ERBB2 phosphorylation and trastuzumab sensitivity of breast cancer cell lines.

Breast cancers that overexpress the ERBB2 tyrosine kinase receptor may be treated with the recombinant humanized monoclonal anti-ERBB2 antibody trastuzumab (herceptin). However, resistance to this targeted therapy is frequent. We have determined the response of 18 breast tumor cell lines to trastuzumab and compared it with the ERBB2 phosphorylation status using antibodies directed against tyrosine residue 1248. We show that sensitivity to trastuzumab is frequently associated with the expression of a phosphorylated ERBB2 protein.

Correlated break at PARK2/FRA6E and loss of AF-6/Afadin protein expression are associated with poor outcome in breast cancer.

Common fragile sites (CFSs) are regions of chromosomal break that may play a role in oncogenesis. The most frequent alteration occurs at FRA3B, within the FHIT gene, at chromosomal region 3p14. We studied a series of breast carcinomas for break of a CFS at 6q26, FRA6E, and its associated gene PARK2, using fluorescence in situ hybridization on tissue microarrays (TMA). We found break of PARK2 in 6% of cases. We studied the PARK2-encoded protein Parkin by using immunohistochemistry on the same TMA. Loss of Parkin was found in 13% of samples but was not correlated with PARK2 break. PARK2 break but not Parkin expression was correlated with prognosis. Alteration of PARK2/FRA6E may cause haplo-insufficiency of one or several telomeric potential tumor suppressor genes (TSG). The AF-6/MLLT4 gene, telomeric of PARK2, encodes the Afadin scaffold protein, which is essential for epithelial integrity. Loss of Afadin was found in 14.5% of cases, and 36% of these cases showed PARK2 break. Loss of Afadin had prognostic impact, suggesting that AF-6 may be a TSG. Loss of Afadin was correlated with loss of FHIT expression, suggesting fragility of FRA6E and FRA3B in a certain proportion of breast tumors.

WNT pathway and mammary carcinogenesis: loss of expression of candidate tumor suppressor gene SFRP1 in most invasive carcinomas except of the medullary type.

Secreted Frizzled-related protein 1 (SFRP1) encodes a member of a protein family that contains a cysteine-rich domain similar to the WNT-binding site of Frizzled receptors and regulates the WNT pathway. The WNT pathway is frequently altered in human cancers. We have defined the pattern of SFRP1 mRNA expression in the progression of breast cancer. We show that SFRP1 is expressed in the epithelial component of normal breast, in the in situ component of ductal carcinomas and is lost in more than 80% of invasive breast carcinomas except the medullary type. Loss of SFRP1 expression is correlated with the presence of hormonal receptors. Conversely, the maintenance of SFRP1 in carcinomas is correlated with the presence of lymphoplasmocytic stroma. No significant association was observed between SFRP1 status and the level of apoptosis in tumoral cells.

Loss of heterozygosity and linkage analysis in breast carcinoma: indication for a putative third susceptibility gene on the short arm of chromosome 8.

We have analysed losses of heterozygosity (LOH) at eight markers from the p12-p22 region of human chromosome 8 in a panel of 113 breast tumors. LOH were detected in almost half of the tumors. The most frequently deleted region included microsatellite (CA)n repeats markers D8S258, D8S133 and D8S259, located at 8p12-p22, while markers NEFL and LPL appeared less frequently altered. In parallel, linkage analysis was performed using the same informative markers, to test for the involvement of chromosome 8p loci in familial breast cancer. Positive cumulative multipoint lod score of 2.51 at theta = 0.0 was obtained with markers NEFL and D8S259. These results suggest that region 8p12-p22 carries at least one tumor suppressor gene involved in sporadic and perhaps also in familial breast cancer.

Multiple sites of loss of heterozygosity on chromosome arms 3p and 3q in human breast carcinomas.

Loss of heterozygosity (LOH) at loci from both arms of chromosome 3 were shown to occur in a high proportion of breast carcinomas. Six regions of consistent loss were identified, and were variably involved in the tumors. This suggests that multiple potential tumor suppressor genes involved in mammary carcinogenesis are present on chromosome 3.

Prognostic value of simultaneous expression of p21 and mdm2 in breast carcinomas treated by adjuvant chemotherapy with antracyclin.

One hundred and sixty-two breast carcinomas treated by adjuvant chemotherapy were investigated in immunohistochemistry for expression of p53 and two wild-type p53-regulated induced proteins, mdm2 and p21/waf1. p21 and mdm2 were expression stongly correlated with Ki67 but not with survival. The p53+/p21+, p53+/p21- and p53+/mdm2- phenotypes were associated with the worst prognosis. The p53+/p21+/ mdm2+ tumors were associated with a better outcome than the other phenotypes, they may be tumors expressing wild-type p53 and p21, and a form of mdm2 that might lead to the stabilization of p53. It is suggested that p21/mdm2 expression should be investigated in all cases of p53 positive breast cancer.

FGF7 protein expression in human breast carcinomas.

FGFs, especially FGF7, are thought to play an important role in the stroma-epithelium interactions that take place in several tissues, including the prostate and mammary glands. Using an immunohistochemistry approach, FGF7 protein expression was measured in a series of 80 human breast carcinomas. FGF7 was found to be expressed in two-thirds of cases studied, either in the stroma or in the tumour cells. Although it did not reach significance level, a tendency was noted for FGF7 expression to be associated with the most differentiated and least aggressive tumours.

