Aberrant expression of maspin in idiopathic inflammatory bowel disease is associated with disease activity and neoplastic transformation.

BACKGROUND: Maspin is both overexpressed in tumors and inflammation, implicating a possible role in bridging inflammation and neoplasia. Idiopathic inflammatory bowel disease (IBD) and IBD-associated dysplasias and carcinomas represent a prototype for studying the relationship between chronic inflammatory states and neoplasia. AIM OF STUDY: To investigate expression of maspin in IBD and IBD-associated dysplasia and colorectal carcinoma. METHODS: Immunohistochemical labeling of maspin was examined using tissue microarrays constructed from archival biopsy and resection tissue from 90 patients with 125 histologically defined lesions including 30 with inactive chronic IBD, 51 with active chronic IBD, 4 IBD-associated foci with epithelial changes indefinite for dysplasia (IFD), 7 with IBD-associated low-grade epithelial dysplasia (LGD), 8 with IBD-associated high grade epithelial dysplasia (HGD), and 25 with IBD-associated invasive colorectal adenocarcinomas. RESULTS: Maspin was expressed in 47/51 (92%) active chronic IBD lesions, which was significantly higher than both inactive chronic IBD (13/30, 43%) and normal mucosa (1 of 9, 11%) (p < 0.01); in particular, the diffuse pattern of maspin expression was significantly higher in active IBD (41/51, 80%), compared with inactive IBD (5/30, 17%) and normal mucosa (0%) (p < 0.01). In the multistage progression model of colitis-associated neoplasia, aberrant labeling was observed at the earliest stages, with 3/4 (75%) IFD foci, 6/7 (86%) LGD, and 8/8 (100%) HGD specimens expressing maspin, virtually always in a diffuse pattern. Expectedly, 22/25 (88%) of invasive IBD-associated cancers overexpressed maspin, including 21 with diffuse labeling. CONCLUSIONS: Maspin is significantly overexpressed in both active IBD and colitis-associated dysplasia compared to either inactive IBD or normal colonic mucosa, suggesting a potential role in disease "flare" as well as neoplastic progression. Targeting maspin for control of disease activity and cancer prophylaxis may be a promising novel therapeutic strategy for IBD.

A subset of pancreatic adenocarcinomas demonstrates coamplification of topoisomerase IIalpha and HER2/neu: use of immunolabeling and multicolor FISH for potential patient screening andtreatment.

We sought to identify the frequency of amplification of the topoisomerase IIalpha gene (TOP2A) in pancreatic cancer and determine the usefulness of TOP2A immunolabeling in screening for TOP2A and human epidermal growth factor receptor (HER)2/neu amplification. We examined 55 pancreatic adenocarcinoma specimens for TOP2A immunolabeling and identified TOP2A protein expression in all specimens with a nuclear labeling index (NLI; positive nuclei/total nuclei x 100) of 5% to 80%. Normal pancreatic ductal epithelium, proposed to give rise to pancreatic adenocarcinoma, did not demonstrate detectable TOP2A expression. In a subset of specimens selected for fluorescence in situ hybridization analysis of TOP2A and HER2/neu amplification using a recently developed multicolor probe, 7 of 8 lesions with an NLI of 25% or more demonstrated TOP2A amplification, in contrast with 2 of 14 lesions with a TOP2A NLI of less than 25%. In 8 of 9 TOP2A-amplified cases, coamplification of HER2/neu was present, suggesting a potential relationship between TOP2A and HER2/neu in pancreatic adenocarcinoma. We propose that TOP2A immunolabeling be used in conjunction with a newly developed multicolor probe to screen patients with pancreatic adenocarcinoma to determine the best potential therapeutic modalities, such as TOP2A inhibitors, trastuzumab, or both.

RPL38, FOSL1, and UPP1 are predominantly expressed in the pancreatic ductal epithelium.

