Background:Breast cancer is heterogeneous and the existing prognostic classifiers are limited in accuracy, leading to unnecessary treatment of numerous women. B-cell lymphoma 2 (BCL2), an antiapoptotic protein, has been proposed as a prognostic marker, but this effect is considered to relate to oestrogen receptor (ER) status. This study aimed to test the clinical validity of BCL2 as an independent prognostic marker.Methods:Five studies of 11 212 women with early-stage breast cancer were analysed. Individual patient data included tumour size, grade, lymph node status, endocrine therapy, chemotherapy and mortality. BCL2, ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) levels were determined in all tumours. A Cox model incorporating the time-dependent effects of each variable was used to explore the prognostic significance of BCL2.Results:In univariate analysis, ER, PR and BCL2 positivity was associated with improved survival and HER2 positivity with inferior survival. For ER and PR this effect was time dependent, whereas for BCL2 and HER2 the effect persisted over time. In multivariate analysis, BCL2 positivity retained independent prognostic significance (hazard ratio (HR) 0.76, 95% confidence interval (CI) 0.66–0.88, P<0.001). BCL2 was a powerful prognostic marker in ER− (HR 0.63, 95% CI 0.54–0.74, P<0.001) and ER+ disease (HR 0.56, 95% CI 0.48–0.65, P<0.001), and in HER2− (HR 0.55, 95% CI 0.49–0.61, P<0.001) and HER2+ disease (HR 0.70, 95% CI 0.57–0.85, P<0.001), irrespective of the type of adjuvant therapy received. Addition of BCL2 to the Adjuvant! Online prognostic model, for a subset of cases with a 10-year follow-up, improved the survival prediction (P=0.0039).Conclusions:BCL2 is an independent indicator of favourable prognosis for all types of early-stage breast cancer. This study establishes the rationale for introduction of BCL2 immunohistochemistry to improve prognostic stratification. Further work is now needed to ascertain the exact way to apply BCL2 testing for risk stratification and to standardise BCL2 immunohistochemistry for this application.

Podocalyxin is a CD34-related cell surface molecule with anti-adhesive qualities. We probed a tissue microarray (n ‫؍‬ 272) linked to long-term outcome data and found that podocalyxin was highly overexpressed in a distinct subset of invasive breast carcinomas (n ‫؍‬ 15; 6%). Univariate disease-specific (P < 0.01) and multivariate regression (P < 0.0005) analyses indicated that this overexpression is an independent indicator of poor outcome. Forced podocalyxin expression perturbed cell junctions between MCF-7 breast carcinoma cells, and it caused cell shedding from confluent monolayers. Therefore, podocalyxin overexpression is a novel predictor of breast cancer progression that may contribute to the process by perturbing tumor cell adhesion.

Prognostically relevant cluster groups, based on gene expression profiles, have been recently identified for breast cancers, lung cancers, and lymphoma. Our aim was to determine whether hierarchical clustering analysis of multiple immunomarkers (protein expression profiles) improves prognostication in patients with invasive breast cancer. A cohort of 438 sequential cases of invasive breast cancer with median follow-up of 15.4 years was selected for tissue microarray construction. A total of 31 biomarkers were tested by immunohistochemistry on these tissue arrays. The prognostic significance of individual markers was assessed by using Kaplan-Meier survival estimates and log-rank tests. Seventeen of 31 markers showed prognostic significance in univariate analysis (P < 0.05) and 4 markers showed a trend toward significance (P < 0.2). Unsupervised hierarchical clustering analysis was done by using these 21 immunomarkers, and this resulted in identification of three cluster groups with significant differences in clinical outcome. 2 analysis showed that expression of 11 markers significantly correlated with membership in one of the three cluster groups. Unsupervised hierarchical clustering analysis with this set of 11 markers reproduced the same three prognostically significant cluster groups identified by using the larger set of markers. These cluster groups were of prognostic significance independent of lymph node metastasis, tumor size, and tumor grade in multivariate analysis (P ‫؍‬ 0.0001). The cluster groups were as powerful a prognostic indicator as lymph node status. This work demonstrates that hierarchical clustering of immunostaining data by using multiple markers can group breast cancers into classes with clinical relevance and is superior to the use of individual prognostic markers.

CD10 is a zinc-dependent peptidase (metalloproteinase), which degrades a variety of bioactive peptides. Earlier studies suggested that CD10 expression in tumor stroma is associated with biological aggressiveness of the tumor. To date, only one study has addressed the clinical significance of stromal CD10 expression in invasive carcinoma of the breast. The aim of this confirmatory study is to evaluate stromal CD10 expression in breast carcinoma and to examine associations between CD10, clinicopathological variables, and patient outcome. Tissue microarrays, containing 438 cases of invasive breast carcinoma and 15 cases of ductal carcinoma in situ with 15 years median follow-up time, were assembled. CD10 expression was assessed by immunohistochemistry and scored as negative, weak and strong. Nonparametric correlational tests, univariate and multivariate survival analyses were performed. Stromal CD10 was preferentially expressed in invasive compared to noninvasive breast cancers (P ¼ 0.003). There were correlations between stromal CD10 expression and higher tumor grade (P ¼ 0.01) and estrogen receptor (ER) negative status (P ¼ 0.002). There was no correlation between CD10 and lymph node status, tumor size, histological subtype, progesterone receptors, and Her2 status. Stromal CD 10 expression was associated with decreased long-term disease-specific and overall survival in the entire cohort (Po0.01), and in lymph node negative (Po0.05), but not lymph node positive subset of patients. It approached prognostic significance in multivariate analysis (P ¼ 0.06) when lymph node status, tumor size, ER and Her2 were considered in the same model; and was associated with a relative risk of death of 2.8, compared to relative risk of 2.4 for lymph node positive status. Thus, stromal CD10 expression in invasive carcinoma of the breast is associated with ER negativity, higher tumor grade and decreased survival and constitutes a potential prognostic marker and a target for development of novel therapies. Modern Pathology (2007) 20, 84-89.

