Connecting cyto-nano-architectural attributes and epithelial molecular expression in oral submucous fibrosis progression to cancer.

OBJECTIVE: Problems in pre-cancer diagnosis complicate cancer theragnosis as well as life expectancy. There is uncertainty regarding malignant transformation of oral submucous fibrosis (OSF), an oral pre-cancer with dysplastic (OSFWD) and non-dysplastic (OSFWT) subtypes. Understanding the structural, molecular and physical aspects of epithelial homeostasis may be useful. MATERIALS AND METHODS: Histopathological grading of biopsy sections was performed using H&E staining. Alterations in epithelial surface architecture in different groups was evaluated using scanning electron microscopy (SEM). The expression of crucial epithelial genes (p63, CK-5/6, CK-10, E-cadherin and ß-catenin) was studied by immunohistochemistry, Western blot and RT-PCR analysis. RESULTS: SEM observations revealed that the surface epithelial ridge pattern became thick and dense, and pit pattern gradually decreased in OSFWD and oral squamous cell carcinoma (OSCC). p63, ΔNp63 and CK-5/6 were up-regulated in OSFWD and OSCC but down-regulated in OSFWT. CK-10 was down-regulated in OSFWD compared to OSFWT. Cytoplasmic expression of E-cadherin and ß-catenin was elevated in dysplastic and cancerous conditions. Moreover, statistical correlation between SEM features (ridges and pits) and molecular attributes demonstrated a significant positive relationship between the ridge-to-pit ratio and p63 population density (r=0.85) and the ridge-to-pit ratio and CK-5/6 intensity (r=0.63). CONCLUSIONS: Molecular changes related to epithelial progressive maturation and cellular proliferation are correlated with concomitant alteration of epithelial surface architecture which helps to predict the malignant potentiality of OSF.

Investigation of CPD and HMDS sample preparation techniques for cervical cells in developing computer-aided screening system based on FE-SEM/EDX.

This paper investigated the effects of critical-point drying (CPD) and hexamethyldisilazane (HMDS) sample preparation techniques for cervical cells on field emission scanning electron microscopy and energy dispersive X-ray (FE-SEM/EDX). We investigated the visualization of cervical cell image and elemental distribution on the cervical cell for two techniques of sample preparation. Using FE-SEM/EDX, the cervical cell images are captured and the cell element compositions are extracted for both sample preparation techniques. Cervical cell image quality, elemental composition, and processing time are considered for comparison of performances. Qualitatively, FE-SEM image based on HMDS preparation technique has better image quality than CPD technique in terms of degree of spread cell on the specimen and morphologic signs of cell deteriorations (i.e., existence of plate and pellet drying artifacts and membrane blebs). Quantitatively, with mapping and line scanning EDX analysis, carbon and oxygen element compositions in HMDS technique were higher than the CPD technique in terms of weight percentages. The HMDS technique has shorter processing time than the CPD technique. The results indicate that FE-SEM imaging, elemental composition, and processing time for sample preparation with the HMDS technique were better than CPD technique for cervical cell preparation technique for developing computer-aided screening system.

Specific intraepithelial localization of mast cells in differentiated vulvar intraepithelial neoplasia and its possible contribution to vulvar squamous cell carcinoma development.

AIMS: The aetiology of vulvar squamous cell carcinomas (SCC) that are not causally associated with high-risk human papillomavirus remains largely elusive. The aim of this study was to analyse the inflammatory response in its presumed precursor lesions, lichen sclerosus (LS) and differentiated vulvar intraepithelial neoplasia (dVIN), and provide evidence that dVIN is a likely precursor of vulvar SCC. METHODS AND RESULTS: Immunohistochemical analyses for CD4+, CD8+, CD20+, CD68+, S100+ and tryptase-positive immune cells were performed and quantified in LS (n = 7), dVIN (n = 19), SCC (n = 11), and normal vulvar tissue (n = 8). The subepithelial inflammatory response in dVIN and SCC was comparable, but absent in LS. Abundant intraepithelial mast cells were observed in dVIN only, and confirmed by electron microscopy, toluidine blue staining and cKIT expression. Adjacent keratinocytes displayed increased proliferation as determined by MIB-1 positivity. Electron microscopy revealed intraepithelial mast cell degranulation. Intraepithelial mast cells were not or infrequently observed in vulvar hyperplasia (n = 13), condylomata acuminata (n = 5), keratinocytic intraepidermal neoplasia of sun-exposed skin (n = 15), epidermal hyperplasia of head and neck (n = 12), and psoriasis (n = 3). CONCLUSIONS: These data indicate that dVIN can be recognized by intraepithelial mast cells and that they might promote the progression of dVIN to SCC.