OBJECTIVES: Establishing more effective treatment of pancreatic cancer requires an understanding of the molecular events leading to the onset and progression of this disease. The biology of tumorigenesis may be better understood if cell type-specific genes in the pancreas are more recognized. This recognition may be as important as discovering a disease-responsible gene. Identification of a ductal epithelium-specific gene can contribute not only to our knowledge of pancreatic tumorigenesis, tumor marker discovery, and effective drug targeting but also is crucial for making a reliable animal model. METHODS: We used the x-Profiler engine online to compare the SAGE (Serial Analysis of Gene Expression) libraries derived from 2 short-term cultures of normal human ductal epithelial cells from the pancreas against 34 other SAGE libraries generated from other normal human tissues to identify the best candidate gene specific for the ductal epithelium of the pancreas. RESULTS: We identified 3 genes, ribosomal protein L38 (RPL38), uridine phosphorylase (UPP1), and FOS-like antigen-1 (FOSL1), predominantly expressed in the pancreatic ductal epithelium. The expression patterns of these 3 genes were confirmed by virtual Northern analysis, semi-quantitative RT-PCR, and in situ hybridization. CONCLUSION: Although the expressions of these 3 genes are not completely restricted to the ductal epithelium of the pancreas, we showed that they have more specific expression patterns than CK19 and MUC1. We also demonstrated that all 3 genes are highly expressed in a panel of pancreatic cancer cell lines and can potentially be useful in tumor targeting or as tumor markers.

HLA-G upregulation in pre-malignant and malignant lesions of the gastrointestinal tract.

HLA-G belongs to the nonclassical MHC class Ib group of molecules and has been implicated in mediating immune-responsiveness in various cancerous and non-cancerous cell types. We have examined HLA-G expression in a number of human gastrointestinal malignancies, including pancreatic ductal adenocarcinoma, ampullary cancer, biliary cancer, and colorectal cancer by immunolabeling analysis. We used indices of <5% (negative), 6-25%, 26-50%, 51-75%, and >75% (diffuse) to subclassify lesions based on percentage of positive cell labeling. Across all cancer subtypes, 52-79% of lesions demonstrated expression of HLA-G, with up to 33% of lesions demonstrating diffuse (>75%) expression. In addition, we utilized the neoplastic progression model of colorectal cancer to evaluate HLA-G protein expression in normal colon, tubulovillous adenomas, invasive cancer, and liver metastases arising from colorectal cancer. Focal HLA-G expression was detected in regions of normal colon adjacent to sites of adenomatous and cancerous lesions, as well as in all stages of cancer progression. Overall, the percentage of diffusely (>75%) labeled lesions appeared increased in preneoplastic and neoplastic conditions, as compared to normal colon. Specifically, tubulovillous adnenomas demonstrated pronounced diffuse labeling in 58% of lesions examined. No correlation with HLA-G expression and CD4+ or CD8+ T cells was identified. We propose that HLA-G expression is upregulated in a large percentage of gastrointestinal lesions and may serve to mediate immune-responsiveness in certain instances.

Identification and analysis of precursors to invasive pancreatic cancer.

Histologically distinct noninvasive precursor lesions have been recognized in the pancreas for close to a century. The recent development of a consistent reproducible nomenclature and classification system for these lesions has been a major advance in the study of these noninvasive precursors. The "pancreatic intraepithelial neoplasia" or PanIN system was developed at a National Cancer Institutes sponsored think tank in Park City, Utah. Numerous studies have now demonstrated that genetic alterations in cancer-associated genes are more common in higher grade PanIN lesions then they are in lower grade PanIN lesions, and that higher grade PanIN lesions have many of the same genetic alterations that are found in invasive ductal adenocarcinomas of the pancreas. Thus, just as there is a progression in the colorectal of adenomas to invasive adenocarcinoma, so too is there a progression in the pancreas of histologically low-grade PanIN, to high-grade PanIN to invasive ductal adenocarcinoma.

Immunohistochemistry and in situ hybridization in pancreatic neoplasia.

There are many types of pancreatic neoplasms. Pathologic examination, which includes both routine (e.g., hematoxylin-and-eosin staining) and ancillary (e.g., immunohistochemistry and in situ hybridization) techniques, is essential in correctly typing a pancreatic neoplasm. This chapter focuses on the use of immunohistochemistry and in situ hybridization in the differentiation of pancreatic neoplasms. The materials and methods of these two techniques are described in detail.

Systemic BCNU enhances the efficacy of local delivery of a topoisomerase I inhibitor against malignant glioma.

