Cytodiagnosis of endometrial carcinoma and hyperplasia on imprint smears with additional immunocytochemistry using Ki-67 and p53 biomarkers.

OBJECTIVE: It is said to be difficult to interpret the different endometrial lesions by cytomorphology; however, evaluation of the microarchitecture of the cell clumps and application of immunocytochemistry can improve diagnostic accuracy. The aim of the present study was to evaluate cytolomorphological features and correlate them with the histological diagnosis of benign and malignant endometrial lesions, and to investigate certain immunocytochemical biomarkers to achieve a more accurate cytodiagnosis. METHODS: In the present study, we graded the cytomorphology on imprint smears of 35 low-grade endometrial endometrioid carcinomas compared with 23 cases of endometrium ranging from disordered proliferative to benign hyperplastic. Additionally, 10 cases of high-grade endometrial carcinoma and 11 cases of atrophic endometrium were evaluated. Ki-67 and p53 biomarkers were applied to the cytological smears. RESULTS: A total cytological score less than six, resulting from nuclear overlapping, nuclear/cytoplasmic ratio, the presence of a branched pattern, vesicular cytoplasm and loss of cohesiveness, distinguished all the cases of disordered proliferative and benign hyperplastic endometrium from low-grade endometrioid carcinomas of endometrium (P < 0.0001). The application of different cut-off values for Ki-67 and p53 helped differentiate certain endometrial lesions in our study. The integration of the immunocytochemical score of Ki-67 and p53 into the cytological score resulted in a final score that was also diagnostically useful. CONCLUSIONS: The results of the present study demonstrated that evaluation of certain cytological features along with specific immunocytochemical findings could improve the accuracy of endometrial cytodiagnosis but our findings need to be tested in a routine clinical situation, using pre-operative cytological samples, to ascertain whether the diagnostic criteria are reproducible.

Estimation of maspin's subcellular localization in invasive ductal breast cancer via light microscopy and computerized image analysis: a comparative study.

PURPOSE: Contradictory results have been reported concerning the role of maspin and its cellular distribution in breast cancer. The purpose of this study was to examine the subcellular localization (nuclear-cytoplasmic) of maspin in breast cancer and to compare the evaluation of maspin immunostaining via light microscopy (LM) to the estimation via computerized image analysis (CIA) system. We also examined correlations between maspin expression and several clinicopathological parameters. METHODS: The sample consisted of 48 primary invasive ductal carcinomas (IDC) of the breast. Maspin immunostaining was quantified and graded via LM by two pathologists, separately in the nuclear and cytoplasmic compartments. Total maspin expression was also estimated via CIA system. Univariate non-parametric statistics and stepwise multivariate ordinal logistic regression were performed. RESULTS: Both maspin components (nuclear and cytoplasmic) were closely associated with each other (p<0.001). Total maspin score was positively and closely associated with nuclear maspin (p<0.001) and cytoplasmic maspin (p<0.001). Total maspin , nuclear maspin and cytoplasmic maspin did not correlate significantly with either age, grade, T, N and M status, stage, micro vessel density (MVD) (CD34), ki-67, p53, estrogen receptor (ER) and HER-2 status, or with any of the 4 groups of the molecular classification. The only factor that showed a borderline inverse correlation with nuclear maspin (p=0.059) was progesterone receptors (PR) positivity. CONCLUSION: The cytoplasmic and nuclear fractions of maspin seem to be closely interwoven. Evidently, both mutually intertwined counterparts were independently reflected upon the total maspin levels measured by CIA. Future studies should ideally encompass all three approaches (nuclear, cytoplasmic, total) adopted herein.

c-MYC and h-TERT co-expression in colon adenocarcinoma: a tissue microarray digitized image analysis.