Preneoplastic and neoplastic changes in the Leydig cells population in mice exposed to low doses of cadmium.

The morphological consequences of chronic exposition to low doses of cadmium (Cd) in the Leydig cells population were investigated in 40 sexually mature male mice at morphological and ultrastructural levels. Animals were orally exposed to cadmium (0.015 g/L of CdCl(2) in drinking water) for 3, 6, 12 and 18 months and then sacrificed, samples were collected for toxicological, light and electron microscope studies. Vascular lesions were evident from 6 months of Cd exposure, the severity of the morphological changes observed in the testicular vases were highly and clearly correlated to the time of exposure to Cd. The severity of the Leydig cells morphological changes were increasing along the time of exposure. Presence of cytoplasm vacuolization and degenerative images of the cells were frequent after 12 months of Cd exposure. Also two Leydig cells tumours after 12 and 18 months Cd exposure were presented. These results indicate that prolonged exposures to low doses of Cd are able to induce morphological damage on the Leydig cells.

Confocal laser endomicroscopy.

Endomicroscopy is a newly developed imaging modality, which provides in vivo histology during ongoing endoscopy. This review characterizes the currently available endomicroscopic systems and reflects the clinical value of endomicroscopy for different diseases. Endomicroscopy can be used to discover histology of the mucosal layer at cellular and subcellular resolution. Furthermore, endomicroscopy can be used to observe physiologic and pathophysiologic changes, which offer a newly available insight into the pathogenesis of different diseases. The diagnostic possibilities of endomicroscopy are extensive and highly valuable for every day practice. However, the era of endomicroscopy has just started and it can be anticipated that its role will significantly increase in the future.

Pancreatic cancer and precursor pancreatic intraepithelial neoplasia lesions are devoid of primary cilia.

Primary cilia have been proposed to participate in the modulation of growth factor signaling pathways. In this study, we determined that ciliogenesis is suppressed in both pancreatic cancer cells and pancreatic intraepithelial neoplasia (PanIN) lesions in human pancreatic ductal adenocarcinoma (PDAC). Primary cilia were absent in these cells even when not actively proliferating. Cilia were also absent from mouse PanIN cells in three different mouse models of PDAC driven by an endogenous oncogenic Kras allele. Inhibition of Kras effector pathways restored ciliogenesis in a mouse pancreatic cancer cell line, raising the possibility that ciliogenesis may be actively repressed by oncogenic Kras. By contrast, normal duct, islet, and centroacinar cells retained primary cilia in both human and mouse pancreata. Thus, arrested ciliogenesis is a cardinal feature of PDAC and its precursor PanIN lesions, does not require ongoing proliferation, and could potentially be targeted pharmacologically.

[Helicobacter pylori infection and changes of cell gap junction of gastric epithelial cells in patients with gastric cancer and precancerous lesion].

