussion of the "classic" precursor entities, this review will also address some of the recent diagnostic controversies for these lesions, in particular features that distinguish IPMNs from PanIN lesions. Finally, the potential clinical impact of recognizing these precursor lesions in the context of early detection of pancreatic cancer will be discussed.

Invasive pancreatic cancer is thought to develop through a series of noninvasive duct lesions known as pancreatic intraepithelial neoplasia (PanIN). We used cDNA microarrays interrogating 15,000 transcripts to identify 49 genes that were differentially expressed in microdissected early PanIN lesions (PanIN-1B/2) compared with microdissected normal duct epithelium. In this analysis, a cluster of extrapancreatic foregut markers, including pepsinogen C, MUC6, KLF4, and TFF1, was found to be up-regulated in PanIN. Up-regulation of these genes was further validated using combinations of real-time reverse transcription-PCR, in situ hybridization, and immunohistochemistry in a total of 150 early PanIN lesions from 81 patients. Identification of these gastrointestinal transcripts in human PanIN prompted assessment of other foregut markers by both semiquantitative and realtime reverse transcription-PCR, revealing similar upregulation of Sox-2, Gastrin, HoxA5, GATA4/5/6, Villin and Forkbead 6 (FoxI1). In contrast to frequent expression of multiple gastric epithelial markers, the intestinal markers intestinal fatty acid binding protein, CDX1 and CDX2 were rarely expressed either in PanIN lesions or in invasive pancreatic cancer. Hedgehog pathway activation induced by transfection of immortalized human pancreatic ductal epithelial cells with Gli1 resulted in up-regulation of the majority of foregut markers seen in early PanIN lesions. These data show frequent up-regulation of foregut markers in early PanIN lesions and suggest that PanIN development may involve Hedgehog-mediated conversion to a gastric epithelial differentiation program.

Using microarrays, we have screened for genes reactivated by drugs that modify epigenetic mechanisms in pancreatic cancer cells. One of the genes identified was tissue factor pathway inhibitor 2 (TFPI-2), which encodes for a broad-spectrum serine proteinase inhibitor that negatively regulates the extracellular matrix degradation, an essential step in tumor invasion and metastasis. We therefore investigated the expression and methylation patterns of the TFPI-2 gene in pancreatic adenocarcinoma, and determined its role in tumor growth and invasion. In contrast to its abundant expression in normal pancreas, TFPI-2 mRNA was undetectable in a high fraction of pancreatic cancer cell lines and in primary pancreatic ductal neoplasms (IPMNs). Loss of TFPI-2 expression was associated with aberrant hypermethylation of its promoter CpG island. Treatment with the phorbol ester (PMA), known to stimulate the TFPI-2 promoter activity, augmented the TFPI-2 expression in cell lines with unmethylated or partially methylated TFPI-2, but failed to induce the expression in cell lines that harbored fully methylated TFPI-2. Aberrant methylation of TFPI-2 was also detected in 73% (102/140) of pancreatic cancer xenografts and primary pancreatic adenocarcinomas, was more likely in older patients with pancreatic cancer, and significantly correlated with progression of IPMNs (P = 0.0002). Restored expression of the TFPI-2 gene in nonexpressing pancreatic cancer cells resulted in marked suppression in their proliferation, migration, and invasive potential in vitro. We thus conclude that epigenetic inactivation of TFPI-2 is a common mechanism that contributes to the aggressive phenotype of pancreatic ductal adenocarcinoma.

Methylthioadenosine phosphorylase (MTAP) plays an important role in the salvage pathway for the synthesis of adenosine. Novel chemotherapeutic strategies exploiting the selective loss of MTAP function in cancers have been proposed. The MTAP gene, on chromosome 9p21, is frequently included within homozygous deletions of the p16(INK4A)/CDKN2A gene. Biallelic deletions of the p16(INK4A)/CDKN2A gene are found in 40% of pancreatic cancers, suggesting that the MTAP gene may be frequently inactivated in pancreatic cancer and that selected patients with pancreatic cancer may benefit from therapies targeting this loss. We immunolabeled six xenografted pancreatic cancers with known MTAP and p16(INK4A)/CDKN2A gene status and found that immunolabeling mirrored gene status. Loss of expression of both MTAP and p16 was observed only in those pancreatic cancers with homozygous deletions that encompassed both the MTAP and p16(INK4A)/CDKN2A genes. We then immunolabeled a series of 320 microarrayed infiltrating pancreatic adenocarcinomas, 35 biliary adenocarcinomas, 54 ampullary cancers, and 35 noninvasive intraductal papillary mucinous neoplasms. Immunolabeling for MTAP was lost in 91 of the 300 (30%) evaluable pancreatic cancers, nine of 54 (17%) ampullary cancers, four of 33 (12%) biliary cancers, and in one of 35 (3%) IPMNs. All neoplasms with loss of MTAP labeling also demonstrated loss of p16 labeling. These results suggest that MTAP expression is lost in similar to 30% of infiltrating pancreatic cancers and in a lower percentage of other periampullary neoplasms, that this loss is the result of homozygous deletions encompassing both the MTAP and p16(INK4A)/CDKN2A genes. Thus, pancreatic cancer is a promising cancer type in which to explore novel chemotherapeutic strategies to exploit the selective loss of MTAP function.