PURPOSE: To investigate the ability of systemically delivered BCNU to enhance the activity of either systemically delivered irinotecan (CPT-11) or locally delivered camptothecin from a biodegradable polymer for treatment of an intracranial 9L gliosarcoma. METHODS: We used a single systemic dose of BCNU on treatment day 1 in combination with systemic doses of CPT-11 on treatment days 1-5 and 8-12 against an intracranial rat 9L gliosarcoma model implanted into female Fischer 344 rats. We also used the same systemic dose of BCNU given on treatment day 1, followed by a local dose of a 20% loaded camptothecin biodegradable polymer implanted on the same day. RESULTS: Two doses of CPT-11 (10 and 60 mg/kg) were delivered systemically against intracranial 9L. Neither dose showed an increase in survival compared to controls ( P>0.2 for 10 mg/kg and P=0.17 for 60 mg/kg). Systemic delivery of CPT-11 (10 mg/kg per day) in combination with systemic BCNU (15 mg/kg) did not show a significant effect on survival compared to systemic BCNU alone ( P>0.2), even at the maximally tolerated systemic dose of CPT-11 (60 mg/kg per day; P=0.06). The combination of systemic BCNU (15 mg/kg) and intracranial delivery of camptothecin (20% loaded polymer), however, significantly extended survival compared to systemic BCNU alone ( P<0.001) and compared to intracranial delivery of camptothecin alone ( P=0.01). CONCLUSIONS: In a 9L gliosarcoma model, systemic delivery of CPT-11 showed no benefit in survival when delivered alone or in combination with systemic BCNU, because CPT-11 is unable to cross the blood-brain barrier in cytotoxic levels. When cytotoxic levels of a topoisomerase I inhibitor are delivered directly to the brain tumor via a biodegradable polymer, however, the systemic delivery of the alkylating agent BCNU significantly enhances the antitumor effects of camptothecin in a 9L gliosarcoma model.

p16 Inactivation in pancreatic intraepithelial neoplasias (PanINs) arising in patients with chronic pancreatitis.

Patients with long-standing chronic pancreatitis are thought to be at increased risk of developing pancreatic ductal adenocarcinoma, but the mechanism for this increased risk is unknown. Since increasing evidence supports the notion that infiltrating pancreatic ductal adenocarcinomas arise from pancreatic intraepithelial lesions (PanINs), we sought to determine if patients with chronic pancreatitis harbor PanINs with alterations in tumor suppressor genes that are associated with infiltrating pancreatic ductal adenocarcinoma. We identified 122 patients with a diagnosis of chronic pancreatitis and 29 patients with a well-differentiated pancreatic endocrine tumor that underwent pancreatic surgery at the Johns Hopkins Hospital from 1985 to 1999. PanINs from each resection specimen were identified, graded, counted, and correlated with smoking and alcohol history. The expression patterns of p16 and Smad4 were determined in a subset of PanINs by immunohistochemistry, and the pattern of labeling compared with that seen in PanINs associated with infiltrating adenocarcinoma of the pancreas as identified in prior studies, and to PanINs associated with pancreatic endocrine tumor. Duct lesions were present in 80 of the 122 pancreata with chronic pancreatitis (66%). Of 405 duct lesions identified in the chronic pancreatitis group, 7.6% were reactive changes, 65.5% were PanIN-1A, 18% were PanIN-1B, 7.4% were PanIN-2, and 1.5% were PanIN-3. Within the pancreatic endocrine tumor group, 22 PanINs were identified: 15 PanIN-1A, 4 PanIN-1B, and 3 PanIN-2. There were significantly fewer high-grade PanINs in the pancreata with chronic pancreatitis than in pancreata with pancreatic adenocarcinoma (P < 0.0001). Within the chronic pancreatitis group, the 80 patients with PanINs were significantly older than the 42 patients without PanINs (mean age 57.0 +/- 14.1 years vs. 50.9 +/- 14.7 years, P = 0.01). Smoking history was not associated with PanIN prevalence or grade, but patients who reported a history of excessive alcohol consumption had fewer PanINs (25 of 44 harbored PanINs, 57%) than those who did not (54 of 74, 73%, P = 0.07). In the chronic pancreatitis group, 0% of PanIN-1A, 11% of the PanIN-1B, 16% of the PanIN-2, and 40% of the PanIN-3 lesions showed loss of p16 expression, whereas all of the PanINs from patients with an pancreatic endocrine tumor retained p16 expression. All of the PanINs analyzed from patients with chronic pancreatitis retained normal Smad4 expression. We conclude that a significant minority of PanINs arising in patients with chronic pancreatitis show loss of p16 expression. This alteration, common to pancreatic cancer-associated PanINs, may contribute to the predisposition of patients with chronic pancreatitis to develop pancreatic ductal adenocarcinoma.