OBJECTIVE: To observe the changes of cell gap junction ultrastructure of gastric epithelial cells in patients with gastric cancer(GC) and precancerous lesion(PL),and to investigate the relation between these changes and H.pylori infection. METHODS: Seventy patients with GC, 88 with PL, and 33 with chronic superfial gastritis (CSG) were studied. H.pylori was detected by rapid urease test,basic fuchsin stain and 14C-urea breath test. The CagA gene of H.pylori was determined by polymerase chain reaction(PCR).The cell gap junction ultrastructure was observed under transmission electronic microscope. RESULTS: Length of junction/unit perimeter of gastric epithelial cells in patients with PL was smaller than that in CSG patients, and the smallest width of the intercellular space was bigger than that in CSG patients. The number of cell junction, the number of junction/unit perimeter, and the length of junction/unit perimeter in patients with GC were all smaller than those in patients with CSG or PL, and its smallest width of the intercellular space was bigger than that in patients with CSG. In patients with GC, the number of cell junction, the number of junction/unit perimeter and the length of junction/unit perimeter in CagA+ H.pylori group were smaller than those in CagA(-) H.pylori group, and its smallest width of the intercellular space was bigger than that in CagA(-) H.pylori group. In PL patients, the intercellular space decreased, and the length of cell junction of gastric epithelial cells became bigger after H.pylori eradication. The length of junction/unit perimeter in patients of H.pylori eradication was bigger than that in patients without eradication, and the smallest width of the intercellular space was smaller than that in patients without eradication. CONCLUSION: The changes of cell gap junction of gastric epithelial cells in patients with GC and PL are associated with H.pylori infection especially CagA+ H.pylori infection. Eradication of H.pylori can promote the formation of cell junction.

A scanning electron microscopic study of the dysplastic epithelia adjacent to oral squamous cell carcinoma.

By light microscopy, the dysplastic oral epithelia due to the neoplastic processes are similar to epithelial changes due to the inflammatory processes. Scanning electron microscopy may elucidate the different surface changes between the two. The aim of this study was to examine the surface appearances of the dysplastic oral epithelia adjacent to oral squamous cell carcinoma to see if there are any surface changes. A total of 2 specimens, one specimen from each patient with oral squamous cell carcinoma, were used for this study. Each specimen was divided in two. One half was prepared for light microscopy and the other half was prepared for scanning electron microscopy. Light microscopically, the epithelia showed mild dysplasia. By scanning electron microscopy, the keratinized cells showed irregular microridges surrounding pits, which were variable and irregular in size and shape, and the nonkeratinized cells showed parallel microridges with irregularly widened intervals between each microridge. Irregular, broad, and partly swollen microridges and irregular short, stubby surface projections were also seen. The oral epithelia adjacent to oral squamous cell carcinoma showed mild dysplasia light microscopically but appeared abnormal by scanning electron microscopy. The abnormal epithelial cells showed pleomorphism, irregular and disoriented microridges, and abnormal surface microstructures.

Performance analysis of different wavelet feature vectors in quantification of oral precancerous condition.

This paper presents an automatic method for classification of progressive stages of oral precancerous conditions like oral submucous fibrosis (OSF). The classifier used is a three-layered feed-forward neural network and the feature vector, is formed by calculating the wavelet coefficients. Four wavelet decomposition functions, namely GABOR, HAAR, DB2 and DB4 have been used to extract the feature vector set and their performance has been compared. The samples used are transmission electron microscopic (TEM) images of collagen fibers from oral subepithelial region of normal and OSF patients. The trained network could classify normal fibers from less advanced and advanced stages of OSF successfully.

Nuclear morphometry in columnar cell lesions of the breast: is it useful?

AIMS: To evaluate the nuclear morphometric features of breast columnar cell lesions (CCLs) observed on mammotome core biopsies, to determine if there are significant measurable differences between those with atypia and those without. Correlation with follow-up open excision specimens was made. METHODS: Mammotome core biopsies performed on patients that contained CCLs were derived from the departmental case files. Histological material was reviewed and foci of CCLs demarcated for nuclear morphometric assessment, which was accomplished using an imaging system. Nuclear parameters studied were nuclear area and perimeter, circularity factor and feret's diameter. Statistical analysis used the GraphPad Prism software, with p<0.05 indicating significance. RESULTS: On examination of core biopsies of 40 patients with CCLs, 8 lesions were benign, 4 showed atypical lobular hyperplasia, 8 showed CCLs with nuclear atypia, 19 disclosed atypical ductal hyperplasia (ADH) and 1 showed ductal carcinoma in situ (DCIS). The nuclear area, perimeter and feret's diameter of CCLs with atypia were significantly greater than those without (p = 0.04, 0.03 and 0.019, respectively), whereas no difference was observed in the circularity factor. Follow-up open excision biopsy specimens in 24 patients showed upgrading to DCIS in 40% of cases diagnosed initially with ADH on core biopsy compared with 20% of CCLs with atypia. CONCLUSIONS: Nuclear morphometry in CCLs confirms nuclear size as the key parameter in the assessment of nuclear atypia. Whether it can be potentially used as an adjunctive tool depends on the establishment of appropriate cut-offs.

