DNA image cytometry in the differential diagnosis of endometrial hyperplasia and adenocarcinoma

OBJECTIVE: To test the value of DNA image cytometry in the differential diagnosis of hyperplastic endometrial lesions and endometrial carcinoma on a series of 153 cases of simple hyperplasia (n = 71), complex hyperplasia (n = 28), complex atypical hyperplasia (n = 11) and endometrial adenocarcinoma (n = 43). STUDY DESIGN: Monolayer smears were prepared from three 50-micron-thick sections by a cell separation technique and were stained according to Feulgen. The DNA content of 250 epithelial cells, chosen randomly, was determined using a TV image analysis system (CM-1, Hund, Wetzlar, Germany). The DNA content of 30 lymphocytes served as an internal standard for the normal diploid value in every case. Different DNA cytometric parameters and the mean nuclear area were calculated. RESULTS: Cases of adenocarcinoma and complex atypical hyperplasia (n = 54) were defined as clinically "positive" as these patients are normally treated by hysterectomy. The remaining cases of simple and complex hyperplasia (n = 99) were interpreted as clinically "negative" as conservative therapy is usually preferred. Requesting a specificity of > 90%, high sensitivity rates were calculated for ploidy imbalance (94%), mean ploidy (91%), diploid deviation quotient (91%), DNA stemline ploidy (87%) and 2c deviation index (85%), based on suitable thresholds. Entropy (76%), 5c exceeding events (63%), mean nuclear area (48%) and 9c exceeding events (6%) revealed lower sensitivity values. 5c Exceeding events (P = .0117) and mean nuclear area (P = .0392) were helpful in differentiating between atypical hyperplasia and endometrial carcinoma as the data distribution was significantly different with the U test. CONCLUSION: Our results indicate that DNA single cell cytometry is a highly relevant tool in the differential diagnosis of endometrial lesions and could be used as a complementary diagnostic method, especially in histomorphologically difficult cases.     

甲状腺肿瘤细胞核DNA含量测定对病理诊断的意义

应用真彩色医学图像分析技术, 对９０ 例甲状腺肿瘤（其中甲状腺腺瘤１０ 例, 不典型腺瘤１５ 例,乳头状腺癌２５ 例, 滤泡癌１５ 例, 髓样癌１５ 例, 未分化癌１０ 例） 细胞核ＤＮＡ含量进行了分析。结果显示,甲状腺腺瘤组与各型甲状腺癌比较均有显著性差异（Ｐ＜ ００１）,甲状腺腺瘤组同不典型腺瘤组比较无统计学意义（Ｐ＞ ００５）。甲状腺癌随组织分化程度的不同, ＤＮＡ 含量明显增加, 多为高倍异倍体细胞, ＤＮＡ直方图明显右移, 峰值主要位于≥５Ｃ处; 甲状腺腺瘤组ＤＮＡ含量较低, 多为低倍整倍体细胞, ＤＮＡ 直方图峰值位于２Ｃ－ ４Ｃ处; 不典型腺瘤组ＤＮＡ含量介于上述二者之间, ＤＮＡ 直方图逐渐右移。表明ＤＮＡ倍性程度与肿瘤的增殖程度呈正相关, 高倍异倍体细胞随肿瘤恶性程度的增高而增多。作者认为ＤＮＡ原位图像定量分析可为甲状腺肿瘤的诊断、分级及早期发现癌变趋势提供一个可靠的参考指标     

Overexpression of CD90 (Thy-1) in Pancreatic Adenocarcinoma Present in the Tumor Microenvironment

