Joop J.T.M. Heuvel

Expression of the transcription factor Ets-1 is an independent prognostic marker for relapse-free survival in breast cancer.

The transcription factor Ets-1 regulates the expression of several angiogenic and extracellular matrix remodeling factors, and might be implicated in disease progression of breast cancer. In the present study, the prognostic value of Ets-1 expression was assessed by quantitative real-time fluorescence RT–PCR in 123 sporadic primary breast cancer samples of patients with a median follow-up time of 62 months. Ets-1 expression levels correlated significantly with VEGF and PAI-1 in the same tissue. In univariate (P=0.0011) and multivariate (P=0.005) analyses, Ets-1 expression showed significant prognostic value for relapse-free survival. Ets-1 is a strong, independent predictor of poor prognosis in breast cancer. This seems – at least in part – to be attributable to its role in transcriptional regulation of factors involved in angiogenesis (VEGF), and extracellular matrix remodeling (PAI-1).

Mammaglobin Is Associated With Low-Grade, Steroid Receptor-Positive Breast Tumors From Postmenopausal Patients, and Has Independent Prognostic Value for Relapse-Free Survival Time

PURPOSE The tumor mRNA expression levels of mammaglobin, a novel breast-specific and breast cancer-associated marker, were correlated with disease outcome in 280 patients with primary breast cancer. PATIENTS AND METHODS Mammaglobin expression levels were assessed by quantitative reverse transcriptase polymerase chain reaction in frozen tumor tissue from breast cancer patients with a median age of 60 years (range, 30 to 88 years) and a median follow-up of 85 months (range, 2 to 169 months). RESULTS High expression levels were associated with low-grade tumors (P =.018), with positive estrogen and progesterone receptor status (P <.001), and postmenopausal status (P =.010). In the analysis of all patients, low tumor mammaglobin expression levels predicted an early relapse both in Cox univariate (hazard ratio [HR], 0.52; 95% CI, 0.34 to 0.79; P =.002) and multivariate regression analyses corrected for the traditional prognostic factors (HR, 0.55; 95% CI, 0.35 to 0.88; P =.012). The association of mammaglobin expression with the rate of relapse was particularly favorable in patients who received adjuvant tamoxifen treatment (HR, 0.35; 95% CI, 0.17 to 0.71; P =.004). CONCLUSION These results demonstrate that the assessment of the tumor mRNA expression level of the breast-specific protein mammaglobin could be useful to stratify patients for individual adjuvant treatment strategies.

Tribbles homolog 3 denotes a poor prognosis in breast cancer and is involved in hypoxia response.

IntroductionHypoxia in solid tumors is associated with treatment resistance, resulting in poor prognosis. Tribbles homolog 3 (TRIB3) is induced during hypoxia and is involved in multiple cellular pathways involved in cell survival. Here, we investigated the role of TRIB3 in breast cancer.MethodsTRIB3 mRNA expression was measured in breast tumor tissue from 247 patients and correlated with clinicopathological parameters and clinical outcome. Furthermore, we studied TRIB3 expression regulation in cell lines, xenografts tissues and human breast cancer material using Reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Finally, the effect of small interfering RNA (siRNA) mediated TRIB3 knockdown on hypoxia tolerance was assessed.ResultsBreast cancer patients with low, intermediate or high TRIB3 expression exhibited a mean disease free survival (DFS) of 80 (95% confidence interval [CI] = 74 to 86), 74 (CI = 67 to 81), and 63 (CI = 55 to 71) months respectively (P = .002, Mantel-Cox log-rank). The prognostic value of TRIB3 was limited to those patients that had received radiotherapy as part of their primary treatment (n = 179, P = .005) and remained statistically significant after correction for other clinicopathological parameters (DFS, Hazard Ratio = 1.90, CI = 1.17 to 3.08, P = .009). In breast cell lines TRIB3 expression was induced by hypoxia, nutrient starvation, and endoplasmic reticulum stress in an hypoxia inducible factor 1 (HIF-1) independent manner. TRIB3 induction after hypoxia did not increase with decreasing oxygen levels. In breast tumor xenografts and human breast cancer tissues TRIB3 co-localized with the hypoxic cell marker pimonidazole. The induction of TRIB3 by hypoxia was shown to be regulated via the PERK/ATF4/CHOP pathway of the unfolded protein response and knockdown of TRIB3 resulted in a dose-dependent increase in hypoxia sensitivity.ConclusionsTRIB3 is independently associated with poor prognosis of breast cancer patients, possibly through its association with tumor cell hypoxia.

