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Correspondence to: Russell G. Postier, PO Box 26901, Dept. of Surgery, U. of Oklahoma Health Sciences Center, Oklahoma City, OK 73190. E-mail: Russell-Postier@ouhsc.edu
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Summary |
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Computer-assisted analysis of DNA ploidy and nuclear morphology were used to elucidate changes in the cell nucleus that occur during the development of experimental pancreatic cancer. Ductal pancreatic adenocarcinoma was induced in 49 Syrian hamsters by SC injection of N-nitrosobis (2-oxopropyl) amine; twenty hamsters served as controls. Groups of animals were sacrificed every 4 weeks for 20 weeks and adjacent sections of pancreatic tissue were H&E and Feulgen-stained for light microscopy and computer assisted cytometry. Pancreatic ductal cells were classified as normal, atypical, or malignant; tissue inflammation (pancreatitis) was also noted when present. DNA ploidy and nuclear morphology evaluation (Markovian analysis) identified an atypical cell stage clearly distinguishable from either normal or malignant cells; pancreatitis preceded this atypia. The DNA ploidy histogram of these atypical cells revealed a major diploid peak and a minor aneuploid peak. The receiver operator characteristic curve areas for a logistic regression model of normal vs atypical cells was 0.94 and for atypical vs malignant was 0.98, numbers indicative of near-perfect discrimination among these three cell types. The ability to identify an atypical cell population should be useful in establishing the role of these cells in the progression of human pancreatic adenocarcinoma. (J Histochem Cytochem 51:303309, 2003)
Key Words: pancreatic cancer, DNA ploidy, Markovian analysis, premalignant neoplastic lesion, pancreatitis, atypia, adenocarcinoma
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Introduction |
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TUMOR FORMATION in epithelial tissue is caused by multiple genetic mutations in proto-oncogenes and tumor suppressor genes due to the combined effects of mutagens and mitogens. In colorectal cancer, much has been learned about oncogenesis by studying a premalignant neoplastic lesion, the neoplastic polyp (
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Materials and Methods |
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Experimental Protocol
Eight-week-old female Syrian golden hamsters were divided into an experimental group of 49 and a control group of 20 animals. All animals were housed in a sealed room and offered hamster chow and water ad libitum. The experimental animals were given 10 mg/kg BOP SC each week for 20 weeks (
Tissue Preparation
A portion of the splenic and duodenal pancreatic segments were frozen. The remainder were placed in 10% buffered formalin for 24 hr and then prepared for light microscopy, deoxyribonucleic acid (DNA) analysis, and Markovian analysis using the CAS 200 computerized microscope (Becton Dickinson; Mountain View, CA) to determine surrogate end-point biomarkers (
Histology
Normal pancreatic ducts were lined by a single epithelial layer ranging from low cuboidal to tall columnar cells. The criterion for hyperplasia in this study was ductal epithelium two or more layers thick without atypia. Ductal atypia was defined as cellular loss of polarity and/or nuclear changes of hyperchromatism, irregularity of nuclear membrane, and an increased nuclear:cytoplasmic ratio. The term "carcinoma in situ" was used to describe atypia in hyperplastic ductular epithelium, and invasive disease was described as adenocarcinoma.
DNA Measurements and Markovian Analysis
The nuclei of Feulgen-stained cells were evaluated for DNA ploidy and for a specific array of morphometrical characteristics, including 26 size, shape, and texture features, using a Cell Measurement Program (Cell Analysis Systems; Elmhurst, IL) combined with the CAS 200 (
Multiple Logistic Regression and Receiver Operating Characteristics
The Markovian texture features were evaluated for relevance using backwards stepwise logistic regression analysis (SAS System software version 6.10). The resulting models were evaluated for discriminatory power (AccuRoc software; Accumetric, Montreal, Canada) by receiver operator characteristic (ROC) curves for continuous variables (
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Results |
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Pathology
Control animals, given only the carrier for BOP (saline), demonstrated normal pancreatic histology throughout the 22-week study period. In contrast, chronic pancreatitis was found in three of the six animals studied after 4 weeks of BOP administration (Table 1). Two of the three with pancreatitis also had ductal hyperplasia and the other was found to have ductal atypia. There were no detectable abnormalities in the pancreatic tissue from the other three hamsters of this 4-week group. At 8 weeks, the group response induced by the carcinogen was found to be similar to the response of the 4-week group in that three of the six hamsters had developed chronic pancreatitis and the pancreatic tissue from the remaining three was normal. However, evaluation of the tissue from one of the three affected hamsters revealed invasive, focal, ductal adenocarcinoma occurring concomitantly with an area of ductal atypia in the opposite pancreatic lobe.
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The cell profile of the BOP-challenged animals significantly changed over the next 4 weeks. After 12 weeks all six animals were afflicted with chronic pancreatitis (Table 1). Ductal adenocarcinoma in situ was also found in one of these animals, and ductal atypia had developed in three of this group. After 16 weeks of exposure to BOP, chronic pancreatitis accompanied by invasive ductal adenocarcinoma was histologically established in all of the animals. Lymph node involvement occurred in one animal. In addition, ductal atypia was detected in the opposite lobe of three of the hamsters.
