BACKGROUND: The product of Wilms' tumor suppressor gene (WT1), a nuclear transcription factor, regulates the expression of the insulin-like growth factor (IGF) and transforming growth factor (TGF) systems, both of which are implicated in breast tumorigenesis and are known to facilitate angiogenesis. In the present study, WT1 allelic integrity was examined by Loss of Heterozygosity (LOH) studies in infiltrating breast carcinoma (n=60), ductal carcinoma in situ (DCIS) (n=10) and benign breast disease (n=5) patients, to determine its possible association with tumor progression.

METHODS: LOH at the WT1 locus (11p13) as determined by PCR-RFLP for Hinf1 restriction site and was subsequently examined for its association with intratumoral expression of various growth factors i.e. TGF-beta1, IGF-II, IGF-1R and angiogenesis (VEGF and Intratumoral micro-vessel density) in breast carcinoma.

RESULTS: Six of 22 (27.2%) genetically heterozygous of infiltrating breast carcinoma and 1 of 4 DCIS cases showed loss of one allele at WT1 locus. Histologically, the tumors with LOH at WT1 were Intraductal carcinoma (IDC) and were of grade II and III. There was no correlation in the appearance of LOH at WT1 locus with age, tumor stage, menopausal status, chemotherapy status and lymph node metastasis. The expression of factor IGF-II and its receptor, IGF-1R was significantly higher in carcinoma having LOH at WT1 locus. A positive correlation was observed between the TGF-beta1, VEGF expression and IMD scores in infiltrating carcinoma.

CONCLUSIONS: The current study indicates that the high frequency of loss of allelic integrity at Wilms' tumor suppressor gene-1 locus in high-graded breast tumors is associated with aggressiveness of the tumor.

3.

Our previous studies revealed that Wilms' tumor 1 (WT-1) protein was highly expressed in breast myoepithelial (ME) and endothelial cells. As the human breast tissue is rich in ME cells and blood vessels, our current study intended to assess whether WT-1 immunohistochemistry may have dual usages in evaluation of the ME cells and micro-vessel density. Consecutive sections were prepared from breast tumors with co-existing normal, hyperplastic, and neoplastic components. Consecutive sections were immunostained for WT-1 and a panel of ME and endothelial cell markers. From each case, 4-5 randomly selected duct clusters were photographed, and the percentages of positive cells for these molecules were compared. Similar to ME cell marker CD10 and smooth muscle actin (SMA), WT-1 expression was preferentially seen in ME cells, and over 90% of WT-1 positive ME cells were immunoreactive to CD10 and SMA. Distinct WT-1 expression was also seen in endothelial cells, and over 90% of WT-1 positive endothelial cells were positive for blood vessel specific markers. With tumor progression, the percentage and intensity of WT-1 positivity decreased in ME cells, whereas increased in endothelial cells. These finding suggest that WT-1 immunohistochemistry may be used to assess both the ME cells and micro-vessel density.

4.

Our recent studies revealed that focal alterations in breast myoepithelial cell layers significantly impact the biological presentation of associated epithelial cells. As pregnancy-associated breast cancer (PABC) has a significantly more aggressive clinical course and mortality rate than other forms of breast malignancies, our current study compared tumor suppressor expression in myoepithelial cells of PABC and non-PABC, to determine whether myoepithelial cells of PABC may have aberrant expression of tumor suppressors. Tissue sections from 20 cases of PABC and 20 cases of stage, grade, and age matched non-PABC were subjected to immunohistochemistry, and the expression of tumor suppressor maspin, p63, and Wilms' tumor 1 (WT-1) in calponin positive myoepithelial cells were statistically compared. The expression profiles of maspin, p63, and WT-1 in myoepithelial cells of all ducts encountered were similar between PABC and non-PABC. PABC, however, displayed several unique alterations in terminal duct and lobular units (TDLU), acini, and associated tumor tissues that were not seen in those of non-PABC, which included the absence of p63 and WT-1 expression in a vast majority of the myoepithelial cells, cytoplasmic localization of p63 in the entire epithelial cell population of some lobules, and substantially increasing WT-1 expression in vascular structures of the invasive cancer component. All or nearly all epithelial cells with aberrant p63 and WT-1 expression lacked the expression of estrogen receptor and progesterone receptor, whereas they had a substantially higher proliferation index than their counterparts with p63 and WT-1 expression. Hyperplastic cells with cytoplasmic p63 expression often adjacent to, and share a similar immunohistochemical and cytological profile with, invasive cancer cells. To our best knowledge, our main finings have not been previously reported. Our findings suggest that the functional status of myoepithelial cells may be significantly associated with tumor aggressiveness and invasiveness.