PURPOSE: c-MYC oncogene is frequently deregulated by amplification in colon adenocarcinoma. c-MYC also activates telomerase by inducing expression of its catalytic subunit (h-TERT). Furthermore, telomerase activation plays a crucial role in tumorigenesis by sustaining cellular immortality. Our aim was to evaluate the significance of c-MYC and h-TERT co-expression in colon adenocarcinoma. METHODS: Sixty paraffin embedded primary colon adenocarcinomas were cored at 1.5 mm diameter and transferred to one microarray block. Immunohistochemistry was performed using anti-h-TERT, and c - MYC antibodies. A quantitative digitized macro was performed to evaluate their expression. RESULTS: c-MYC and h-TERT overexpression was observed in 27 (45%) and 28 (46.6%) cases, respectively. Co-over expression of those genes was observed in 17 (28.3%) cases and found to be statistically significant (p=0.001). The results also showed a strong association between c-MYC and grade of differentiation of the examined neoplasms (p=0.0217rpar;. CONCLUSION: Simultaneous c-MYC and h-TERT deregulation is a relatively frequent genetic event in colon adenocarcinoma. Because c-MYC overexpression is correlated with progressive disease - due to colon adenocarcinoma dedifferentiation - inhibition of its activity combined with h-TERT regulated expression is a new target for novel therapeutic regimens.

PTEN expression in non small cell lung carcinoma based on digitized image analysis.

PURPOSE: HER2 depended signalling pathway is dereg-ulated in a subset of non small cell lung carcinoma (NSCLC). The tumor suppressor gene PTEN (10q21) regulates the HER2/PI3K/Akt signalling pathway. Our aim was to evaluate PTEN protein expression in NSCLC based on a quantitative analysis method correlating also the results with clinicopathological parameters. METHODS: Protein expression was determined by immunohistochemistry (IHC) in 61 paraffin-embedded cases of patients with NSCLC. Digital image analysis (staining intensity levels) was performed in the corresponding immunostained slides. RESULTS: Loss of PTEN expression was observed in 24 (39.34%) cases, low expression in 29 (47.54%) and overexpression in 8 (13.12%) cases. Multivariate analysis determined that PTEN overexpression was associated with lower risk to develop metastases (p=0.05). CONCLUSION: PTEN deregulation is a relatively frequent genetic event in NSCLC, associated with progressive metastatic process in those patients. Because of binding to the ErbB2 receptor, trastuzumab stabilizes and activates PTEN gene, and loss of its expression negatively affects the response rates in such patients.

Improvement of skin-graft survival after autologous transplantation of adipose-derived stem cells in rats.

BACKGROUND: Skin grafts are frequently used for a variety of indications in plastic and reconstructive surgery. Their necrosis is a common complication, while different therapies have been proposed. Currently, adipose-derived stem cells (ASCs) hold great promise for their angiogenic potential and role during tissue repair. In this study, autologous transplantation of ASCs was used in skin grafts in rats to determine if it increases angiogenesis, skin-graft survival and wound healing. METHODS: ASCs were isolated, cultured, labelled with fluorescent dye and injected under full-thickness skin grafts in 10 rats (group 1), while 10 others served as controls (group 2). Skin grafts were analysed after 1 week. Collagen's framework was assessed with Masson's trichrome stain and angiogenesis with von Willebrand factor (vWF) immunohistochemistry. In addition, immunohistochemical staining intensity of vascular endothelial growth factor (VEGF) and transforming growth factor b3 (TGFb3) was assessed in all grafts. RESULTS: Mean area of graft necrosis was significantly less in group 1 than in group 2 (6.12% vs. 32.62%, p<0.01). Statistically significant increase of microvessel density, collagen density, VEGF and TGFb3 expression was noted in group 1 compared with group 2 (all: p<0.01). CONCLUSIONS: These findings suggest that autologous ASCs transplantation increases full-thickness skin-graft survival and shows promise for use in skin-graft surgery. This might be both due to in situ differentiation of ASCs into endothelial cells and increased secretion by ASCs of growth factors, such as VEGF and TGFb3 that enhance angiogenesis and wound healing.