Histological type and syncytial growth pattern affect E-cadherin expression in a multifactorial analysis of a combined panel of sporadic and BRCA1-associated breast cancers.

E-cadherin is a prominent factor in maintaining the epithelial architecture, and loss of its normal function is considered to be a key element in cancer invasion. In breast cancer, correlation between alteration of E-cadherin expression and histological type has been reported, but associations with other parameters remain uncertain. To refine these findings and to explore the biological significance of features thought to result from alterations of cell-to-cell adhesion systems, rare in sporadic cases but more frequent in BRCA1-associated breast cancers (BRCA1-BCs), we investigated E-cadherin expression by immuno-histochemistry in a combined panel of 214 breast cancers enriched in hereditary cases (176 sporadic cases and 38 BRCA1-BCs). Following multivariate statistical analysis using a logistic regression model, only 2 parameters were significantly associated with loss of E-cadherin expression: lobular histological type (p < 0.0001), in agreement with previous results, and syncytial growth pattern (SGP) (p = 0.01). The latter result provides a biological basis for SGP, the cardinal feature of medullary breast carcinoma.

Prognosis of breast-carcinoma lymphagenesis evaluated by immunohistochemical investigation of vascular-endothelial-growth-factor receptor 3.

Very few studies have yet addressed the question of the existence and role of lymphagenesis in tumor growth; it is generally overshadowed by the greater emphasis placed on the blood vascular system. Monoclonal antibodies against vascular endothelial-growth-factor receptor 3 (VEGFR3) have been shown to provide a specific antigenic marker for lymphatic endothelium. By comparison with the microvascular count (MVC), we investigated the prognostic value of the microlymphatic count (MLC) in a series of 60 cases of 2-cm-diameter breast carcinomas. The mean value of MVC was 72.5 and of MLC, 40.5. There was no quantitative correlation between these 2 parameters. The MVC but not the MLC had a prognostic value in overall survival. Neither the MLC nor the MVC had any correlation with axillary-lymph-node invasion.

Angiogenesis as a prognostic marker in breast carcinoma with conventional adjuvant chemotherapy: a multiparametric and immunohistochemical analysis.

It has now been clearly established that quantitative immunohistochemical methods applied to tumour angiogenesis under suitable quality control conditions are a powerful prognostic tool for use in the initial assessment of breast carcinomas. Appropriate parameters for predicting the aggressiveness of tumours and their sensitivity to treatment are, however, still required. To determine whether the microvessel count (MVC) may serve to predict the chemotherapeutic response, a retrospective study was carried out on a series of 162 patients with breast carcinoma, who were all treated with the same standard adjuvant chemotherapy. Angiogenesis was assessed by performing CD31 immunostaining and MVC per mm2. Several other factors such as P53, ERBB2, BCL2, and Ki67 were also measured, and their prognostic value was compared with that of the MVC. The MVC was not found to be correlated with any of the other prognostic parameters, but turned out to be of great prognostic value whatever the threshold value chosen, which suggests that it is continuously valid at all levels. The median value of the MVC (43.5 per mm2) divided this series into two significantly different prognostic categories, in terms of both disease-free survival (P = 0.0002) and overall survival (P = 0.037). Univariate analysis showed that most of the parameters analysed were of prognostic value regarding the disease-free survival, namely grade (P = 0.029), mitotic index (P = 0.049), size (P = 0.015), oestrogen receptors (P = 0.022), progesterone receptors (P = 0.018), P53 (P = 0.0045), ERBB2 (P = 0.046), and Ki67 (P = 0.0008). As regards overall survival, grade and ERBB2 showed a loss of prognostic value. In multivariate analysis on disease-free survival, the MVC was the most accurate prognostic factor (RR = 2.64), followed by Ki67 (RR = 2.06) and P53 (RR = 1.69). With respect to overall survival, the MVC ranked third among the prognostic parameters analysed. Standard chemotherapy did not reduce the high prognostic value of the MVC performed on tumour angiogenesis. This suggests that the MVC may predict the degree of resistance to chemotherapy. Patients with high levels of angiogenesis, particularly node-negative patients, might therefore be able to benefit from adjuvant therapy of another kind.

Chromosome region 8p11-p21: refined mapping and molecular alterations in breast cancer.

Several genes, most of them unknown, of the short arm of chromosome 8 are involved in malignant diseases. Numerous studies have implicated a portion of the 8p11-p21 region as the location of one or more tumor suppressor genes involved in a variety of human cancers, including breast cancer. We and others have reported linkage analyses suggesting the presence of a putative breast cancer susceptibility gene. Furthermore, several oncogenes of the 8p11-p12 region are involved in reciprocal translocations in myeloproliferative and myelodysplastic disorders and in amplification in breast cancer. To facilitate the analysis of the 8p11-p21 region and the cloning of candidate oncogenes and tumor suppressor genes, a high-resolution physical and transcriptional map was established with 39 yeast artificial chromosomes and 94 markers, including so-called sequence-tagged sites and expressed sequence-tagged sites derived from either known genes or expressed sequence tags corresponding to unidentified transcripts. In addition, four novel transcripts were identified and localized precisely within the map. This transcription map provides a detailed description of gene order for the 8p11-p21 region and will be helpful in the identification of candidate genes for diseases. From this basis, we refined the mapping of two types of molecular alterations that occur at 8p11-p21 in sporadic breast cancers, i.e., amplification and deletion.