5.

BACKGROUND: The Wilms' tumor suppressor gene, wt1, encodes a zinc-finger protein, WT1, that functions as a transcription regulator. Previous studies have suggested a contradictory role for WT1 in breast cancer development.

MATERIALS AND METHODS: MCF10AT3B cells, a cell line derived from a xenograft model of progressive human proliferative breast disease, were used to study WT1 function in early development of breast cancer. A stable cell line was established from MCF10AT3B cells that ectopically expressed the Wilms' tumor suppressor, WT1. Western blot analysis, in vitro and in vivo growth assays were used to study the effects of constitutive WT1 expression on malignant progression of MCF10AT3B cells.

RESULTS: WT1 expression had a profound effect on several aspects of the cell cycle machinery and inhibited estrogen-stimulated and nonstimulated cell growth in vitro. In nude mice, WT1 expression strongly suppressed estrogen-stimulated tumorigenesis of neoplastigenic MCF10AT3B cells.

CONCLUSION: WT1 plays an important role in maintaining normal growth of mammary epithelial cells and dysregulated WT1 expression may contribute to breast cancer development.

Current literature suggests that strong WT1 expression in a carcinoma of unknown origin virtually excludes a breast primary. Our previous pilot study on WT1 expression in breast carcinomas has shown WT1 expression in approximately 10% of carcinomas that show mixed micropapillary and mucinous morphology (Mod Pathol 2007;20(Suppl 2):38A). To definitively assess as to what subtype of breast carcinoma might express WT1 protein, we examined 153 cases of invasive breast carcinomas. These consisted of 63 consecutive carcinomas (contained 1 mucinous tumor), 20 cases with micropapillary morphology (12 pure and 8 mixed), 6 micropapillary 'mimics' (ductal no special type carcinomas with retraction artifacts), 33 pure mucinous carcinomas and 31 mixed mucinous carcinomas (mucinous mixed with other morphologic types). Overall, WT1 expression was identified in 33 carcinomas, that is, 22 of 34 (65%) pure mucinous carcinomas and in 11 of 33 (33%) mixed mucinous carcinomas. The non-mucinous component in these 11 mixed mucinous carcinomas was either a ductal no special type carcinoma (8 cases) or a micropapillary component (3 cases). WT1 expression level was similar in both the mucinous and the non-mucinous components. The degree of WT1 expression was generally weak to moderate (>90% cases) and rarely strong (<10% cases). None of the breast carcinoma subtype unassociated with mucinous component showed WT1 expression.

7.

Serous papillary adenocarcinoma of the female genital organs and invasive micropapillary carcinoma of the breast have close histologic similarities. Thus, when these cancers occur synchronously or metachronously in the same patient, it is difficult to determine the primary site. We examined 23 serous papillary adenocarcinomas (16 ovarian, 5 endometrial, and 2 peritoneal) and 37 invasive micropapillary carcinomas of the breast (12 pure and 25 mixed types) on immunohistochemical expression of Wilm's tumor antigen-1 (WT1), CA125, and gross cystic disease fluid protein-15 (GCDFP-15), which have been reported to be useful in the differential diagnosis of primary ovarian carcinomas versus metastatic breast cancer to the ovary. The positive rates of WT1, CA125, and GCDFP-15 in serous papillary adenocarcinomas were 78%, 78%, and 0%, respectively, and the corresponding rates in invasive micropapillary carcinomas were 3%, 40%, and 38%. The CA125-positive rate of invasive micropapillary carcinoma was higher than the rate reported for other types of breast carcinomas. We consider CA125 to be not always useful in the differential diagnosis of serous papillary adenocarcinoma and invasive micropapillary carcinoma. Although the positive rate of WT1 was significantly higher in serous papillary adenocarcinoma than in invasive micropapillary carcinoma, WT1 expression in endometrial serous papillary adenocarcinoma was infrequent (20%). WT1 and GCDFP-15 could be useful markers for the differential diagnosis of ovarian and peritoneal serous papillary adenocarcinoma versus breast invasive micropapillary adenocarcinoma. However, the availability of GCDFP-15 is limited because of the low positive rate of GCDFP-15 in invasive micropapillary carcinomas.