Novel markers of pancreatic adenocarcinoma in fine-needle aspiration: mesothelin and prostate stem cell antigen labeling increases accuracy in cytologically borderline cases.

The interpretation of pancreas fine-needle aspiration (FNA) is extremely difficult given the cytologic overlap of neoplastic and reactive processes. Using serial analysis of gene expression, we have discovered 2 new markers of pancreatic adenocarcinoma, mesothelin and prostate stem cell antigen (PSCA), and confirmed their specificity by immunohistochemical labeling. Here we evaluate the potential contribution of immunohistochemical labeling of mesothelin and PSCA to the interpretation of pancreas FNAs. Thirty pancreas FNAs with follow-up data were reviewed. Unstained cell block sections from these aspirates labeled for mesothelin and PSCA using immunohistochemistry were compared with initial cytologic diagnoses and with follow-up diagnoses. On follow-up, 19 patients proved to have cancer, and 11 did not. Initial cytologic diagnosis of malignancy correlated with carcinoma on follow-up in 12 of 12 cases, and initial benign cytologic diagnosis correlated with benign follow-up in 8 of 9 cases (sensitivity, 92%; specificity, 100%). Six of the 9 patients with suspicious cytology were found to have a carcinoma on follow-up. PSCA labeling was present in 16 of the 19 patients who ultimately were proven to have carcinoma; PSCA labeling was absent in 10 of the 11 lesions proven to be benign (sensitivity, 84%; specificity, 91%). Mesothelin labeling was present in 13 of the 19 patients who ultimately were proven to have carcinoma; mesothelin labeling was absent in 10 of the 11 lesions proven to be benign (sensitivity, 68%; specificity, 91%). Five of the 6 cytologically suspicious cases with malignant follow-up labeled for either PSCA or mesothelin (83%), and 2 of the 6 cases labeled for both markers. None of the 3 suspicious cases with benign follow-up labeled for either PSCA or mesothelin. Increasingly, molecular techniques are identifying potential cancer markers that may have diagnostic utility. In this study, immunohistochemical labeling for 2 of these markers, PSCA and mesothelin, appears highly specific for pancreatic adenocarcinoma in FNA specimens and useful in categorizing cytologically suspicious lesions.

Analysis of anaphase figures in routine histologic sections distinguishes chromosomally unstable from chromosomally stable malignancies.

A classification of neoplasms as chromosomally stable vs. unstable is of importance in assessing inherited risks but is primarily defined in cell lines where rates of instability can be directly measured. The confirmation of such a mechanistic theory in primary tissues is desirable. We examined anaphase figures in histologic slides to evaluate such criteria. The process of chromosomal instability (CIN) involves mis-segregation of chromosomes at anaphase in some or most instances, whereas chromosomally stable tumors should lack these particular abnormalities. Anaphase bridges are associated with chromosome mis-segregation and have occasionally been observed in colorectal neoplasia and in sarcomas, but have not been considered for use as a diagnostic tool. We assembled series of sarcomas, colorectal and pancreatic carcinomas belonging to two groups: those associated with or lacking chromosomal instability. Among the chromosomally stable tumors, all lacked anaphase bridges, while anaphase bridges were found in most sarcomas and carcinomas having complex genomic alterations. These results confirm a mechanistic explanation involving chromosome mis-segregation for primary human cancers having aneuploidy/CIN. A basic mechanistic distinction between neoplasms with CIN and those with minimal chromosomal changes is supported. Most importantly, our data suggest a diagnostic utility for the examination of anaphase bridges in the routine practice of pathology with a high potential to impact clinical decision-making processes regarding colorectal cancer treatment.

Atheroembolism in cardiac surgery.