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Rearrangements of the neuregulin (NRG1) gene have been implicated in breast carcinoma oncogenesis. To determine the frequency and clinical significance of NRG1 aberrations in clinical breast tumors, a breast cancer tissue microarray was screened for NRG1 aberrations by fluorescent in situ hybridization (FISH) using a two-color split-apart probe combination flanking the NRG1 gene. Rearrangements of NRG1 were identified in 17/382 cases by FISH, and bacterial artificial chromosome array comparative genomic hybridization was applied to five of these cases to further map the chromosome 8p abnormalities. In all five cases, there was a novel amplicon centromeric to NRG1 with a minimum common region of amplification encompassing two genes, SPFH2 and FLJ14299. Subsequent FISH analysis for the novel amplicon revealed that it was present in 63/262 cases. Abnormalities of NRG1 did not correlate with patient outcome, but the novel amplicon was associated with poor prognosis in univariate analysis, and in multivariate analysis was of prognostic significance independent of nodal status, tumor grade, estrogen receptor status, and human epidermal growth factor receptor (HER)2 overexpression. Of the two genes in the novel amplicon, expression of SPFH2 correlated most significantly with amplification. This amplicon may emerge as a result of breakpoints and chromosomal rearrangements within the NRG1 locus.

The translocation t(12;15)(p13;q25), in which the ETV6 gene from chromosome 12 is rearranged with the NTRK3 gene from chromosome 15, has recently been identified in secretory breast carcinoma (SBC). This fusion gene was initially described in congenital fibrosarcoma and congenital mesoblastic nephroma. The biological consequence of this translocation is the expression of a chimeric protein tyrosine kinase with potent transforming activity. To assess the frequency of t(12;15)(p13;q25) in breast cancer, we developed complementary probe sets (fusion and split-apart probes) for the detection of this translocation by fluorescence in situ hybridization (FISH) in paraffin-embedded, formalin-fixed tissue sections. We tested four histologically confirmed cases of SBC for the presence of the ETV6-NTRK3 gene fusion and then applied the FISH assay to tissue microarrays (TMAs) in order to screen 481 cases of formalin-fixed, paraffin-embedded invasive breast carcinomas of various histologic subtypes. Three of the four cases of SBC revealed fusion signals. Of the 481 cases in the TMAs, 202 gave signals of sufficient quality for screening by FISH, and only one case showed fusion signals in most or all of the tumor cells. On review of the histology of this case, SBC was confirmed. On the other hand, none of the fusion-negative breast cancers revealed SBC histology. In all cases, the results from the fusion and split-apart FISH assays for the ETV6-NTRK3 fusion genes were concordant. Our data suggest that the ETV6-NTRK3 fusion gene is a specific genetic alteration in SBC.

BACKGROUNDThe clinical significance of coexpression of type 1 growth factor receptor (T1GFR) family members remains largely unknown. The objective of the current study was to determine the frequency and the possible prognostic effect of coexpression of HER‐1, HER‐2, HER‐3, and HER‐4 by breast carcinoma.METHODSTissue microarrays were constructed using clinically annotated formalin‐fixed, paraffin‐embedded tumor samples from 242 patients with invasive breast carcinomas with a median 15‐year follow‐up. The levels of TIGFR family members (HER‐1–HER‐4) were measured by immunohistochemistry. K‐means clustering algorithm, as well as univariate (Kaplan–Meier, log‐rank test) and multivariate (Cox regression) survival analyses were applied to the data set.RESULTSUsing univariate analysis, expression of HER‐1, HER‐2, and HER‐3, but not HER‐4, was significantly associated with decreased patient disease‐specific survival (P < 0.05). Kaplan–Meier survival analysis showed that coexpression of ≥ 2 of HER‐1, HER‐2, and HER‐3 in any combination was associated with reduced patient disease‐specific survival compared with single marker expression or no expression (35% vs. 65% vs. 78% 10‐year survival rates, P = 0.001). Using multivariate analysis, expression of ≥ 2 of HER‐1, HER‐2, and HER‐3 was independent of lymph node status and tumor size.CONCLUSIONSIn a cohort of patients with breast carcinoma, the authors observed T1GFR family member coexpression (HER‐1, HER‐2, and HER‐3) to have a negative synergistic effect on patient outcome, independent of tumor size or lymph node status. Thus, coexpression of T1GFR family members identified a subset of patients with a poor disease prognosis who may potentially benefit from therapy simultaneously targeting several T1GFR family members. Cancer 2005. © 2005 American Cancer Society.