Nikita Makretsov

BCL2 in breast cancer: a favourable prognostic marker across molecular subtypes and independent of adjuvant therapy received

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.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.

Overexpression of the Anti-Adhesin Podocalyxin Is an Independent Predictor of Breast Cancer Progression

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.

Coexpression of the type 1 growth factor receptor family members HER-1, HER-2, and HER-3 has a synergistic negative prognostic effect on breast carcinoma survival

The 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.

A fluorescence in situ hybridization study of ETV6-NTRK3 fusion gene in secretory breast carcinoma

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.

The presence of stromal mast cells identifies a subset of invasive breast cancers with a favorable prognosis

Tissue microarrays containing 348 cases of invasive breast carcinoma were studied by immunohistochemical staining for CD-117, CD-3, CD-20, CD-68, Her2, estrogen receptor protein, and progesterone receptor protein, and results were correlated with patient outcome. Hormone receptor status (both estrogen receptor and progesterone receptor) correlated with a good outcome while Her2 overexpression was associated with a poor outcome. The presence of mast cells in the stroma, as demonstrated by positive c-kit (CD-117) staining, correlated with a good prognosis (P=0.0036). On subset analysis, this association between the presence of mast cells and favorable prognosis was present in the node-negative patients (P=0.018). The presence of mast cells showed an inverse correlation with the presence of CD-68 positive macrophages. No correlation was observed between the presence of mast cells and either B-cells (CD20-positive) or T-cells (CD3-positive). The presence of stromal mast cells was of prognostic significance independent of nodal status and tumor size (P=0.02). When the multivariate analysis was expanded to include tumor grade, estrogen receptor status and Her2 status, as well as tumor size and nodal status, the presence of stromal mast cells approached significance as an independent prognostic indicator.

Tissue microarray analysis of neuroendocrine differentiation and its prognostic significance in breast cancer

The aim of this study was to detect neuroendocrine differentiation (NE), to determine its association with major clinicopathological parameters of breast cancer, and to study the prognostic significance of NE differentiation in a group of breast carcinomas by using tissue microarray (TMA) methodology. NE differentiation was studied by using 3 markers, synaptophysin (Syn), chromogranin A (ChA), and neuron-specific enolase (NSE) in a group of 334 patients with breast carcinoma. TMA blocks were made by using duplicate 0.6-mm-diameter tissue cores from each paraffin block. Results of immunostaining were scored on a 4-point scale, that is, as negative, weak, intermediate, and strong immunoreactivity. Positive staining of breast cancers for any of the 3 NE markers was detected in 19.5% of cases. Expression of a single marker was present in 16.2% of cases, and expression of 2 or 3 markers in combination was detected in 3.3% of cases. There was no statistically significant correlation of NE phenotype with tumor morphology, except mucinous carcinoma (3 of 6 cases positive), estrogen receptor, progesterone receptor, or nodal status. A weak correlation was noted between synaptophysin staining and higher tumor grade (P = 0.029). Analysis of disease-specific and overall survival based on up to 20 years of follow-up data showed a correlation between NSE expression and improved disease-specific (P = 0.043) and overall survival (P = 0.03) in univariate but not in multivariate analysis. The expression of Syn and ChA, as well as coexpression of multiple NE markers, had no prognostic significance.

Expression of the Insulin-Like Growth Factor I Receptor and Urokinase Plasminogen Activator in Breast Cancer Is Associated with Poor Survival Potential for Intervention with 17-Allylamino Geldanamycin

Urokinase plasminogen activator (uPA) expression in breast cancer is associated with relapse and a reduction in disease-specific survival. Thus, efforts are under way to identify uPA inhibitors. By screening a chemical library of >1000 compounds, 17-allyaminogeldanamycin (17AAG) was identified as a potent inhibitor of uPA by the National Cancer Institute and is now in Phase I clinical trials. At this time, it remains unclear how 17AAG blocks uPA; one possibility is through disruption of the insulin-like growth factor I receptor (IGF-IR) pathway. This would be consistent with studies from our laboratory showing that activation of IGF-IR results in the induction of uPA protein. In the study described herein, we observed that IGF-IR and uPA were highly expressed in 87 and 55% of breast cancer by screening tumor tissue microarrays representing 930 cases. A significant proportion (52.1% = 354 of 680 cases, P < 0.0001) of the patients had tumors expressing both proteins. uPA alone (P = 0.033) or in combination with IGF-IR (P = 0.0104) was indicative of decreased disease-specific survival. Next, we demonstrated that treating MDA-MB-231 cells with increasing concentrations of 17AAG resulted in IGF-IR degradation (IC50 = 1.0 μm) and blocked signal transduction through the Akt and mitogen-activated protein kinase pathways. Finally, we found that 17AAG had a robust inhibitory effect on the production of uPA mRNAand protein in the presence of IGF-I. Thus, our study raises the possibility that 17AAG could prove to be an effective therapeutic agent for a large number of breast cancer patients by inhibiting the IGF-IR and ultimately uPA.

Stromal CD10 expression in invasive breast carcinoma correlates with poor prognosis, estrogen receptor negativity, and high grade

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=0.003). There were correlations between stromal CD10 expression and higher tumor grade (P=0.01) and estrogen receptor (ER) negative status (P=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 (P<0.01), and in lymph node negative (P<0.05), but not lymph node positive subset of patients. It approached prognostic significance in multivariate analysis (P=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.

NRG1 gene rearrangements in clinical breast cancer: identification of an adjacent novel amplicon associated with poor prognosis

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.

Identification of prognostically relevant and reproducible subsets of endometrial adenocarcinoma based on clustering analysis of immunostaining data

Panels of immunomarkers can provide greater information than single markers, but the problem of how to optimally interpret data from multiple immunomarkers is unresolved. We examined the expression profile of 12 immunomarkers in 200 endometrial carcinomas using a tissue microarray. The outcomes of groups of patients were analyzed by using the Kaplan–Meier method, using the log-rank statistic for comparison of survival curves. Correlation between clustering results and traditional prognosticators of endometrial carcinoma was examined by either Fishers exact test or χ2-test. Multivariate analysis was performed using a proportional hazards method (Cox regression modeling). Seven of the 12 immunomarkers studied showed prognostic significance in univariate analysis (P<0.05) and 1 marker showed a trend toward significance (P=0.06). These eight markers were used in unsupervised hierarchical clustering of the cases, and resulted in identification of three cluster groups. There was a statistically significant difference in patient survival between these cluster groups (P=0.0001). The prognostic significance of the cluster groups was independent of tumor stage and patient age on multivariate analysis (P=0.014), but was not of independent significance when either tumor grade or cell type was added to the model. The cluster group designation was strongly correlated with tumor grade, stage, and cell type (P<0.0001 for each). Interlaboratory reproducibility of subclassification of endometrial adenocarcinoma by hierarchical clustering analysis was verified by showing highly reproducible assignment of individual cases to specific cluster groups when the immunostaining was performed, interpreted, and clustered in a second laboratory (κ=0.79, concordance rate=89.6%). Unsupervised hierarchical clustering of immunostaining data identifies prognostically relevant subsets of endometrial adenocarcinoma. Such analysis is reproducible, showing less interobserver variability than histopathological assessment of tumor cell type or grade.