BACKGROUND: Atheroembolism is a recognized complication of cardiac surgery, but its incidence and various outcomes have not been completely described. A retrospective study was undertaken to better characterize the syndrome. METHODS: Records of 49,377 autopsies and surgical specimens from the Johns Hopkins Hospital between 1973 and 1995 were reviewed. Three hundred twenty-seven patients (0.7%) had an identifiable atheroembolism on histologic examination. Of these patients, 29 (0.2%) had undergone a cardiac surgical procedure within 30 days of autopsy or surgical resection. Patient charts and pathology specimens were reviewed for operative findings, postoperative outcomes, and histology. RESULTS: Six of the 29 patients (21%) had atheroembolism to the heart, 7 patients (24%) had embolism to the central nervous system, 19 patients (66%) had embolism to the gastrointestinal tract, 14 patients (48%) had embolism to one or both kidneys, and 5 patients (17%) had embolism to a lower extremity. Sixteen patients (55%) had atheroembolism in two or more areas. In 6 patients (21%), death was directly attributable to atheroembolism, including intraoperative cardiac failure from coronary embolism (n = 3), massive stroke (n = 2), and extensive gastrointestinal embolization (n = 1). CONCLUSIONS: Atheroembolism in cardiac surgery has a broad spectrum of clinical presentations, including devastating injuries and death. Although the true incidence is probably underestimated in this retrospective study, the high attendant mortality and morbidity of atheroembolism have been documented. Improvements in outcome are likely to be associated with preoperative identification of patients at high risk, modifications of perfusion technique, and interventions to minimize secondary thrombosis and progressive organ ischemia.

Role of the DPC4 tumor suppressor gene in adenocarcinoma of the ampulla of Vater: analysis of 140 cases.

The K-ras oncogene is activated in approximately 90% of pancreatic adenocarcinomas, and the DPC4 (MADH4/SMAD4) tumor suppressor gene is inactivated in approximately 55% of pancreatic adenocarcinomas. The contributions of these genetic alterations to the development of adenocarcinoma of the ampulla of Vater have not been fully established. One hundred forty surgically resected ampullary adenocarcinomas (76 with associated adenomas with high-grade dysplasia) were immunohistochemically labeled for the DPC4 gene product, and in 85 cases the results were correlated with the status of the K-ras oncogene from previously reported data. The results were correlated with clinical outcome and with other pathologic predictors of prognosis. Complete loss of Dpc4 labeling was identified in 34% (95% confidence interval [CI]: 26%, 43%) of the invasive carcinomas and in none (upper 95% CI: 6%) of the associated adenomas. Focal loss of Dpc4 was seen in three (4%; 95% CI: 1%, 14%) of the areas of high-grade dysplasia. Complete loss of Dpc4 expression was seen in 28/77 intestinal-type tumors, in 17/46 pancreaticobiliary-type tumors, and in 0/10 colloid carcinomas. Activating point mutations in the K-ras gene were identified in 40% of the invasive cancers. There was no correlation between K-ras gene mutations and Dpc4 expression and no correlation between these variables and survival. The overall 5-year survival rate was 38%. Lymph node metastases were associated with shorter survival (P =.03). Loss of Dpc4 expression occurs in approximately one third of invasive ampullary cancers but is not seen in adenomas; thus, loss of Dpc4 expression occurs late in ampullary carcinogenesis. Although ampullary and pancreatic adenocarcinomas share histologic and molecular features, ampullary carcinomas are less likely to show loss of Dpc4 expression or K-ras gene mutations.

Pancreaticoduodenectomy (Whipple resections) in patients without malignancy: are they all 'chronic pancreatitis'?

Pancreaticoduodenectomy (Whipple resection) has evolved into a safe procedure in major high-volume medical centers for the treatment of pancreatic adenocarcinoma and refractory chronic pancreatitis. However, some Whipple resections performed for a clinical suspicion of malignancy reveal only benign disease on pathologic examination. We evaluated the frequency of such Whipple resections without tumor in a large series of pancreaticoduodenectomies and classified the diverse pancreatic and biliary tract diseases present in these specimens. Of 442 Whipple resections performed during 1999-2001, 47 (10.6%) were negative for neoplastic disease and, in 40 cases, had been performed for a clinical suspicion of malignancy. Most Whipple resections revealed benign pancreatic disease, including 8 (17%) alcohol-associated chronic pancreatitis, 4 (8.5%) gallstone-associated pancreatitis, 1 (2.1%) pancreas divisum, 6 (12.8%) "ordinary" chronic pancreatitis of unknown etiology, and 11 (23.4%) lymphoplasmacytic sclerosing pancreatitis. In particular, patients with lymphoplasmacytic sclerosing pancreatitis were all thought to harbor malignancy, whereas only 13 of 19 (68.4%) of Whipple resections showing histologically "ordinary" forms of chronic pancreatitis were performed for a clinical suspicion of malignancy. Benign biliary tract disease, including three cases of primary sclerosing cholangitis, two cases of choledocholithiasis-associated chronic biliary tract disease, and four fibroinflammatory strictures isolated to the intrapancreatic common bile duct, was a common etiology for clinically suspicious Whipple resections (22.5% of cases). Pancreatic intraepithelial neoplasia (PanIN) was a common finding among all pancreata, whether involved by pancreatitis or histologically normal. Overall, PanIN 1A/1B was present in 68.1%, PanIN 2 in 40.4%, and PanIN 3 in just 2.1%. These findings indicate that "benign but clinically suspicious" Whipple resections are relatively common in high-volume centers (9.2%) and reveal a diverse group of clinicopathologically distinctive pancreatic and biliary tract disease.