Chronic pancreatitis and ductal adenocarcinoma were observed in the tissue taken from every animal in the groups exposed to BOP for 2022 weeks before tissue collection (Table 1). An additional focus of ductal atypia was found in the pancreas of one animal at this time point. Chronic pancreatitis was first detected after 4 weeks of exposure to BOP and was present in all animals at 12 weeks; this condition persisted in all animals throughout the 22 week study. Ductal atypia and hyperplasia were initially observed at 4 weeks and were recurrent and associated with in situ and invasive ductal adenocarcinoma in a portion of the animals from the eighth through the twenty-second week. Invasive ductal adenocarcinoma was present in all animals beginning at the sixteenth week of BOP exposure.
Description of Histopathology
Representative pancreatic tissue from hamsters that were not challenged with BOP is shown in Fig 1A. The acini are normal and back-to-back, and the small duct is lined with a low cuboidal epithelium and is normal. The tissue in Fig 1B is typical of the chronic pancreatitis induced by BOP in this study, presenting with acinar loss, chronic inflammation, and the presence of ductal ectasia. The tissue classified as atypical hyperplasia is shown in Fig 1C. This pancreatic tissue was characterized by hyperplasia of ductal epithelium, as manifested by crowding of the nuclei, many mitotic figures, and mild hyperchromasia. Pancreatic adenocarcinoma from hamsters administered BOP is shown in Fig 1D. The micrograph representing this tissue is distinguished by the presence of a nodule of poorly differentiated ductal carcinoma and malignant cells that are jumbled and crowded with irregular hyperchromatic nuclei. Interestingly, these cells are surrounded by fibrosis and inflammation.
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DNA Content
The pancreatic tissue from all control animals throughout the 22-week experimental protocol generated normal DNA histograms (Fig 2A), consistent with the benign histological evaluation (Fig 1A). Pancreatic ductal cells from animals challenged with BOP that appeared normal by light microscopy also had normal distributions of DNA mass. Significantly, eight of nine animals with BOP-induced ductal atypia (Fig 1C) were found to have an aneuploid peak in the near diploid region (Fig 2B), and three of these had an elevated G2/M-phase proliferative fraction. No hyperploid cells were detected in this group of animals. All 27 of the animals with ductal adenocarcinoma (Fig 1D) were found to have higher peaks, more aneuploid peaks, or both (Fig 2C) than the animals with ductal atypia. Hyperploid cells (>5C) were detected in five of the 27 adenocarcinomas.
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Markovian Analysis
Multiple logistic regression with backward stepwise elimination of variables was used to determine a subset of the 26 Markovian texture features (Table 2) which best discriminated between normal and atypical cells and between atypical and malignant cells from hamster pancreatic tissue. The criterion for elimination of features was p>0.10. Contrasting the normal and atypical cells revealed a combination of 14 of the features that significantly discriminated between the types (Table 2). Fig 3A demonstrates that the area under the ROC curve corresponding to the resulting model was 0.94 (95% CI, 0.920.97) . The possible range is 0.51.0, with 1.0 being perfect discrimination between groups of cells and 0.5 being discrimination no better than random selection. A combination of 17 of the features was found to discriminate between the atypical and malignant pancreatic cells (Table 2). The area under the ROC curve for these cells was 0.98 (95% CI, 0.970.99), as shown in Fig 3B. These numbers are highly significant and discriminatory, demonstrating distinct morphological differences between normal and atypical cells and between atypical and malignant cells in this hamster model of BOP-induced pancreatic cancer.
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Discussion |
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In this study we have evaluated a BOP-elicited premalignant lesion in the Syrian hamster pancreatic adenocarcinoma model. This lesion contains atypical ductal cells as observed by light microscopy, and a DNA histogram that shows an aneuploid peak in the near diploid range with an elevated G2/M-phase proliferative fraction. Pancreatic cancer in humans and in BOP-treated hamsters is believed to arise from pancreatic ductal cells in a progression from cellular atypia to adenocarcinoma (
Recent studies in hamster pancreatic adenocarcinoma cell lines have generated data demonstrating that p16, a tumor suppressor gene, and the associated p15 gene are homozygously deleted in these lines, implying that these deletions may be important in hamster tumorigenesis (
Studies in humans have shown that pancreatic ductal cells retrieved either at endoscopic retrograde pancreatography or by duodenal intubation techniques and evaluated for cytologic abnormalities or the presence of mutations in the K-ras gene can be used to diagnose pancreatic cancer (
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Acknowledgments |
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Supported by the Presbyterian Health Foundation, the Department of Veterans Affairs Medical Research Service, and the University of Oklahoma Department of Surgery Research Funds.
Received for publication April 1, 2002; accepted August 30, 2002.
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