CD90 (Thy-1) plays important roles in oncogenesis and shows potential as a candidate marker for cancer stem cells (CSCs) in various malignancies. Herein, we investigated the expression of CD90 in pancreatic adenocarcinoma (PDAC), with a comparison to normal pancreas and non-malignant pancreatic disease, by immunohistochemical (IHC) analysis of tissue microarrays containing 183 clinical tissue specimens. Statistical analysis was performed to evaluate the correlation between CD90 expression and the major clinicopathological factors after adjustment of age and gender. The IHC data showed that CD90 was significantly overexpressed in PDAC and its metastatic cancers as compared to chronic pancreatitis and benign islet tumors, while it was negative in normal pancreas and 82.7% of adjacent normal pancreas tissues. The abundant CD90 expression was predominantly present in PDAC stroma, such as fibroblasts and vascular endothelial cells, which could serve as a promising marker to distinguish pancreatic adenocarcinoma from normal pancreas and non-malignant pancreatic diseases. Double immunostaining of CD90 with CD24, a CSC marker for PDAC, showed that there was little overlap between these two markers. However, CD90⁺ fibroblast cells were clustered around CD24⁺ malignant ducts, suggesting that CD90 may be involved in the tumor-stroma interactions and promote pancreatic cancer development. Furthermore, CD90 mostly overlapped with α-smooth muscle actin (αSMA, a marker of activated pancreatic stellate cells (PSCs)) in PDAC stroma, which demonstrated that CD90⁺ stromal cells consist largely of activated PSCs. Double immunostaining of CD90 and a vascular endothelial cell marker CD31 demonstrated that CD90 expression on vascular endothelial cells was significantly increased in PDACs as compared to normal pancreas and non-malignant pancreatic diseases. Our findings suggest that CD90 could serve as a promising marker for pancreatic adenocarcinoma where desmoplastic stroma plays an important role in tumor growth and angiogenesis.     

Influence of section thickness, mean nuclear diameter and nuclear crowding on DNA ploidy in histologic sections of melanocytic skin lesions

OBJECTIVE: To determine the influence of section thickness, nuclear diameter (MND) and area percentage of nuclei (a measure of nuclear crowding) on histologic DNA ploidy assessed by image cytometry (ICM) of primary melanocytic skin neoplasms (MSNs). STUDY DESIGN: Initially a feasibility study was performed to determine if comparable DNA ploidy histograms could be obtained from cell disaggregates and tissue sections. Following this, DNA ICM was performed on Feulgen-stained tissue sections (4, 6, 8 and 10 microns thick) from 30 primary MSNs (20 benign, 10 malignant) with nuclear diameters from 5.6 to 8.6 microns. Area percentage of nuclei was assessed in all cases at all section thicknesses. RESULTS: The feasibility study produced comparable results for cytocentrifuge and tissue section preparations. For sectioned MSNs, DNA ploidy histograms from 4-micron sections had a higher coefficient of variation of the 2c peak than those from 6-, 8- and 10-micron sections. Ten-micrometer sections had marked overlapping of nuclei, and only small numbers of cells could be measured, giving inadequate results. MND and area percentage of nuclei did not have an important influence on the results. CONCLUSION: Adequate DNA ploidy profiles can be obtained by DNA ICM on 6- and 8-micron-thick histologic sections of MSNs, provided that a strict measurement protocol is followed.     

Fine needle aspiration cytology of neuroendocrine tumors of the pancreas. A cytologic, immunocytochemical and electron microscopic study.

We report the fine needle aspiration cytology findings in six cases of neuroendocrine tumor of the pancreas. Three cases were from the pancreas, two from hepatic metastases and one from a peripancreatic lymph node metastasis. The cytologic features that permitted a preoperative diagnosis of pancreatic neuroendocrine tumor were: a cellular aspirate; numerous isolated cells and irregular, loose, dyshesive cellular aggregates; minimal nuclear pleomorphism; infrequent mitoses; fine, evenly dispersed nuclear chromatin with occasional inconspicuous nucleoli; a scant-moderate amount of granular, amphophilic, well-defined cytoplasm; clustering of tumor cells around segments of capillaries; and rosette formation. The differential diagnosis includes cells derived from normal pancreatic acini, islet cell hyperplasia, acinic cell carcinoma, well-differentiated pancreatic adenocarcinoma, metastatic small cell undifferentiated carcinoma of the lung, pancreatic small cell anaplastic carcinoma and malignant lymphoma. The application of immunocytochemistry to cytologic smears can be easily and reliably performed to confirm the neuroendocrine nature of the tumor and identify the specific type of polypeptide hormone or hormones produced by these tumors. Four aspirates showed immunoreactivity for chromogranin, and one was positive for gastrin. Cells of a lipid-rich neuroendocrine tumor were negative for chromogranin; however, the tissue section contained neuron specific enolase, and neurosecretory granules were demonstrated by electron microscopy.     