Telomere shortening is nearly universal in pancreatic intraepithelial neoplasia.

A multistep model of carcinogenesis has recently been proposed for pancreatic ductal adenocarcinomas. In this model, noninvasive precursor lesions in the pancreatic ductules accumulate genetic alterations in cancer-associated genes eventually leading to the development of an invasive cancer. The nomenclature for these precursor lesions has been standardized as pancreatic intraepithelial neoplasia or PanIN. Despite the substantial advances made in understanding the biology of invasive pancreatic adenocarcinomas, little is known about the initiating genetic events in the pancreatic ductal epithelium that facilitates its progression to cancer. Telomeres are distinctive structures at the ends of chromosomes that protect against chromosomal breakage-fusion-bridge cycles in dividing cells. Critically shortened telomeres can cause chromosomal instability, a sine qua non of most human epithelial cancers. Although evidence for telomeric dysfunction has been demonstrated in invasive pancreatic cancer, the onset of this phenomenon has not been elucidated in the context of noninvasive precursor lesions. We used a recently described in situ hybridization technique in archival samples (Meeker AK, Gage WR, Hicks JL, Simon I, Coffman JR, Platz EA, March GE, De Marzo AM: Telomere length assessment in human archival tissues: combined telomere fluorescence in situ hybridization and immunostaining. American Journal of Pathology 2002, 160:1259-1268) for assessment of telomere length in tissue microarrays containing a variety of noninvasive pancreatic ductal lesions. These included 82 PanIN lesions of all histological grades (24 PanIN-1A, 23 PanIN-1B, 24 PanIN-2, and 11 PanIN-3) that were selected from pancreatectomy specimens for either adenocarcinoma or chronic pancreatitis. Telomere fluorescence intensities in PanIN lesions were compared with adjacent normal pancreatic ductal epithelium and acini (62 of 82 lesions, 76%), or with stromal fibroblasts and islets of Langerhans (20 of 82 lesions, 24%). Telomere signals were strikingly reduced in 79 (96%) of 82 PanINs compared to adjacent normal structures. Notably, even PanIN-1A, the earliest putative precursor lesion, demonstrated a dramatic reduction of telomere fluorescence intensity in 21 (91%) of 23 foci examined. In chronic pancreatitis, reduction of telomere signal was observed in all PanIN lesions, whereas atrophic and inflammatory ductal lesions retained normal telomere length. Telomere fluorescence intensity in PanIN lesions did not correlate with proliferation measured by quantitative Ki-67-labeling index or topoisomerase IIalpha expression. Thus, telomere shortening is by far the most common early genetic abnormality recognized to date in the progression model of pancreatic adenocarcinomas. Telomeres may be an essential gatekeeper for maintaining chromosomal integrity, and thus, normal cellular physiology in pancreatic ductal epithelium. A critical shortening of telomere length in PanINs may predispose these noninvasive ductal lesions to accumulate progressive chromosomal abnormalities and to develop toward the stage of invasive carcinoma.

Smad4 overexpression in hepatocellular carcinoma is strongly associated with transforming growth factor beta II receptor immunolabeling.