The significance of DNA image cytometry and Edmondson-Steiner grading on prognosis after curative resection of hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is the commonest primary cancer of the liver. Hepatic resection remains the main curative option, although the incidence of disease recurrence in the remaining hepatic parenchyma is high and accounts for the leading cause of death post resection. For this reason, the need to identify prognostic factors which may determine treatment response and survival is of paramount importance. In this study we assessed whether DNA image cytometry and Edmondson-Steiner grading could be used as prognostic factors in a cohort of patients with HCC undergoing radical hepatic resection. METHODS: Forty-four patients with HCC who underwent radical resection were retrospectively analyzed. Histological grading according to Edmondson and Steiner and DNA ploidy using DNA image cytometry, were the two parameters analyzed. Pearson's x(2) or Fisher's exact tests were used to test for any associations between categorical variables. Univariate semi-parametric Cox proportional hazard regression models were used to assess the effect of explanatory variables on death. All reported p values were based on two-sided tests and compared to a significance level of 0.05. RESULTS: In univariate Cox regression analysis, adverse survival outcome was strongly associated with high DNA score and advanced histological grading. Patients with ploidy score >2.2 had 3.95 times higher probability of death, as compared to those with ploidy score ≤ 2.2. Edmondson-Steiner grades III and IV were also associated with 20.49 and 34.47 higher probability of death respectively as compared to grade I. CONCLUSION: Our results validate the prognostic significance of DNA image cytometry and Edmondson-Steiner grading following curative resection of HCC.

Increased vein wall apoptosis in varicose vein disease is related to venous hypertension.

OBJECTIVES: The study aimed to evaluate a wide range of apoptotic markers in the vein wall of patients with superficial chronic venous disease (SCVD) compared with normal veins. DESIGN: This was an observational study. METHODS: Vein specimens were obtained from 19 patients suffering from SCVD. From each patient, a specimen of the proximal part of the great saphenous vein (GSV), a specimen of the distal part of the vein and a specimen of a varicose tributary were obtained. Immunohistochemical analysis was used to localise the expression of BAX, p53, Caspase 3, BCL-2, BCL-6, BCL-xs, BCL-xl and Ki-67. Vein specimens from 10 healthy GSVs were used as controls. RESULTS: Saphenous vein specimens from patients with SCVD showed increased BAX, Caspase 3, BCL-xl and BCL-xs (p < 0.01 for all) and Ki-67 (p = 0.02) compared with healthy GSVs. In the venous disease group, GSV specimens from the distal ankle area showed increased BAX (p < 0.01) and BCL-xs (p = 0.031) compared with varicose tributaries specimens, which subsequently showed increased BAX (p = 0.044), Caspase 3 (p = 0.028) and BCL-xs (p = 0.037) compared with specimens from the proximal GSV. In addition, in the venous disease group, specimens from distal GSV showed increased BAX (p < 0.01), Caspase 3 (p = 0.019) and BCL-xs (p = 0.014) compared with the proximal GSV. CONCLUSION: Varicose veins exhibit increased apoptotic activity, by means of increased BAX, Caspase 3, BCL-xl and BCL-xs, compared with normal veins. Patients with varicose vein disease show increased apoptosis in the distal saphenous trunk compared with the proximal saphenous trunk, suggesting an association between chronic venous hypertension and apoptosis.

Deregulation of EGFR/VEGF/HIF-1a signaling pathway in colon adenocarcinoma based on tissue microarrays analysis.