Development and retention of phenotypically specialized cells in pituitary allografts in the hamster (Mesocricetus auratus)

Summary We used immunohistochemistry to identify cells present in pituitary allografts in the hamster. Hypophyses removed from neonatal hamsters or adenohypophyses removed from adult females were placed beneath renal capsules of hypophysectomized adult females. Serum PRL, LH, and GH concentrations were measured at two, five, and eight weeks after placement of allografts. Allografts were removed after eight weeks and stained for cells containing PRL, LH, FSH, GH, or ACTH. Allografts did not release LH or GH. Those of adult adenohypophyseal tissue released significantly more PRL. The morphology of allografts of neonatal hypophyseal tissue resembled that of the adult adenohypophysis in situ. Lactotrophs, corticotrophs, somatotrophs and LH-cells were observed; very few FSH-cells were present. Allografts of adult adenohypophyseal tissue contained pituitary cells, numerous cavities, often enclosing lymphoid cells, and fibrous tissue. Atypical lactotrophs were the numerically dominant cells in these allografts; all other cells were present. The LH-cells outnumbered FSH-cells. These observations suggest that: (a) development of normal adenohypophyseal morphology can occur in an ectopic position; (b) intracellular hormones are present in cells in an ectopic site; (c) development and retention of intracellular FSH is more dependent on occupation of the normal position of the adenohypophysis than is retention of intracellular LH; and (d) release of PRL occurs from atypical cells in allografts of adult adenohypophyseal tissue.     

Proteomics portrait of archival lesions of chronic pancreatitis

Chronic pancreatitis is a chronic inflammatory disorder of the pancreas. The etiology is multi-fold, but all lead to progressive scarring and loss of pancreatic function. Early diagnosis is difficult; and the understanding of the molecular events that underlie this progressive disease is limited. In this study, we investigated differential proteins associated with mild and severe chronic pancreatitis in comparison with normal pancreas and pancreatic cancer. Paraffin-embedded formalin-fixed tissues from five well-characterized specimens each of normal pancreas (NL), mild chronic pancreatitis (MCP), severe chronic pancreatitis (SCP) and pancreatic ductal adenocarcinoma (PDAC) were subjected to proteomic analysis using a "label-free" comparative approach. Our results show that the numbers of differential proteins increase substantially with the disease severity, from mild to severe chronic pancreatitis, while the number of dysregulated proteins is highest in pancreatic adenocarcinoma. Important functional groups and biological processes associated with chronic pancreatitis and cancer include acinar cell secretory proteins, pancreatic fibrosis/stellate cell activation, glycoproteins, and inflammatory proteins. Three differential proteins were selected for verification by immunohistochemistry, including collagen 14A1, lumican and versican. Further canonical pathway analysis revealed that acute phase response signal, prothrombin activation pathway, and pancreatic fibrosis/pancreatic stellate cell activation pathway were the most significant pathways involved in chronic pancreatitis, while pathways relating to metabolism were the most significant pathways in pancreatic adenocarcinoma. Our study reveals a group of differentially expressed proteins and the related pathways that may shed light on the pathogenesis of chronic pancreatitis and the common molecular events associated with chronic pancreatitis and pancreatic adenocarcinoma.     

Microvascular density in chronic pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic adenocarcinoma represent two pathologic phenomena with marked production of connective tissue stroma containing numerous small blood vessels. The aim of this study was to characterise quantitatively the vascular supply of pancreatic adenocarcinoma and fragments of the periductal tissue collected from patients with chronic pancreatitis. The study material included 18 cases of pancreatitis and 22 cases of pancreatic ductal adenocarcinoma. Microvessels were marked using monoclonal anti-CD34 antibodies. The number of blood vessels in the fibrous stroma was significantly higher in the chronic pancreatitis samples compared to the pancreatic carcinoma group (mean vessel count 298 and 194 vessel/mm2; median 251 and 187 vessel/mm2 respectively; p<0.01). Distributions of the vascular diameter in both studied groups were very similar. The obtained results suggest that the development of vascular network accompanying chronic pancreatitis is more effective in some parts of pancreas compared to angiogenic intensity in pancreatic adenocarcinoma.     

Is agNOR and DNA ploidy analysis useful for evaluating thyroid neoplasms?