Despite the biological and clinical importance of the interaction between the chemokine receptor CXCR4 and its ligand CXCL12 (SDF-1 alpha) in human cancers, little is known about transcriptional regulation of the CXCR,4 gene. Although aberrant hypermethylation in cancer has been described typically in genes with tumor-suppressor properties, this epigenetic alteration has also been observed to affect potential cancer-promoting genes. We now demonstrate that DNA methylation influences CXCR4 expression in human pancreatic cancer. Gene expression profiling and reverse transcription-PCR identified a significant proportion of pancreatic cancer cell lines that displayed little or no CXCR4 mRNA expression. Using methylation-specific PCR, combined bisulfite restriction analysis, and bisulfite sequencing, we found the 5' CpG islands of the CXCR4 gene to be unmethylated in normal pancreas, whereas promoter hypermethylation was detected in 45% (9 of 20) of pancreatic cancer cell lines and in 46% (46 of 100) of primary pancreatic adenocarcinomas. There was a significant inverse correlation between methylation and mRNA expression level of CXCR4 (P = 0.008) in a large panel of pancreatic cancer cell lines. Constitutive as well as inducible expression of CXCR,4 could be restored in methylated cell lines pharmacologically using epigenetic modifying drugs. These findings demonstrate the first evidence for epigenetic regulation of CXCR4 in human cancers, providing new insights into the role of CXCR4/CXCL12 interactions in tumor progression.

Mucinous cystic neoplasms (MCNs) of the pancreas are uncommon neoplasms usually located in the body or tail of the pancreas and usually in females (>90% of cases). Clinically, they are often misdiagnosed as non-neoplastic pseudocysts leading to failed opportunities for curative resection. To better understand the biology of MCNs and to identify markers of the disease, we performed global gene expression profiling of MCNs using oligonucleotide microarrays. Using laser capture microdissection applied to frozen sections, RNA was extracted from the neoplastic epithelium of MCNs, from the adjacent 'ovarian-type' stroma of MCNs, from histologically normal pancreatic ductal epithelium, from pancreatic acinar tissue and from fibrous stroma in pancreata affected by chronic pancreatitis. Each RNA sample was subjected to two rounds of linear amplification followed by hybridization with U133A gene chips (Affymetrix). The expression patterns of selected genes were confirmed by quantitative RT-PCR and by immunohistochemistry using tissue microarrays containing 19 resected MCNs. A total of 114 known genes were overexpressed in the neoplastic epithelium compared to normal pancreatic ductal epithelium (>3-fold) including S100P, PSCA, c-myc, STK6/STKI5, cathepsin E and pepsinogen C. Activation of the Notch pathway in the epithelial component of MCNs was evident by the demonstration of overexpression of Jagged1 and the downstream Notch pathway member Hes1. In the 'ovarian-type' stroma, several genes involved in estrogen metabolism were overexpressed including STAR and ESR1 genes. Some of the genes identified as overexpressed in these neoplasms may be useful as markers that can distinguish MCNs from non-neoplastic pancreatic cystic lesions.

We developed the LigAmp assay for sensitive detection and accurate quantification of viruses and cells with single-base mutations. In LigAmp, two oligonucleotides are hybridized adjacently to a DNA template. One oligonucleotide matches the target sequence and contains a probe sequence. If the target sequence is present, the oligonucleotides are ligated together and detected using real-time PCR. LigAmp detected KRAS2 mutant DNA at 0.01% in mixtures of different cell tines. KRAS2 mutations were also detected in pancreatic duct juice from patients with pancreatic cancer. LigAmp detected the K103N HIV-1 drug resistance mutation at 0.01% in plasmid mixtures and at similar to0.1% in DNA amplified from plasma HIV-1. Detection in both systems is linear over a broad dynamic range. Preliminary evidence indicates that reactions can be multiplexed. This assay may find applications in the diagnosis of genetic disorders and the management of patients with cancer and infectious diseases.

Invasive pancreatic ductal adenocarcinoma is an almost uniformly fatal disease. Several distinct noninvasive precursor lesions can give rise to invasive adenocarcinoma of the pancreas, and the prevention, detection, and treatment of these noninvasive lesions offers the potential to cure early pancreatic cancers. Noninvasive precursors of invasive ductal adenocarcinoma of the pancreas include pancreatic intraepithelial neoplasias (PanINs), intraductal papillary mucinous neoplasms (IPMNs), and mucinous cystic neoplasms. Diagnostic criteria, including a distinct ovarian-type stroma, and a consistent nomenclature are well established for mucinous cystic neoplasms. By contrast, consistent nomenclatures and diagnostic criteria have been more difficult to establish for PanINs and IPMNs. Because both PanINs and IPMNs consist of intraductal neoplastic proliferations of columnar, mucin-containing cells with a variable degree of papilla formation, the distinction between these two classes of precursor lesions remains problematic. Thus, considerable ambiguities still exist in the classification of noninvasive neoplasms in the pancreatic ducts. A meeting of international experts on precursor lesions of pancreatic cancer was held at The Johns Hopkins Hospital from August 18 to 19, 2003. The purpose of this meeting was to define an international acceptable set of diagnostic criteria for PanINs and IPMNs and to address a number of ambiguities that exist in the previously reported classification systems for these neoplasms. We present a consensus classification of the precursor lesions in the pancreatic ducts, PanINs and IPMNs.