PURPOSE: Overexpression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in colon adenocarcinoma (CA) is a frequent event, whereas specific deregulation mechanisms in the corresponding signaling pathways remain under investigation. Our aim was to co-evaluate their expression correlated to the hypoxia inducible factor 1alpha (HIF-1a), which activates the transcription of VEGF gene. METHODS: 60 paraffin-embedded primary CAs were cored at 1.5 mm diameter and transferred to the microarray block. Immunohistochemistry (IHC) was performed using anti-EGFR, -VEGF, and -HIF 1a monoclonal antibodies. Concerning EGFR, quantitative evaluation was based on a semi-automated analysis system. Chromogenic in situ hybridization (CISH) was performed using EGFR gene and chromosome 7 centromeric probes. RESULTS: Protein overexpression was observed in 13/60 (21.6%), 45/60 (75%) and 7/60 (11.6%) cases regarding EGFR, VEGF, and HIF 1a, respectively. CISH analysis detected 4/60 (6.6%) EGFR gene amplified cases, whereas chromosome 7 aneuploidy was identified in 11/60 (18.3%) cases. Significant associations raised correlating stage to chromosome 7 (p=0.024), HIF 1a expression to tumor anatomical location (p=0.019) and also VEGF to HIF 1a expression (p=0.001), whereas EGFR expression was not associated to EGFR gene copies. CONCLUSION: According to our results, chromosome 7 instability is correlated to advanced disease, whereas a significant subset of CAs demonstrates an alternative, non- HIF 1a depended mechanism of VEGF overexpression. Furthermore, EGFR protein overexpression does not predict a specific gene deregulation mechanism.

Increased angiogenesis and mast cells in the centre compared to the periphery of vitiligo lesions.

The larger number of T-lymphocytes in the periphery of vitiligo lesions and their association with angiogenesis are reported. The objective of this study was to investigate angiogenesis, VEGF and mast cell in vitiligo lesions. Specimens of 30 patients' biopsies, from lesional and perilesional nondepigmented skin were stained for mast cells, CD34 and VEGF. The evaluation was made by image analysis and the measured variables were statistically analyzed. A significantly increased number of CD34 and VEGF positive vessels and mast cells were detected in the centre of the lesion than in the periphery (p < 0.0001, p < 0.0001 and p = 0.001). There was a positive correlation of CD34, VEGF and mast cell number between the centre and the periphery of the lesions (r = 0.877, p < 0.0001; r = 0.946, p < 0.0001 and r = 0.863, p < 0.0001, respectively). The increased angiogenesis and mast cell numbers in the area where lymphocyte number is lower may be explained with the stepwise inflammatory process in vitiligo.

Microvessel density (MVD) and cyclooxygenase-2 (COX-2)/ beta-catenin interaction are associated with relapse in patients with transitional carcinoma receiving adjuvant chemotherapy with paclitaxel/carboplatin: a hellenic cooperative oncology group (HECOG) study.

BACKGROUND: Cycloxygenase (COX)-2 has been associated with proliferation, apoptosis and angiogenesis in urothelial cancer. The prognostic significance of COX-2 in patients who received adjuvant chemotherapy for urothelial cancer was examined. PATIENTS AND METHODS: Expression of COX-2, p53, ki67, beta-catenin, vascular endothelial growth factor (VEGF) and microvessel density (MVD) were studied retrospectively in 59 patients with urothelial cancer (pT3, pT4, N+) who had undergone surgery. The patients had subsequently received adjuvant chemotherapy. RESULTS: Thirty-eight out of 59 cases (64%) were positive for COX-2. COX-2 was not associated either with progression-free survival (PFS) or overall survival (OS). MVD levels > or =47 were associated with longer median PFS compared with lower levels (not reached vs. 13 months [95% CI: 8-18], p=0.048). The median PFS for patients with beta-catenin nuclear accumulation and COX-2 expression was 6 months (95% CI: 4-7) compared with 19 months (95% CI: 14-23) for neither or only one of these factors (p=0.018). CONCLUSION: MVD may be a useful indicator of relapse in high-risk urothelial cancer treated with adjuvant chemotherapy.

The relationship of endothelin-1 and inducible nitric oxide synthase at several stages of the anastomotic healing process.