OBJECTIVE: To correlate the subjective AgNOR counting method and DNA content with histologic diagnoses of thyroid cancer and invasion. STUDY DESIGN: Eighty-one consecutive cases of thyroid carcinoma were selected for DNA and AgNOR analysis. The diagnoses were: papillary carcinoma (n = 40), follicular carcinoma (n = 31), Hürthle cell adenocarcinoma (n = 4), and undifferentiated carcinoma (n = 6). Seven normal thyroids were used as controls. DNA quantitative measurement was performed with Vidas 2.0 software (Kontron Bildanalyse, Munich, Germany) connected to an MPM 210 photometer microscope (Carl Zeiss, Oberkochen, Germany). The DNA index was obtained using histograms. Counting the NORs was performed by subjectively counting the NORs in 200 malignant cells. RESULTS: DNA ploidy analysis showed all Hürthle cell adenocarcinomas, 21 (67%)follicular tumors, 23 (57%) papillary tumors and 4 (67%) undifferentiated carcinomas to be aneuploid. DNA analysis correlated with histologic type of the tumor (p = 0.032). There was no statistical significance to the AgNOR counting variables studied. Statistical analysis showed correlation between ploidy and histologic diagnosis, but not AgNOR counting, to have prognostic value. CONCLUSION: DNA ploidy is more useful than subjective counting of NORs as an adjunct method for thyroid lesion analysis.     

胃癌细胞DNA含量、AgNOR计数与其生物学特性关系的研究

本文使用图像分析技术测定了正常胃粘膜、萎缩性胃炎、不典型增生及胃癌共８９例细胞ＤＮＡ相对含量及ＤＮＡ倍体分布;并用银染方法对８９例细胞核仁形成区嗜银蛋白进行定量分析。结果显示：不典型增生往往可观察到与胃癌相似的ＤＮＡ核型即高异倍体的出现,高异倍体的出现可能是重要的癌前标志;在胃粘膜病变中,ＡｇＮＯＲ值随病变异型程度的加重而递增,各组间差异均有非常显著性意义（Ｐ＜０．０１）。早期癌、浸润癌和转移癌间,以及癌转移过程中粘膜层、肌层、淋巴结内癌细胞ＤＮＡ含量无明显差异;ＡｇＮＯＲｓ计数均值差异亦无非常显著性意义（Ｐ＞０．０５）。随癌细胞分化程度升高,ＤＮＡ含量增多及异倍体出现率升高;各期胃癌除粘激腺癌与分化型腺癌差异无显著性意义外,余各型胃癌ＡｇＮＯＲｓ值随分化程度升高而增多,差异有显著性意义（Ｐ＜０．０５或Ｐ＜０．０１）。因此,ＤＮＡ含量及倍体分析,ＡｇＮＯＲｓ计数与胃癌的生物学行为密切相关,可作为胃癌早期诊断,癌前病变预测以及组织分级的一种重要指标。     

Atypical Mesothelial Cells Associated With Eosinophilic Pleural Effusions: Nuclear Dna Content and Immunocytochemical Staining Reaction With Epithelial Markers

The nuclear DNA contents of atypical mesothelial cells from five patients who had an eosinophilic pleural effusion (EPE) were studied by the use of DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) DNA staining. Analysis of the nuclear DNA content revealed a polyploid pattern, with a major peak in the tetraploid region. Using an immunocytochemical technique, the atypical mesothelial cells showed a positive reaction for cytokeratin. In contrast carcinoembryonic antigen (CEA) was always negative in these cells. It is suggested that the atypical mesothelial cells with EPE had a higher rate of proliferation than did the normal mesothelial cells.     

Increased B Cell-Activating Factor Promotes Tumor Invasion and Metastasis in Human Pancreatic Cancer