Three-dimensional analysis of isolated hexosaminidase-altered aberrant crypts from colons of 1,2-dimethylhydrazine-treated rats.

Aberrant crypt foci, consisting of morphologically irregular crypts, are thought to be precancerous lesions for colorectal cancers. For analysis of individual crypts, F344 rats were administered weekly subcutaneous injections of 1,2-dimethylhydrazine ten times and sacrificed at experimental weeks 10 and 20 with 5-bromo-2'-deoxyuridine (BrdU) incorporation 1 h before the sacrifice. Isolated colonic crypts were classified into hexosaminidase-altered aberrant crypts (HAACs) and hexosaminidase-preserved normal-appearing crypts (HPNCs) and stereopaired images (tilt angle, 6 degrees ) were taken with a scanning electron microscope for three-dimensional analyses. While HPNCs showed symmetrical fission at the base, HAACs exhibited abnormal budding in the middle of the crypt body. At week 10, average BrdU labeled cells per crypt for DMH-treated HPNCs and HAACs were 4.9 +/- 1.0 and 18.7 +/- 2.2 (P < 0.0001), respectively, while the value for non-treated control crypts was 14.7 +/- 0.8/crypt. BrdU-positive cell numbers per unit crypt length (100 microm) in HPNCs and HAACs were 1.75 +/- 0.37 and 5.99 +/- 0.70 (P < 0.0001), respectively, while that for the control was 6.65 +/- 0.35 (P < 0.02 vs. HAAC). At the 20-week time point, the numbers per crypt were 4.0 +/- 0.8, 10.1 +/- 1.6, and 27.4 +/- 2.4, respectively, the control value being significantly higher than the others (P < 0.0001). The figures per unit length were 1.72 +/- 0.35, 2.92 +/- 0.42, and 13.39 +/- 1.11 (P < 0.0001 vs. HAAC and HPNC), respectively. BrdU incorporating cells were distributed in the bottom third of the crypt columns in HAACs, but only 18% in the HPNCs, providing evidence of hyperplasia. HAACs could be good surrogate indicators of carcinogen exposure, at least some of which may be related to colon carcinogenesis.

Scanning electron microscopy of colonic lesions in 1,2-dimethylhydrazine-treated rats.

The surface morphology of late colonic lesions in F344 rats treated with 1,2-dimethylhydrazine was studied by scanning electron microscopy. At week 31 after carcinogen treatment, the surface epithelial characteristics of different types of lesions observed in the colonic mucosa were compared, namely classic elevated aberrant crypt foci (ACF), flat lesion and gross tumour. Classic elevated ACF were easily observed as structures with enlarged crypts elevated from the background mucosa. When the various ACF were compared, or when the ACF were compared with the background mucosa, no ultrastructural differences, or differences in the density of goblet cells were found. The flat lesion showed an epithelium without goblet cells and crypts with small openings harbouring a large number of loose, undefined, dysplastic epithelial cells. These changes appeared to be linked to the malignant development since they were also characteristic of the examined tumour.

A novel wavelet neural network based pathological stage detection technique for an oral precancerous condition.

AIM: To describe a novel neural network based oral precancer (oral submucous fibrosis; OSF) stage detection method. METHOD: The wavelet coefficients of transmission electron microscopy images of collagen fibres from normal oral submucosa and OSF tissues were used to choose the feature vector which, in turn, was used to train the artificial neural network. RESULTS: The trained network was able to classify normal and oral precancer stages (less advanced and advanced) after obtaining the image as an input. CONCLUSIONS: The results obtained from this proposed technique were promising and suggest that with further optimisation this method could be used to detect and stage OSF, and could be adapted for other conditions.

Exploratory study of ovarian intraepithelial neoplasia.