Tissue inhibitors of metalloproteinase expression in human breast cancer: TIMP-3 is associated with adjuvant endocrine therapy success.

Tissue inhibitors of matrix metalloproteinase (TIMPs) may be involved in tumour growth, apoptosis, angiogenesis, invasion, and the development of metastases. This study has evaluated the association of the expression levels of the TIMP forms 1, 2, 3, and 4, measured by quantitative real‐time RT‐PCR, with classical clinicopathological characteristics, ie age, menopausal status, tumour size, histological grade, number of involved lymph nodes, and steroid hormone receptor status, and with disease progression and treatment sensitivity in 273 breast cancer patients. The mRNA levels of TIMP‐1 and TIMP‐2 were not associated with any known clinicopathological tumour feature. TIMP‐3 and TIMP‐4 levels were significantly higher in steroid hormone receptor‐positive samples, although the levels of TIMP‐4 were much lower than those of the other TIMPs. Only TIMP‐3 predicted relapse‐free survival (RFS) time differently depending on post‐surgical treatment as, in particular, the interaction of TIMP‐3 with endocrine therapy (p = 0.008, HR = 0.24, 95% CI = 0.09–0.69) contributed significantly to RFS in multivariate Cox regression analysis. In subgroup analyses, the 107 patients treated with tamoxifen differed greatly in prognosis after dichotomization by the median TIMP‐3 level (p = 0.0003). Thus, high tumour levels of the matrix metalloproteinases inhibitor and pro‐apoptotic factor TIMP‐3 are associated with successful tamoxifen treatment of patients with breast cancer. Copyright

Complexes between urokinase-type plasminogen activator and its receptor in blood as determined by enzyme-linked immunosorbent assay

Complexes between urokinase‐type plasminogen activator (uPA) and its receptor (uPAR) were assessed in plasma and serum from 39 breast cancer patients and from 20 healthy individuals, applying a recently developed enzyme‐linked immunosorbent assay (ELISA) for the analysis of these complexes in tumor tissue extracts. The assay is based on a combination of rabbit polyclonal anti‐uPA antibodies for catching and a mouse anti‐uPAR monoclonal antibody (MAb) for detection. The specificity of the assessment of uPA:uPAR complexes was verified by simultaneous analysis of the individual blood samples in corresponding non‐sense ELISA formats, in which either the anti‐uPA catching antibody or the anti‐uPAR detecting antibody was substituted with an irrelevant antibody. Assessment of native uPA:uPAR complexes was ascertained by demonstrating the absence of any de novo formation of uPA:uPAR complexes in plasma and serum during the sample incubation step in the ELISA, as verified by the use of a peptide antagonist for uPAR. Plasma and serum samples contained almost identical levels of uPA:uPAR complexes. The levels of uPA:uPAR complexes were found to be significantly lower in serum from breast cancer patients compared to the serum of healthy donors, while the levels of (total) uPAR in plasma from breast cancer patients were significantly higher than in plasma from the healthy controls. In addition, the free, uncomplexed uPAR levels, estimated by subtraction of uPA:uPAR complex levels from (total) uPAR levels, were significantly elevated in plasma as well as in serum from breast cancer patients compared to healthy individuals. The uPA:uPAR complex levels were highly comparable to the uPA levels analyzed in the same plasma and serum samples, indicating that most if not all of the uPA present in these samples is complexed with uPAR. Int. J. Cancer 77:236–242, 1998.© 1998 Wiley‐Liss, Inc.