AIM: The aim of this study was to investigate the interaction between the endothelin-1 (ET-1) and inducible NO synthase (iNOS) in anastomotic healing. METHODS: The expression of ET-1 and iNOS were investigated by immunohistochemistry in a rat end-to-end arterial anastomotic model. The aorta of 50 male Wistar rats was exposed, then transversely divided and re-anastomosed. The animals were sacrificed immediately after the operation (group A, control group), after 24 h (group B), on 7th postoperative day (group C), on 30th day (group D) and at 6 months (group E). Intima and media thickness and their ratio of the anastomotic segments in each group were calculated from computer digitized images of the individual sections. ET-1 and iNOS expression were measured on a semiquantitative scale ranging from 0 to 3. RESULTS: ET-1 was expressed from endothelial and smooth muscle cells (SMCs), while iNOs was expressed from SMCs and inflammatory cells. An intense expression of ET-1 was demonstrated mainly at 1 week and to a lesser degree at 1 month. Yet, at 6 months this expression was significantly weakened (P<0.001). In contrast, an intense iNOS expression was identified at 24 h, substantially regressing at statistical significant lower levels after 1 week (P<0.001). Bivariate correlation test showed a positive correlation between ET-1 and iNOS expression. CONCLUSION: ET-1 appears to play an important role in intimal thickening during anastomotic healing, especially in the late period of the process. Although there is a positive correlation between ET-1 and iNOS production, the activity of the latter is relatively limited after the first postanastomosis week.

Evaluation of caspase-3 and caspase-8 deregulation in tongue squamous cell carcinoma, based on immunohistochemistry and computerised image analysis.

AIMS: To investigate the potential role of caspase-3 and caspase-8 protein expression in the biological behaviour of tongue squamous cell carcinoma. MATERIALS AND METHODS: We conducted immunohistochemical analyses of 87 specimens of primary tongue squamous cell carcinoma, using monoclonal anti-caspase-3 and anti-caspase-8 antibodies. A digital image analysis assay was also performed in order to evaluate the results. RESULTS: Reduced expression of caspase-8 and -3 proteins was observed in 30/87 (34.5 per cent) and 79/87 (90.5 per cent) cases, respectively. Cox regression analysis showed no prognostic significance for the association between overall protein expression of either marker and survival probability (p = 0.174 for caspase-3; p = 0.608 for caspase-8). Interestingly, the size of the examined tumours was strongly correlated with survival status (p = 0.024). CONCLUSIONS: Simultaneous deregulation of caspase-8 and -3 is a frequent event in tongue squamous cell carcinoma. Activation of caspase-3, which is predominantly down-regulated, may be a crucial process for induction of apoptosis and response to therapeutic strategies.

Nuclear fractal dimension as a prognostic factor in oral squamous cell carcinoma.

Strong theoretical reasons exist for using fractal geometry in measurements of natural objects, including most objects studied in pathology. Indeed, fractal dimension provides a more precise and theoretically more appropriate approximation of their structure properties and especially their shape complexity. The aim of our study was to evaluate the nuclear fractal dimension (FD) in tissue specimens from patients with oral cavity carcinomas in order to assess its potential value as prognostic factor. Relationships between FD and other factors including clinicopathologic characteristics were also investigated. Histological sections from 48 oral squamous cell carcinomas as well as from 17 non-malignant mucosa specimens were stained with Hematoxylin-Eosin for pathological examination and with Feulgen for nuclear complexity evaluation. The sections were evaluated by image analysis using fractal analysis software to quantify nuclear FD by the box-counting method. Carcinomas presented higher mean values of FD compared to normal mucosa. Well differentiated neoplasms had lower FD values than poorly differentiated ones. FD was significantly correlated with the nuclear size. Patients with FD lower than the median value of the sample had statistically significant higher survival rates. Within the sample of patients studied, FD was proved to be an independent prognostic factor of survival in oral cancer patients. In addition this study provides evidence that there are several statistically significant correlations between FD and other morphometric characteristics or clinicopathologic factors in oral squamous cell carcinomas.