B cell-activating factor (BAFF) is a cytokine belonging to the tumor necrosis factor (TNF) superfamily. It has been reported that BAFF is elevated in patients with autoimmune pancreatitis and contributes to the malignant potential of blood cancers and solid tumors. In this study, clinical evidence of increased BAFF levels in patients with pancreatic ductal adenocarcinoma (PDAC) was obtained, and the roles and mechanisms of BAFF in PDAC were clarified in human tissues of PDAC and from in vitro data of PDAC cell lines. Serum levels of BAFF in patients with PDAC were significantly higher than in healthy subjects (p = 0.0121). Patients with UICC stage IV PDAC (T1-4, N0-1, M1) had significantly higher levels of serum BAFF compared to patients with PDAC (p = 0.0182). BAFF was remarkably expressed in infiltrating B lymphocytes surrounding pancreatic cancer in human pancreatic tissues, suggesting that BAFF may play a role in progression of pancreatic cancer. PDAC cell lines were cultured with human recombinant BAFF, and morphology and gene expression were analyzed; pancreatic cancer cells changed to a fibroblast-like morphology, and showed altered gene expression of E-cadherin, vimentin and Snail. These BAFF-induced changes reflect enhanced cell motility and invasion. BAFF-R-overexpressing cell clones confirmed the association between these BAFF-induced changes and epithelial-mesenchymal transition (EMT)-related genes. BAFF was elevated in patients with metastatic advanced PDAC and induced alterations in PDAC cells via regulation of EMT-related genes. Elucidation of the precise role and mechanism of control of BAFF may lead to new therapeutic approaches with the aim of improving pancreatic cancer survival.     

Influence of conjugated and conventional linoleic acid on tumor growth and lipid peroxidation in pancreatic adenocarcinoma in hamster

Conventional linoleic acid (LA) is regarded as a promotor of carcinogenesis. However, the effect of its conjugated derivative on cancer is still unknown. Therefore we investigated the influence of conventional and conjugated LA on tumor growth and lipid peroxidation in a solid model of pancreatic adenocarcinoma in Syrian hamsters. 60 male hamsters were randomized in 4 groups (Gr.) (n=15). Gr. 1 and 2 received 0.5 ml 0.9% sodium chloride subcutaneously (s.c.) once a week while Gr. 3 and 4 were injected 10 mg N-nitrosobis-2-oxopropylamine (BOP)/kg body weight weekly for 12 weeks to induce pancreatic cancer. Gr. 1 and 3 received a diet containing conventional LA, Gr. 2 and 4 were fed a diet of conjugated LA. After 29 weeks all animals were sacrificed, pancreas was weighed and examined macroscopically and histologically. The level of lipid peroxidation and activities of glutathion peroxidase and superoxide dismutase were determined in tumor-free as well as in pancreatic carcinoma tissue. Different diets did not influence the incidence of pancreatic carcinoma, however, pancreas weight was increased by conjugated LA compared to conventional LA. Furthermore both diets decreased the activity of glutathion peroxidase and increased the level of lipid peroxidation in pancreatic intratumoral tissue. The content of conjugated LA in dietary did not influence pancreatic tumor growth in a solid model of pancreatic adenocarcinoma in Syrian hamsters.     

Morphometry in surgical pathology

The potential role of morphometry in surgical pathology is discussed. Specific areas in which morphometry could be helpful are in (1) identifying malignant cells in lesions that are largely composed of benign-appearing cells (e.g., follicular thyroid neoplasms), (2) defining reference points in apparent continua (e.g., in the progression from normal colon to adenoma to adenocarcinoma), (3) distinguishing between benign and malignant lesions with similar appearances (e.g., fibromatosis and fibrosarcoma of the soft tissue) and (4) distinguishing between similar-appearing types of malignant neoplasms (e.g., between small-cell carcinoma of the lung and small-cell lymphoma). Morphometric techniques are already being used in DNA ploidy determinations, which frequently bear prognostic information. The measurement of other nuclear and cellular parameters has been used for both diagnostic and prognostic ends; one example is the relation of nuclear roundness to metastatic potential in prostatic carcinomas. Morphometry is now being increasingly applied to histologic sections, as in the prognostic study of lesion thickness in malignant melanoma and the diagnostic study of glandular architecture in colonic adenoma. The use of morphometry can enhance the observation and interpretation of morphologic features, which, combined with the clinical data and the experience of the pathologist, can lead to greater accuracy and precision in surgical pathology diagnoses.     