Molecular Beacon Reverse Transcription-PCR of Human Chorionic Gonadotropin-β-3, -5, and -8 mRNAs Has Prognostic Value in Breast Cancer

BACKGROUND The beta-subunit of human chorionic gonadotropin (hCG) is encoded by four genes, of which expression of the hCGbeta-3, -5, and -8 genes could have prognostic value in breast cancer. METHODS Applying a new, modified Molecular Beacon reverse transcription-PCR assay, we investigated the prognostic value of the hCGbeta-3, -5, and -8 gene transcripts in 129 sporadic unilateral breast cancer samples from patients with a median follow-up of 62.3 months. RESULTS Expression of hCGbeta-3, -5, -8 was significantly (P = 0.020) associated with relapse-free survival (RFS). In multivariate survival analysis, hCGbeta-3, -5, and -8 maintained prognostic value for RFS, with high expression predicting shorter RFS (P = 0.015; hazard ratio, 2.25; 95% confidence interval, 1.17-4.34). Only 1 of 24 (4%) node-negative patients with low hCGbeta-3, -5, -8 expression relapsed, in contrast to 7 of 26 (27%) patients with high expression (P = 0.046). CONCLUSIONS Expression of hCGbeta-3, -5, -8, which differ by only one nucleotide from other hCGbeta genes, can be assessed by our modified Molecular Beacon assay in breast cancer tissues. Expression of hCGbeta-3, -5, -8 has independent, prognostic value for RFS in breast cancer and may help identify node-negative patients with poor prognosis.

ELISA for complexes of urokinase-type and tissue-type plasminogen activators with their type-1 inhibitor (uPA-PAI-1 and tPA-PAI-1).

An ELISA has been developed for the assessment of complexes between the urokinase‐type (uPA) and the tissue‐type plasminogen (tPA) activators with their inhibitor type‐1 (PAI‐1) in cell‐culture medium and cytosolic extracts of breast tumours. The “4‐stage/2‐site” ELISA involves 2 polyclonal antibodies in the pre‐analyte stage 2 and in the post‐analyte stage. For the specific measurement of the uPA∼PAI‐1 complex, 2 assay formats may be employed, uPA/PAI‐1 and PAI‐1/uPA. This offers an attractive facility for quality‐assessment studies of this kind of assays. Analogously, the tPA∼PAI‐1 complex was assessed using the formats tPA/PAI‐1 and PAI‐1/tPA. Only complexes are able to evoke a signal in their appropriate assay formats. The free component, however, which responds to the capture antibody, could interfere with the binding of the complex molecule, reducing the OD signal. Increasing the coating Ab concentration diminishes the signal‐suppressing effect of the free component. In 15 cell‐culture supernatants, uPA and PAI‐1 concentrations were measured as well as the uPA of PAI‐I complex in different dilutions in 2 assay formats. The differences between the values of complex measured in the 2 assay formats could be accounted for by the free uPA and PAI‐1 concentrations. At dilution 1:10, the measured values obtained in the 2 separate formats differed substantially (correlation coefficient r = 0.641). At dilution 1:20, the differences were already smaller between the values (agreement 0.945). At dilution 1:30, close agreement between the corresponding values was observed (r = 0.971). Extrapolation to infinite dilution of the results obtained resulted in an even closer estimation of the complex concentration. Comparable results have been observed when tPA, PAI‐1 and tPA∼PAI‐1 values were measured in tumour biopsy extracts. Int. J. Cancer 81:598–606, 1999.

E1AF Expression Levels are not Associated with Prognosis in Human Breast Cancer

E1AF is a transcription factor involved in regulation of several metastasis-associated genes, and is associated with overexpression of HER2/neu. We were unable to find a clear prognostic value of E1AF expression in human breast cancer. Furthermore, no association of E1AF levels with HER2/neu mRNA levels, hormone receptor status, histological grade, tumor size, or lymph node involvement was found.