Rates of infiltration by macrophages and dendritic cells and expression of interleukin-18 and interleukin-12 in the chronic inflammatory lesions of Sjögren's syndrome: correlation with certain features of immune hyperactivity and factors associated with high risk of lymphoma development.

OBJECTIVE: To evaluate the expression profile of infiltrating macrophages and dendritic cells (DCs) as well as of interleukin-18 (IL-18) and IL-12 in the minor salivary gland (MSG) lesions of patients with Sjögren's syndrome (SS), and to assess the relationship of these factors with disease parameters. METHODS: Macrophages, DCs, T cells, B cells, proIL-18, mature IL-18, and IL-12 were detected by single- and double-labeling immunohistochemistry in MSG specimens from 21 patients with primary SS (13 of 21 tested for IL-12), 7 patients with secondary SS, and 9 disease control patients. Expression profiles were assessed for correlations with various disease parameters, including adverse predictors of lymphoma development. RESULTS: MSGs from patients with SS (but not from disease controls) manifested increased infiltration by macrophages and DCs, strong expression of IL-18 by macrophages (particularly in B cell-rich areas and in germinal center-like structures in primary SS), and expression of IL-12 by mononuclear cell infiltrates. In primary SS, high infiltration by macrophages correlated with SG enlargement (P = 0.01). The DC infiltration rate correlated positively with the macrophage infiltration rate (P = 0.04), occurrence of SG enlargement (P = 0.03), and presence of C4 hypocomplementemia (P = 0.05), and inversely with serum C4 complement levels (P = 0.001). The rate of infiltration by IL-18-expressing cells correlated positively with biopsy focus scores (P < 0.001), larger infiltrates of macrophages (P = 0.01), DCs (P = 0.01), and B cells (P = 0.02), and SG enlargement (P = 0.02), and negatively with serum C4 complement levels (P = 0.02). The rate of infiltration by IL-12-expressing cells correlated inversely with that by IL-18-expressing cells (P = 0.001), biopsy focus scores (P = 0.003), and SG enlargement (P = 0.01), and positively with serum C4 complement levels (P = 0.05). CONCLUSION: In patients with primary SS, infiltration of the SG by macrophages and DCs and expression of IL-18 and IL-12 appear to play active roles in the expansion and organization of infiltrative injuries and have a correlation with certain predictors of lymphoma development.

Diabetes may increase risk for oral cancer through the insulin receptor substrate-1 and focal adhesion kinase pathway.

In light of recent epidemiological studies that associate diabetes mellitus with increased risk for oral cancer, we investigated in diabetic (type I) and normal rats with induced oral squamous cell carcinoma whether the molecular basis for that putative association involves insulin receptor substrate-1 (IRS-1) and focal adhesion kinase (FAK). Fourteen diabetic and 12 normal rats developed cancer after 4-nitroquinoline-N-oxide treatment, while six diabetic and six normal animals were used as controls. Oral sections were studied using monoclonal antibodies against IRS-1 and FAK proteins. Expression of IRS-1 was significantly higher in diabetic than normal rats, but it decreased in diabetic animals with tumor, especially in more advanced stages. FAK expression was significantly higher in rats with cancer in comparison to the ones without it, regardless the diabetes status. These data suggest that the IRS-1/FAK pathway is altered by diabetes resulting in reduced cell adhesion and possibly increasing risk for oral cancer.

Heregulin, cysteine rich-61 and matrix metalloproteinase 9 expression in human carotid atherosclerotic plaques: relationship with clinical data.

