SUMMARY:Superficial transitional cell carcinomas (TCC) of the urinary bladder have been shown to be monoclonal. However, no combined study of clonality and tumor suppressor genes (TSG) is available to date for muscle-invasive TCC. Forty-four muscle-invasive TCC of the urinary bladder selected from women were included in this study. Tumor cells located above and below the muscularis mucosa zone were systematically microdissected and used for DNA extraction. Hha-I digested and undigested samples were used to study the methylation pattern of androgen receptor alleles and undigested samples were used for microsatellite analysis of TSG (TP53, RB1, WT1, and NF1). Both loss of heterozygosity (LOH) and single nucleotide polymorphism (SNP) analyses were performed using optimized denaturing gradient gel electrophoresis. The expression of p53, pRB, and p21 WAF1 was assessed by immunohistochemistry. Appropriate controls were run in every case. All except two TCC showed a monoclonal pattern with the same allele inactivated in both compartments. Microsatellite analysis of TSG revealed the same LOH/SNP pattern in both tumor compartments in 30 cases (involving more than 1 TSG locus in 8) and genetic heterogeneity in 14 cases. From the latter group, 9 cases expressed more genetic changes in the deep compartment (involving TP53 gene in all cases, WT1 gene in 2, and NF1 in 1), whereas in 4 cases the superficial compartment showed more genetic changes (three involving NF1 and one involving both RB and TP53). No statistical difference in the immunoexpression was detected, although it tended to be higher in the superficial compartment than in the deep compartment. These concordant data in polymorphic DNA regions indicate that bladder-muscle-invasive TCC are monoclonal proliferations with homogeneous tumor cell selection. Heterogeneous tumor cell selection by topography defined two different genetic compartments: superficial, NF1-defective, and deep, TP53-defective. No differences in the immunohistochemical expression were observed, precluding a more extensive clinical application. (Lab Invest 2000, 80:279-289).

The dissemination of tumour cells to the lymph nodes is a complex process involving the formation of new lymph vessels, or lymphangiogenesis, produced by the tumour itself. The main growth factor involved in lymphangiogenesis is vascular endothelial growth factor C (VEGF-C), which is secreted by several different malignant tumours, including melanoma. Not only has VEGF-C expression been found in tumour cells, it has also been detected in tumour stromal cells like macrophages and fibroblasts. This study aimed to determine whether the expression of VEGF-C in tumour and stromal cells in cutaneous melanoma determines lymphangiogenesis and neoplastic dissemination to lymph nodes. We examined cases from 50 patients with melanoma who underwent selective biopsy of the sentinel lymph node. Immunohistochemical study was done with D2-40 to label lymph vessels, and the expression of VEGF-C was evaluated in tumour and stromal cells. Lymph vessel density was greater in sentinel lymph node-positive than in sentinel lymph node-negative cases, though the difference was not significant (P = 0.075). A significant correlation was seen between lymph vessel density and tumour thickness and the presence of ulceration. The main finding was that the expression of VEGF-C in fibroblasts was highly associated with the presence of metastasis in the sentinel node and with the Clark level. However, VEGF-C expression showed no relation in either tumour cells or macrophages with node status or other prognostic factors, such as the Breslow index or Clark level. Our results highlight the relevance of the stroma in tumour progression in cutaneous melanoma and its role in the spread to lymph nodes.

Objectives: To evaluate the role of bcl-2 and apoptotic index in the progression from primary to metastatic breast carcinoma and their influence on prognosis. Methods:bcl-2 expression was examined by immunohistochemistry and apoptotic index by in situ end-labelling in 116 surgical breast carcinomas and lymph node metastases from 50 patients. Results:bcl-2 was observed in 69 cases (59.4%) of primitive carcinomas and 26 cases (65%) of metastatic breast carcinomas and there was agreement of bcl-2 expression between primary and metastatic sites except in 3 cases. bcl-2 expression was significantly associated with several favourable prognostic features, such as small tumour size (p = 0.03) and oestrogen and progesterone-receptor positivity (p < 0.01 and p < 0.001, respectively). A high apoptotic index was significantly associated with a number of poor prognostic factors, including poorly differentiated carcinomas, large tumour size, high Ki67 expression and high mitotic count (p < 0.001 in all cases). The mean apoptotic index was higher in lymph node metastasis than in primary carcinomas (1.19 vs. 0.69, p < 0.01). A low bcl-2 expression and a high apoptotic index were significantly associated with short-relapse free survival rates (p = 0.02 and p < 0.01, respectively), but only apoptotic extent provided independent prognostic information by multivariate analysis. Conclusions: The evaluation of bcl-2 expression and extent of apoptosis may provide useful prognostic information on breast cancer patients; however while increased apoptosis is strongly associated with the progression from primary carcinomas to lymph node metastases, bcl-2 does not seem to play a significant role in this process.

We analyzed serum and tumor samples from 133 patients with operable primary breast cancer to determine the possible relationship between presurgery and postsurgery circulating serum vascular endothelial growth factor (VEGF) levels and tumor-associated macrophage (TAM) numbers, tumor VEGF expression, and other immunohistochemical parameters. A significant positive correlation was observed between the number of TAM and postsurgery circulating VEGF values (P < .05). Moreover, patients with a p53+ tumor had higher postsurgery serum VEGF levels than those with a p53- tumor (P < .05), and tumor p53 overexpression correlated significantly with TAM number (P = .007). We observed no significant association between serum values and tumor VEGF expression. Although the macrophage index was higher in VEGF+ than in VEGF- tumors, the differences were not statistically significant. Our data show a positive interrelation between high circulating VEGF levels, the number of TAM, and p53 overexpression, a relationship that might have an important role in the enhanced angiogenesis processes in breast cancer.

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