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The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Background. Usually, silver stain is needed to differentiate between normal or hyperplastic hypophysis and pituitary adenomas. Many papers reported the lack of reticular fibers network as mandatory for pituitary adenoma diagnosis. Aim. Differences between the architecture of reticular fibers in normal pituitary and pituitary adenomas. Methods. Gordon- Sweet silver staining of pituitary specimens, prelevated during pituitary surgery performed in 138 patients with the endocrine and imagistic diagnosis of pituitary macro-adenomas. Results. Pituitary specimens of pituitary adenomas was confirmed in 133 cases; 3 specimens were with normal pituitary tissue, 1 with pituitary hyperplasia, 1 with pituitary apoplexy. Twelve of 133 pituitary adenomas specimens were associated also with normal pituitary tissue. There was a loss of acinar network of reticular fibres in 115 cases, but the present study describes the persistence of reticular fibers networks in 18 (13.53%) of 133 pituitary adenomas. We identified five distribution patterns of remanent reticular fibers network in pituitary adenomas using silver staining. Conclusion. Persistence of reticular fibers in a small number of pituitary adenomas could be a possible pitfall in the discrimination between the normal hypophysis and pituitary adenoma. [ABSTRACT FROM AUTHOR]

Background. Androgen receptor (AR) plays a crucial role in the homeostasis of mammary tissue. In the present study we investigated the immunohistochemical expression of AR in breast cancer and its relationships with clinico-pathological parameters of prognosis, hormone receptors status and HER2 overexpression. Methods. There were investigated 124 patients admitted with invasive breast cancer, and from these 48 had lymph node metastasis and 9 had distant metastasis. In all specimens there were performed immunohistochemical staining for AR, estrogen receptors (ER), progesterone receptor (PR) and HER2. Results were correlated with pathological findings and the molecular profile of the tumors. Results. The immunohistochemical expression for AR was found in 92 cases. A significant correlation was found between AR expression and pathological type, grade and lymph node status, but not with distant metastasis and the expression of ER, PR and HER2. An inverse correlation was found between AR expression and triple negative breast cancers. Conclusion. Our data suggest that AR is an individual factor of prognosis and is a useful marker to stratify patients with triple negative tumors. AR could be a potential target for specific therapy in individuals excluded from the conventional hormone therapy. [ABSTRACT FROM AUTHOR]

The present study proposes a classification of renal cancer tumor blood vessels according to their morphology and maturation grade. We identified four vascular patterns: reticular, diffuse, fasciculated and trabecular. The reticular pattern was present in 63% of cases, being characterized by the predominance of mature CD34+/SMAct+ tumor vessels, highly interconnected. For this pattern, 74% of cases had vascular invasion, and a significant correlation was observed between tumor grade and immature state of tumor vessels (p = 0.022). The diffuse pattern was observed in 23% of cases and was characterized by non-interconnected vessels predominantly of mature CD34+/SMAct+ type and vascular invasion in 64% of cases. Only 8% of cases, had a fasciculate model of vessels distribution, all of them being of mature type, located in the connective axis of papillary renal tumors. For this pattern vascular invasion was found in 50% of cases. In 6% of cases a trabecular pattern was observed and the lowest rate of vascular invasion was registered. We defined here four distinct vascular patterns in renal cell carcinomas showing a strong impact on vascular invasion. A complete morphological and molecular characterization of tumor vessels would be beneficial in elucidating the mechanisms that underlie the ineffectiveness of antiangiogenic/antitumor therapies.

The molecular phenotypic heterogeneity of mast cells (MCs) makes them attractive as potential therapeutic targets in anti-cancer adjuvant therapy. Mast cell aggregations observed in tumors suggested their involvement in tumor pathogenesis. Despite several studies using mast cell tryptase, MCs' involvement in the progression of prostate tumors has not been demonstrated. The aim of our study was to identify and quantify the phenotypic heterogeneity of MCs in prostate lesions. Our study included 7 cases of normal prostate, 25 cases of benign epithelial hyperplasia and 64 cases of prostate carcinoma. MCs were immunohistochemically assessed using three markers: tryptase, chymase and CD117. Two immunophenotypes of MCs were identified in benign lesions: tryptase+/CD117+/chymase- and tryptase-/chymase+/CD117+, located in peritumoral areas. Intratumoral MC phenotype of malignant lesions was characterized by tryptase+/chymase+/CD117+, while in the peritumoral areas three different MCs phenotypes were identified: tryptase+/chymase+/CD117-, tryptase+/CD117+/chymase- and chymase+/CD117+/tryptase-. Our results suggest the correlation of chymase positive MCs of the peritumoral areas and CD117 positive MCs of the intratumoral areas with tumor grade.