OBJECTIVES: Heregulins (HRGs) are known to induce expression of angiogenic factors such as cysteine rich-61 (CYR61) and collectively to promote neoangiogenesis. Along with extracellular matrix remodelling, mediated by matrix metalloproteinases (MMPs), these factors are important in atherogenesis. The aim of the present study was to investigate HRG, CYR61 and MMP-9 expression and their relationship with clinical and histopathological findings in carotid occlusive disease. MATERIALS AND METHODS: Specimens of human carotid atherosclerotic plaque (n=90) were obtained by endarterectomy. Expression of HRG, CYR61 and MMP-9 was assessed by immunohistochemical and Western blot analysis. Associations between protein expression and degree of carotid stenosis, presence of symptoms, presence of an infarct in CT scan and carotid plaque histopathology were investigated. RESULTS: An increase in HRG, CYR61 and MMP-9 expression was found, particularly in neovascularized regions of the plaques. High HRG expression was associated with the degree of carotid stenosis (p=0.028) and plaque histopathology (p=0.002). More than half of specimens from plaques with >90% stenosis had intense expression of CYR61 (p=0.047). Increased expression of MMP-9 was associated with degree of stenosis and presence of cerebral infarct on CT scan (p=0.05). CONCLUSION: HRG, CYR61 and MMP-9 are highly expressed in human atherosclerotic carotid plaques. The association with the degree of stenosis and/or plaque histopathology implies an involvement in lesion progression.

Effect of green tea on angiogenesis and severity of atherosclerosis in cholesterol-fed rabbit.

BACKGROUND AND AIMS: Since the development of the atherosclerotic plaque requires the growth of new microvessels in the plaque itself (vasa vasorum), we postulated that green tea may exert an anti-atherogenic effect. METHODS AND RESULTS: Thirteen male New Zealand white rabbits were studied for 17 weeks. All rabbits were fed an hypecholesterolemic diet. After 2 weeks of adaptation rabbits were randomly assigned into two groups. Animals in Group A were fed the hypercholesterolemic diet and received plain tap water ad libitum. Animals in Group B were fed with the same diet and furthermore received 2.5% (g/g) green tea for 17 weeks. CONCLUSION: According to our results the atherosclerotic lesions were more severe in Group B than in Group A specimens. Also, the number of VEGF positively stained foam cells and smooth muscle cells of Group B were significantly greater than in Group A. About 30% less plaque was found in Group A than in the control group (Group B). So, our study showed that the consumption of green tea leads to a reduction of atherosclerosis as well as a significant decrease of VEGF expression in the atherosclerotic plaque of rabbit aorta. The hypothesis that probably green tea may produce its anti-atherogenetic effect through an anti-angiogenetic mechanism needs more investigation.

Study of phospho-beta-catenin subcellular distribution in invasive breast carcinomas in relation to their phenotype and the clinical outcome.

Beta-catenin has a crucial role in cell-cell adhesion as well as a signaling role as a member of the Wnt pathway. The aim of this study was to examine the clinicopathological and prognostic value of phosphorylated beta-catenin, as well as its relation to the tumors' phenotype, in breast cancer. Immunohistochemistry was applied on 141 paraffin-embedded breast tissue specimens for the detection of phospho-beta-catenin, ER, PR, c-erbB-2, p53, Ki-67, bcl-2, uPAR and TIMP-1. For each case, a phospho-beta-catenin index was determined by image analysis. Phospho-beta-catenin staining was detected in the cytoplasm and the nucleus of the malignant cells. Cytoplasmic phospho-beta-catenin was statistically higher in carcinomas of smaller tumor size (P = 0.030), lower stage (P = 0.026), decreased Ki-67 and high c-erbB-2 immunoreactivity (P = 0.052 and P = 0.037, respectively). Nuclear phospho-beta-catenin showed a parallel correlation with ER and ERbeta (P = 0.022 and P = 0.043, respectively), bcl-2 (P = 0.042), uPAR in cancer cells (P = 0.041) and TIMP-1, although the correlation was borderline (P = 0.066). Cytoplasmic phospho-beta-catenin was found to be independently correlated with prolonged disease-free and overall survival (P = 0.046 and P = 0.002, respectively), whereas nuclear localization was correlated with a shortened overall survival (P = 0.046). In conclusion, phospho-beta-catenin may have a different involvement in invasive breast carcinomas, according to its subcellular distribution. Nuclear localization seems to be related to an aggressive tumor phenotype, negatively affecting patients' overall survival, whereas cytoplasmic localization is associated with a favorable tumor phenotype and a longer disease-free and overall survival.