PURPOSE: This was an exploratory study to test two hypotheses related to potential epithelial precursors to ovarian cancer: (a) histologically normal ovarian surface epithelium exhibited changes in the nuclear chromatin pattern, which indicate an ovarian abnormality, and (b) such changes were detectable in the ovarian surface epithelium of cancer-free subjects who were at high risk for ovarian cancer. EXPERIMENTAL DESIGN: Ovaries were carefully collected to avoid damage to the surface epithelium. Five-micron-thick histologic sections were cut and stained with H&E. High-resolution images were recorded from the ovarian surface epithelium and from the underlying stroma of ovaries from normal women (10 cases), women at high risk of developing ovarian cancer (7 cases), and histologically normal areas adjacent to ovarian cancer (3 cases). Karyometric features and measurements of nuclear abnormality were computed for 3,390 epithelial nuclei. Discriminant function analyses and unsupervised learning algorithms were employed to define deviations from normal and to identify the subpopulations of nuclei exhibiting these changes. RESULTS: Epithelium from ovaries harboring a malignant lesion had changes in the nuclear chromatin pattern consistent with a second phenotype, which were not visually detected with histopathologic surveillance. This phenotype was also present in the ovaries obtained from women at increased risk of ovarian cancer, suggesting that it may represent a premalignant abnormality. These changes were statistically significant. CONCLUSION: The observed changes in karyometric features were sufficiently distinct to warrant further study as both diagnostic and prognostic biomarkers for early detection and prevention of ovarian cancer.

Ultrastructure and molecular biological changes of chronic gastritis, gastric cancer and gastric precancerous lesions: a comparative study.

AIM: To carry out a comparative study on ultrastructure and molecular biological changes of chronic gastritis (CG), gastric cancer (GC) aand gastric precancerous lesions. METHODS: By the use of histochemical staining, SEM with EDAX, TEM with EDAX, image analysis technique, RIA and chemiluminescence method, gastric mucosa of 168 patients were synchronously analyzed in morphology, trace elements, DNA, cAMP, SOD, (3)H-TdR LCT and serum LPO were also done. RESULTS: The incidence of epithelial nucleoplasmic ratio >1, lobulated nuclei, inter-chromatin aggregation of granules, nucleolar hypertrophy, and the content of DNA, Zn, Cu in nuclei and serum LPO of each group were showed as belows: normal control group (0.0, 0.0, 6.7, 0.0, 12.6+/-2.7, 7.6+/-0.4, 58.4+/-0.3, 2.6+/-0.6), CSG group (5.7, 2.9, 7.4, 2.9, 15.2+/-3.1, 8.1+/-0.5, 58.9+/-0.5, 4.2+/-0.7), CAG group (31.3, 29.7, 45.3, 42.2, 16.5+/-3.1, 8.6+/-0.4, 59.3+/-0.5, 4.5+/-0.6), CA group (100.0, 100.0, 72.2, 50.0, 30.7+/-8.2, 8.8+/-0.3, 59.5+/-0.4, 6.8+/-1.6), ATP(++) group (61.5, 38.5, 23.1, 38.5, 23.5+/-8.9, 8.3+/-0.4, 59.1+/-0.4, 5.1+/-1.2), IM(++)+ATP(++)group (77.8, 55.5, 33.3, 44.4, 25.1+/-7.2, 8.4+/-0.5, 59.5+/-0.4, 6.5+/-1.1), IM(+++)+ATP(++) group (100.0, 100.0, 75.0, 62.5, 28.5+/-9.1, 8.9+/-0.5, 59.7+/-0.4, 7.6+/-0.7), IMII(b) group (100.0, 62.5, 75.0, 50.0, 27.3+/-10.3, 8.6+/-0.3, 59.5+/-0.4, 6.1+/-0.9); whereas the content of Zn, Cu in mitochondria and cAMP, SOD in gastric mucosa, and (3)H-TdR LCT of each group were showen as belows: normal control group (9.2+/-0.5, 58.3+/-0.3, 15.9+/-1.5, 170.5+/-6.1, 1079.7+/-227.4), CSG group (8.6+/-0.5, 57.8+/-0.3, 14.6+/-1.8, 163.3+/-5.6, 867.3+/-240.5), CAG group (8.3+/-0.4, 57.5+/-0.3, 13.4+/-1.8, 161.2+/-4.3, 800.9+/-221.8), CA group (8.9+/-0.4, 57.1+/-0.3, 10.2+/-3.9, 152.2+/-3.8, 325.7+/-186.8), ATP(++) group (9.1+/-0.4, 57.0+/-0.3, 12.4+/-1.8, 161.5+/-3.8, 642.9+/-174.3), IM(++)+ATP(++) group (8.6+/-0.4, 56.9+/-0.3, 12.0+/-2.3, 152.2+/-2.5, 326.3+/-160.3), IM(+++)+ATP(++) group (8.5+/-0.3, 56.8+/-0.2, 10.4+/-0.9, 147.4+/-2.6, 316.1+/-170.7), IMII(b) group (8.6+/-0.3, 56.9+/-0.3, 11.9+/-1.9, 150.0+/-2.8, 318.9+/-145.8), there were significant differences between groups (P<0.05-0.01). CONCLUSION: There was a significant difference between CG and GC in their ultrastructure and molecular biology. Only on the condition of changes of internal environment in combination with the harmful effect of external environment, chronic atrophic gastritis can then develop into gastric cancer. Hence it might have similar epithelial cell ultrastructure and molecular biological changes in ATP(++), IMII(b) and cancer, hence there were similar patterns of occurrence, development and transformation. Recognition of this trend might help to explore problems of prevention and cure.