Prox1 is a key regulator of lymphatic endothelial cell commitment during embryonic development. No correlations between Prox1 and VEGF-C/VEGFR3 expression in cervical cancer has been done until now. The aim of the present study was to evaluate the peculiarities of Prox1, VEGF-C and VEGFR3 expression during uterine cervix neoplasia progression. Material and methods. One hundred and four specimens taken from women with macroscopically detectable lesions were classified by histopathology and analyzed by immunohistochemistry for Prox1, VEGFR3 and VEGF-C expression. Results. The presence of Prox1 nuclear expression was detected starting from CIN2 and CIN3 lesions to microinvasive carcinoma, in the nuclei of lymphatic and venous endothelial cells and scattered stromal cells. All Prox1 positive lymphatic vessels were positive for VEGFR3. A significant correlation was found between expression of VEGF-C in tumor cells and nuclear density of Prox1 positive lymphatic cells (p=0.044). Conclusion. The commitment of Prox1 positive cells through a lymphatic lineage is an early event for cervical neoplastic progression, being present starting with intraepithelial cervical lesions, and is strongly associated with VEGFR3 and VEGF-C expression. These findings suggest an early active lymphangiogenesis during cervical neoplasia progression and explain, in part, the early presence of lymph node metastasis in cervical cancer. By the detection of Prox1 expression in lymphatic and venous endothelial cells, and also in stromal cells, it has been suggested that there are at least three different mechanism of lymph vessel development during cervical neoplasia progression.

The role of estrogen and androgen receptors signaling in breast cancer is widely accepted, but the interrelations between them are not well understood. It was suggested that PSA could be a marker of endogenous balance between androgens and estrogens. In this context, we intended to investigate the potential of relationship between polymorphic tandem repeats (CAG, TA and CA) in AR (androgen receptor), ERalpha (estrogen receptor alpha) and ERbeta (estrogen receptor beta) genes and the immunoexpression of PSA and AR proteins. We assessed also the possible influences of CAG, TA, and CA variables and other available prognostic factors (ER, PR, AR, HER2/neu, PSA expression, and nodal status) on disease-free survival. We assessed the polymorphic tandem repeats lengths by genotyping, followed by high-resolution denaturing polyacrylamide gel electrophoresis in 163 breast cancers. Immunohistochemistry was performed to assess the expressions of AR, PSA, ER, PR and HER2/neu proteins. Our results showed that PSA was correlated with the length of CA repeats in the 3'-untranslated region of ERbeta, shorter CA repeats being correlated with PSA expression (p=0.03). AR immunoexpression was correlated with CAG repeats on AR gene, higher number of repeats being linked to a higher AR immunoexpression (p=0.04). Performing logistic regression to investigate relationships with prognosis, we observed that PSA immunoexpression (p=0.004), the nodal status (p-<0.001) and marginally, longer TA repeats (p=0.05) were correlated with increased disease-free survival. AR expression presented a low statistical value (p=0.054) in predicting evolution and was not entered into the multivariate regression analysis. Altogether, our findings supports the hypothesis that estrogens, through both alpha and beta-receptors variants are mediating the AR signaling pathway.