In the normal liver, the transforming growth factor beta (TGF-beta) signaling pathway plays an important role in inhibiting hepatocyte growth. This effect is mediated through Smad4 (or Dpc4), a tumor-suppressor gene that affects gene transcription and controls cell growth. A loss of Smad4 is associated with carcinoma in a number of other organs, including the pancreas and colon. Despite these facts, several recent studies using cDNA microarrays have surprisingly shown overexpression of Smad4 in hepatocellular carcinoma (HCC). Because Smad4 plays a central role in the TGF-beta signaling pathway, we hypothesized that activation of the TGF-beta signaling pathway may explain Smad4 overexpression. To investigate this, 21 surgically resected HCCs were immunostained with antibodies to Smad4 and TGF-beta receptor II. Tumor and normal liver tissues were stained in all cases, and expression in the tumor was scored in comparison to the nonneoplastic liver. Thirteen hepatic adenomas were also immunostained as a control group. The average age at resection was 58 +/- 16 years for the 17 men and 4 women with HCC. TGF-beta receptor II was weakly expressed in the hepatocyte cytoplasm of all normal livers and was overexpressed in 10 of 21 HCCs. Of these 10 HCCs increased Smad4 immunolabeling was also present in 10 of 10 cases. In contrast, of the 11 of HCCs that did not show TGF-beta overexpression, only 1 showed increased Smad4 immunolabeling. Increased TGF-beta receptor II and Smad4 labeling was associated with a worse nuclear grade and increased mitotic activity. For the hepatic adenomas, the 13 women had an average age at resection of 36 +/- 10 years. Whereas 2 adenomas showed over expression of TGF-beta receptor II, there was no Smad4 overexpression in any case. In conclusion, increased Smad4 protein expression in HCC is tightly linked to overexpression of TGF-beta II receptors and is associated with increased mitoses and a worse nuclear grade. Hepatic adenomas only rarely show overexpression of TGF-beta II receptors and did not show increased Smad4 labeling. The results from this study indicate that Smad4 protein overexpression is present in a subset of HCCs and is strongly correlated with immunostaining for TGF-beta II receptor, findings that may represent activation or dysregulation of the TGF-beta signaling pathway.

The dichotomy in the preinvasive neoplasia to invasive carcinoma sequence in the pancreas: differential expression of MUC1 and MUC2 supports the existence of two separate pathways of carcinogenesis.

Emerging evidence suggests a dichotomy in the dysplasia-CIS-invasive carcinoma sequence in the pancreas. Pancreatic intraepithelial neoplasms (PanINs; small, incidental duct lesions) progress to invasive ductal adenocarcinomas (5-y survival of < 15%), whereas intraductal papillary mucinous neoplasms (large, intraductal tumors with ductal dilatation) are often associated with colloid carcinoma (5-y survival of > 55%). We explored the relationship of these lesions by examining the expression of MUC1 and MUC2, glycoproteins reportedly reflecting "aggressive" and "indolent" phenotypes in pancreas cancer, respectively. Immunohistochemical labeling with MUC1 (clone Ma695) and MUC2 (clone Ccp58) antibodies was performed on PanINs (n = 43), intraductal papillary mucinous neoplasms (n = 74), ductal adenocarcinomas (n = 136), and colloid carcinomas (n = 15). Fifty-four percent of the intraductal papillary mucinous neoplasms expressed MUC2, whereas none of the PanINs did. In contrast, PanINs, especially higher grade lesions, were often positive for MUC1 (61% of PanIN 3), whereas the expression of this glycoprotein was infrequent in intraductal papillary mucinous neoplasms (20%). This dichotomy was further accentuated in the invasive carcinomas with which these two preinvasive pathways are respectively associated: all colloid carcinomas were MUC2+ (100%) and MUC1- (0%), whereas the labeling pattern was the reverse for ductal adenocarcinomas: 63% were MUC1+ and only 1% were MUC2+. These results support a dichotomy in the dysplasia-CIS sequence in the pancreas. Because these two pathways often lead to different types of invasive carcinomas, this is an invaluable model for the study of carcinogenesis. The findings here also support the previous impression that MUC2 (the mucin associated with gel formation) is a marker of the "indolent" pathway (intraductal papillary mucinous neoplasm and colloid carcinoma), whereas MUC1 (the glycoprotein known to have an inhibitory role in cell-cell and cell-stroma interactions as well as in immunoresistance of tumor cells) is a marker of the "aggressive" pathway (PanIN to ductal adenocarcinoma).

Hepatocellular carcinomas show abnormal expression of fibronectin protein.