DNA ploidy and markovian analysis of neoplastic progression in experimental pancreatic cancer.

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.

Elemental sulphur and alkali elutable melanin detected in oral melanosis and malignant melanoma by energy-filtering transmission electron microscopy.

BACKGROUND: The morphology and contents of melanosomes are important features for differentiating melanocyte-derived melanotic lesions such as melanosis and malignant melanoma. METHODS: In this study, we attempted to elucidate the structure of melanin and sulphur content in oral melanosis and malignant melanomas by ultrastructural analysis. RESULTS: In oral melanosis, the essential pathological findings were overproduction of eumelanin and discharge of melanin into keratinocytes. In malignant melanoma in situ, pleomorphic and ellipsoid abnormal melanosomes with an increase in sulphur content and alkali elution rate were detected. In invasive malignant melanoma, the irregular ellipsoid and spheroid melanosomes existing either as discrete bodies or compound melanosomes with furtherly increased sulphur content and alkali elution were detected. CONCLUSIONS: Our findings suggest that abnormal melanosome morphology and high sulphur content are predictive markers for assessment of early or precancerous melanotic lesions and malignant melanoma.

Ultrastructural changes in malignant transformation of oral mucosa.

Transmission electron microscopy (EM) has been used to identify the ultrastructural details of normal and cancerous human oral mucosa. However, inconsistent reports of structural descriptions have rendered transmission EM valueless in the diagnosis of oral squamous cell carcinoma (SCC) or as a prognostic indicator. To identify features of dysplasia for diagnostic purposes, normal mucosa, severe dysplasia, oral SCC and normal margin adjacent to oral SCC were used to compare the ultrastructural features of normal and premalignant oral mucosa and oral SCC. The preparatory stages of dehydration, embedding, cutting and positive staining for transmission EM were modified and tested to improve ultrastructural definition. Thin and discontinuous basal laminas were found in mucosa with severe dysplasia and normal margin adjacent to oral SCC. No basal lamina was identified in oral SCC. This study has shown that there are some ultrastructural changes during malignant transformation of oral mucosa. Together with other laboratory investigative techniques, transmission EM may be helpful in detecting malignant changes in oral mucosa.

Sclerosing polycystic adenosis of parotid gland with dysplasia and ductal carcinoma in situ. Report of three cases with immunohistochemical and ultrastructural examination.

We describe three cases of sclerosing polycystic adenosis (SPA) of the parotid gland, a salivary condition analogous to fibrocystic disease of the breast. For the first time, immunoreactivity for oestrogen and progesterone receptors was demonstrated, suggesting a possible participation of hormone stimulation in its pathogenesis. In addition, all our cases showed foci of dysplasia of the ductal epithelium, which in one case was severe enough to amount to carcinoma in situ. This feature that has not previously been reported in SPA.