In gastric cancer, lymph node metastasis is a major prognostic factor. Tumor lymphangiogenesis promotes metastasis in experimental models, but in human tumors data about the presence and clinical significance of lymphatic vessels in the tumor area are controversial. We investigated 70 patients with advanced-stage gastric carcinoma and the pathological examination showed 40 cases with intestinal subtype and 30 cases with diffuse subtype. Forty three from 70 cases had regional lymph node metastasis. Additional slides were stained with an antibody against podoplanin, and lymphatic microvessel density (LMVD) was evaluated in the tumoral and peritumoral areas. Lymphatic vessels were identified in tumor area in all cases and LMVD was higher in the peritumoral than in the tumor area. Podoplanin-positive vessels in tumor area were usually small, with narrow lumen. A significant correlation was found between LMVD and stage of the tumor (p<0.002) and lymph node metastasis (p<0.031), but not with the pathological subtype and grade of the tumor. We found tumor cells in the lumen of lymphatic vessels in 11 cases, whereas tumor cells expressing podoplanin were found in 4 cases of less differentiated diffuse subtype gastric carcinoma. In conclusion, our results suggest that LMVD predicts tumor stage and lymph node metastasis, and podoplanin-positive tumor cells select a subgroup of tumors with high potential of invasion and metastasis.

The aim of present study was to investigate the relationship between the immunohistochemical expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) assessed by CD31 and endoglin (CD105) in renal cell carcinoma (RCC). Specimens from 45 cases of RCC. were formalin-fixed, paraffin embedded, and sections were stained with H&E. Additional sections from each case were stained for VEGF, CD31, CD105, and alpha smooth muscle cell actin (SMA). VEGF immunohistochemical expression was estimated as negative (0), weak positive (+1), moderate positive (+2), and intense positive (+3). Microvessel density (MVD) was estimated on 5 hot spots (x400) from each case, and the arithmetic media was the final result. MVD was separately calculated on slides stained with CD31 and CD105. The rate between mature and immature blood vessels was calculated on slides stained with CD31/CD105/SMA. Statistic analysis was performed with SPSS10.0. The immunoreaction for VEGF was positive in epithelial cells of the renal tubules, and occasionally, in endothelial cells. In RCC, tumor cells were positive in 34 from 45 cases (75.5%). 11 cases were negative, 14 were slightly positive (+1), 13 moderate (+2), and 7 intense (+3). No relationship was found between the expression of VEGF and pathological form and nuclear grade, excepting for the chromophilic variant (3 cases, all positive). CD31 was positive in all cases, and CD105 in 39 cases. The mean values of MVD on slides stained for CD31 and CD105 were: 31.68 (range 9.8-60.2)/20.66 (range 4.2-52.8). The rate CD31/SMA positive blood vessels was 1/0.62. VEGF was expressed in 75.5% of 45 cases with RCC, and the mean value of MVD CD31/CD105 was 31.68/20.66. The immunohistochemical expression of VEGF does not correlate with MVD performed on slides stained for both CD31 and endoglin. The majority of blood vessels in the tumor area are of mature type, with perivascular cells positive for SMA.

The immunoexpression of prostate-specific antigen in breast cancers has been well established, but the role of this extra-prostate PSA appears to be a complex, poorly understood and of doubtful prognostic value. In this context, our aim was to evaluate PSA in breast carcinomas and to compare the results with several established prognostic markers of breast cancer: estrogen and progesterone receptors status, HER2/neu status, histological type of tumor, grade of differentiation, stage, tumor size, nodal and menopausal status. We have immunohistochemically assessed 53 breast carcinomas for PSA, ER, PR and oncoprotein HER2/neu status. The relationship between the clinical and histopathological markers was analyzed by chi-square test. In the present study PSA was expressed in 60.3% of cases, and we have found a significant correlation with the histological type and HER2/neu negative status in premenopausal women. No statistically significant difference was found between PSA positivity and menopausal status of the patients, nodal status, estrogen and progesterone receptors, HER2/neu status in postmenopausal patients, tumor size or histological grade. We conclude that in our study PSA can not be considered as a valuable independent prognostic factor in breast carcinoma. As long as the majorities of PSA positive carcinomas were small in size, early stage, better and moderately differentiated, HER2/neu negative and 70% of ER/PR positive carcinomas expressed PSA, it might be useful as a marker for a subset of breast cancers with better prognosis, which could respond to endocrine therapy, in correlation with other prognostic markers.