Hepatic expression of the proliferative marker Ki-67 and p53 protein in HBV or HCV cirrhosis in relation to dysplastic liver cell changes and hepatocellular carcinoma.

To evaluate hepatic expression of the nuclear proliferative marker Ki-67 and the p53 oncoprotein in hepatitis B virus (HBV)/HCV cirrhosis in relation to dysplastic liver cell changes and hepatocellular carcinoma (HCC). We studied needle liver biopsies from 107 patients with cirrhosis and no HCC (52 HBV, 55 HCV) who had been assessed for protocol studies, and 57 cirrhotic patients with HCC (40 HBV, 17 HCV). We evaluated small and large cell dysplastic changes along with the expression of Ki-67 and p53 by immunohistochemistry. The labelling index (LI) was defined as the proportion (%) of positive-stained nuclei of the 500 measured. Large and small cell dysplastic changes were observed in 12 and 9% of specimens respectively. Only small cell changes were associated with Ki-67 expression. Ki-67 LI was 5.50 +/- 5.7 in cirrhosis (13.90 +/- 3.84 in those with small cell dysplastic changes vs 4.64 +/- 4.98 in those without, P < 0.01), 10.2 +/- 5.95 in cirrhosis with HCC (P < 0.05) and 18.56 +/- 10 in HCC (P < 0.01). Neither the presence of small cell dysplastic changes nor the expression of Ki-67 was related to severity or aetiology of cirrhosis. Expression of p53 was observed in 30% of the non-tumorous and in 53% of the neoplastic tissue obtained from patients with HCC, with no differences between HCV and HBV. Ki-67 and p53 expression was associated with the tumour grade (P < 0.001). Our observations clearly demonstrate the association between the proliferation activity and the morphological changes in the cirrhotic liver from the non-dysplastic to dysplastic lesion to HCC. They also support the hypothesis that p53 alterations are a rather late event in carcinogenesis and related to HCC grade. And finally, they suggest that the final steps of hepatocarcinogenesis are common and independent of the aetiology of the chronic viral infection.

Angiogenesis in Hodgkin's lymphoma: a morphometric approach in 286 patients with prognostic implications.

The significance of angiogenesis in Hodgkin's lymphoma (HL) is not well defined. The aim of this study was to evaluate various morphometric characteristics of microvessels in lymph node sections of 286 patients with HL at diagnosis and investigate their relationship with clinicopathologic parameters and prognosis. Microvessel density (MVD), total vascular area (TVA) and several size- and shape-related microvascular parameters were quantitated--after anti-CD34 immunohistochemical staining--in the region of most intense vascularization, using image analysis. An increase in microvessel caliber parameters (area, perimeter, major and minor axis length) and a decrease in MVD were noted with increasing stage. An inverse relationship was recorded between MVD and the number of involved sites (NIS) and LDH. In univariate analysis, overall disease-specific survival was adversely affected by MVD and TVA, whereas inferior failure-free survival (FFS) was associated with the presence of more flattened vessel sections. Multivariate analysis disclosed that the extent of angiogenesis (MVD/TVA), age and the NIS independently affected overall survival. Accordingly, FFS was independently linked to the shape of microvessels and albumin levels or the NIS. In conclusion, our data support the view that angiogenesis in HL provides independent prognostic information, requiring the concomitant evaluation of quantitative and qualitative aspects of microvascular network.