Intracellular pH, esterase activity, and DNA measurements of human lung carcinomas by flow cytometry

An important intention of flow cytometric investigations is to obtain biochemical and biophysical information about cells which is suitable for automated tumor diagnosis. In this study, the ploidy status, the intracellular pH value, the intracellular esterase activity, and the cell volume of vital cells and the DNA and cell volume of dead cells were measured in cancerous tissue and normal lung tissue of 30 patients by flow cytometry. The cell samples were simultaneously stained with the pH and esterase indicator dye 1.4-diacetoxy-2,3-dicyanobenzene (ADB) and propidium iodide (PI). The flow cytometric measurements were performed in three-parameter list mode. The data were evaluated on an AT-compatible personal computer with the DIAGNOS1 program system for automated diagnosis of flow cytometric list mode data. Significant differences were found between normal and malignant tissue in DNA ploidy, in the intracellular esterase activity, in the cell, volume and in the percentage of inflammatory cells and parameters of necrosis. DNA-aneuploidy was observed in 38% of the lung carcinomas. The simultaneous detection of DNA-aneuploidy and tumor-associated properties in a multifactorial analysis led to correct automatic tumor diagnosis in 85% of cases. DNA-aneuploidy was found at a significant higher frequency in advanced tumors. Adenocarcinomas displayed DNA-aneuploidy more often (80%) than squamous cell carcinomas (33%).     

Recovery of exocrine pancreas six months following pancreatitis induction with L-arginine in streptozotocin-diabetic rats.

The aim of the present work was to investigate the laboratory and morphologic alterations in the pancreas 6 months after pancreatitis induction with L-arginine (Arg) in normal and streptozotocin (STZ)-diabetic rats. The amylase content of the pancreas was significantly decreased in the Arg-treated groups vs. the control group. No significant changes were observed in the DNA, soluble protein and lipase contents of the pancreas. In the STZ-treated groups, the serum glucose level was significantly elevated, whereas the serum immunoreactive insulin (IRI) level was significantly decreased vs. the control group. In these treated groups, the amylase content of the pancreas was also significantly decreased, but that of trypsinogen was significantly elevated vs. the control group. Histologic sections revealed periductal fibroses, adipose tissue and tubular complexes in the Arg-treated rats, but centroacinar hyperplasia was not observed in these groups. No alterations were observed on histological examination in the diabetic rats vs. normal rats 6 months following pancreatitis induction. In conclusion, a major restitution of the pancreatic enzyme content, but moderate histologic alterations were detected 6 months following pancreatitis induction with Arg. The diabetic state appeared to shift the normal pancreatic enzyme content (decreased amylase and increased trypsinogen) in this long-term study, but not to modify the recovery of the exocrine pancreas 6 months following Arg-induced pancreatitis.     

A method for determining ploidy distributions in liver tissue by stereological analysis of nuclear size calibrated by flow cytometric DNA analysis

A method is presented for determining ploidy distributions in mouse liver from image analysis with stereological estimations of nuclear size in tissue sections. Nuclear profile distributions obtained from profile measurements were subjected to a mathematical unfolding procedure in order to obtain the nuclear size distributions. Based on the assumption that nuclear size increases monotonically with nuclear DNA content, flow cytometric DNA analysis of suspensions of liver cell nuclei was used to calibrate the method, thus yielding the mean nuclear size of each ploidy class, i.e., diploid, tetraploid, and octaploid nuclei. After the size interval for each of the ploidy classes was determined, the method allowed determination of ploidy distributions in mouse liver by stereological image analysis alone. The method was established from combined stereological and flow cytometric measurements on liver tissue representing two different stages of liver regeneration after two-thirds partial hepatectomy, and it was tested against an independent set of data representing a marked increase in the portion of S-phase cells.     

Pancreatic Duct Obstruction in the Pig: Light Microscopy of Chronic Pancreatitis

Morphological changes of pancreatic tissue in young pigs caused by surgical ligation of the main pancreatic duct are described. Nineteen animals from 6 to 7 weeks in age were operated on and necropsied 3 or 6 to 8 weeks later. Twelve pigs developed a pronounced chronic pancreatitis with complete exocrine insufficiency. Of the 7 animals failing to develop ectasia of pancreatic ducts, 2 died due to surgical complications. In addition, 3 pigs were sham-operated and served as controls. In macroscopical studies it was observed that in the pronounced pancreatitis cases the ligated duct was greatly dilated by a clear watery fluid. Only remnants of pale and firm grandular tissues were seen around the ectatic ducts. Microscopically, typical changes of chronic pancreatitis were noted. Complete disappearance of acini was followed by ductular cell proliferations. Glandular tissues were divided into lobuli by fibrotic tissues and fat cells. The wall of the main pancreatic duct was greatly thickened and fibrotic, presenting intensely proliferating ductular cells and round cell infiltrates. Furthermore, enlarged endocrine islets surrounded by connective tissue fibres were seen.