Fibronectin plays an important role in cell-to-cell adhesion, cell migration, and cell signaling. In the liver, fibronectin expression has been studied primarily as a component of the extracellular matrix, but little information is available on the expression of fibronectin protein in the neoplastic cells of hepatocellular carcinomas (HCCs). Twenty-four surgically resected HCCs were immunostained with fibronectin. Tumor and normal liver tissues were concurrently analyzed in all cases, and expression in the tumor was evaluated in comparison to the nonneoplastic liver. The average age at resection was 54 +/- 18 years for the 18 men and 6 women. Twenty-one of the cases were classic HCCs including 6 cases that were well differentiated, 12 cases moderately differentiated, and 3 cases poorly differentiated. The remaining 3 cases were moderately differentiated fibrolamellar carcinomas. In the normal liver, fibronectin labeled the sinusoids and weakly to moderately stained the cytoplasm of hepatocytes. In HCCs, 15/24 showed overexpression of fibronectin in the cytoplasm, 8/24 showed no change from the nonneoplastic liver, and one case showed decreased cytoplasmic staining. In addition, an abnormal membranous staining pattern was noted in 16/24 HCCs. In contrast to the HCCs, none of the three fibrolamellar carcinomas showed increased cytoplasmic or membranous staining. Excluding fibrolamellar carcinoma, increased cytoplasmic staining and/or an abnormal membranous staining was noted in 19/21 (90%) of HCCs. Fibronectin shows abnormal cytoplasmic and/or membranous staining in the majority of HCCs. The implications of fibronectin overexpression are uncertain but may reflect a critical step in tumor genesis.

Cyclooxygenase 2 expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia: an immunohistochemical analysis with automated cellular imaging.

We immunohistochemically examined material from 36 pancreata (adenocarcinomas, 30 lesions; pancreatic intraepithelial neoplasia [PanIN], 65; normal pancreatic ducts, 30) for cyclooxygenase 2 (COX-2) with an automated platform. We analyzed 7 to 10 discrete foci and generated an average percentage of positive cells and average staining intensity for each lesion. These 2 values were then multiplied to create an overall "HistoScore" for each lesion. COX-2 demonstrated considerable heterogeneity of expression between and within cases. The overall average percentage of positive cells in adenocarcinomas was 47.3%; in PanINs, 36.3%; and in normal ducts, 19.2%. COX-2 was expressed in more than 20% of cells in 23 adenocarcinomas (77%), 42 PanINs (65%), and 12 normal ducts (40%). The overall average HistoScore for adenocarcinomas was 6.1; for PanINs, 5.4; and for normal ducts, 3.5. Significant differences in COX-2 expression were demonstrable in adenocarcinomas vs normal ducts, PanINs vs normal ducts, and PanIN 2/3 vs PanIN 1a/1b. In general, the pattern of COX-2 expression increased from normal to PanIN to adenocarcinoma. The up-regulation of COX-2 in a subset of noninvasive precursor lesions makes it a potential target for chemoprevention with selective COX-2 inhibitors.

Immunohistochemical validation of a novel epithelial and a novel stromal marker of pancreatic ductal adenocarcinoma identified by global expression microarrays: sea urchin fascin homolog and heat shock protein 47.

We extended the results of a previous microarray analysis by immunohistochemical validation of differential protein expression in a series of 57 surgically resected infiltrating ductal pancreatic adenocarcinomas. Two representative genes were examined: sea urchin fascin homolog (overexpressed in both cell lines and primary tumors) and heat shock protein 47 (HSP47; overexpressed in primary tumors only). Protein expression also was evaluated in the precursor lesions of pancreatic cancer pancreatic intraepithelial neoplasia (PanIN), and normal ductal epithelium. Fascin expression was seen in the neoplastic cells of 54 (95%) of 57 ductal adenocarcinomas but not in 49 (94%) of 52 adjacent nonneoplastic epithelium. In the multistep pathogenesis of ductal adenocarcinomas, fascin expression seemed to be a late event, usually present in PanINs 2 and 3. HSP47 expression was almost universal and most intense in the ductal adenocarcinoma-associated stromal desmoplasia (57/57), although 37 cases (65%) also expressed HSP47 in the neoplastic epithelium. HSP47 expression was absent in the majority of nonneoplastic pancreata (46 [88%]). Fascin and HSP47 are novel tumor markers with potential diagnostic and therapeutic implications for pancreatic carcinoma. These results establish the usefulness of global expression platforms to identify novel tumor markers.