Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR.

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

The NFkappaB pathway and endocrine-resistant breast cancer.

Endocrine therapy with an estrogen receptor (ER)-targeted antiestrogen, such as tamoxifen, or estrogen ablation by aromatase inhibitors is clinically indicated for the management of all forms of ER-positive breast cancer. However, 30-50% of ER-positive breast cancer cases fail to benefit clinically from endocrine therapy alone, and recent molecular evidence suggests that 'crosstalk' pathways originating from activated receptor tyrosine kinases and/or other proliferative and survival signals may be contributing to this endocrine resistance. Molecular identification and validation of candidate ER crosstalking pathways will likely lead to clinically important prognostic markers and targets for the application of novel therapeutics in combination with standard endocrine agents. This review focuses on a critical survival and proliferation pathway involving activation of nuclear factor-kappaB (NFkappaB), a family of ubiquitously expressed transcription factors that for nearly two decades have been known to be critical regulators of mammalian immune and inflammatory responses, and more recently have been associated with chemotherapy resistance. With the demonstration that activation of NFkappaB is absolutely required for normal mammary gland development, NFkappaB involvment in human breast cancers was initially explored and linked to the development of hormone-independent (ER-negative) breast cancer. Newer clinical evidence now implicates NFkappaB activation, particularly DNA-binding by the p50 subunit of NFkappaB, as a potential prognostic marker capable of identifying a high-risk subset of ER-positive, primary breast cancers destined for early relapse despite adjuvant endocrine therapy with tamoxifen. Furthermore, initial preclinical studies suggest that treatment strategies designed to prevent or interrupt activation of NFkappaB in cell-line models of these more aggressive, ER-positive breast cancers can restore their sensitivity to such standard endocrine agents as tamoxifen.

Understanding the age dependency of breast cancer biomarkers.

This review and study of nearly 4,000 primary breast cancers evaluates the hypothesis that human aging not only increases breast cancer incidence but also alters breast cancer biology. Clinically validated biomarkers were chosen as surrogate measures of genetic instability and tumor growth, invasiveness and metastatic potential. Our results support the premise that breast cancer clinical behavior and biology are significantly affected by patient age, but call into question key aspects of the current cancer-aging paradigm.

BRCA1/2 mutations in Swiss patients with familial or early-onset breast and ovarian cancer.

QUESTIONS UNDER STUDY: Germ-line alterations in BRCA1 and BRCA2 genes account for 30-50% of all forms of familial breast and ovarian cancer syndromes. Specific mutations in specific populations and ethnic groups have been identified in BRCA1 and BRCA2. However, it is not known whether such specific mutations prevail in the Swiss population. METHODS: We started to screen patients with primary breast and ovarian cancer and a strong family history of both cancers by sequencing the full-length coding regions of BRCA1 and BRCA2. RESULTS: With the selection criteria used in this study we identified 19 mutations in the first 38 patients screened (50%). These mutations were either defined as deleterious and resulted in a protein truncation (n = 10) or were defined as unclassified variants (n = 9). One novel truncating mutation was found in BRCA2 and two novel unclassified variants were detected in BRCA1. These three mutations are not described in the BIC and HGMD databanks. CONCLUSIONS: We detected three unknown mutations among 38 patients in a Swiss study of BRCA1/2 mutation patterns. One of these novel mutations is clearly deleterious as it leads to protein truncation at nucleotide 133 of BRCA2.

Prognostic and predictive significance of ErbB-2 breast tumor levels measured by enzyme immunoassay.

PURPOSE: A retrospective analysis to assess the prognostic and predictive clinical value of breast tumor ErbB-2 receptor expression quantified by enzyme immunoassay (EIA), to compare levels measured by EIA with ErbB-2 status determined by immunohistochemistry (IHC), and to correlate receptor content with levels of phosphorylated (Y1248-P) ErbB-2, a measure of functional tyrosine kinase activity. MATERIALS AND METHODS: EIA quantification of ErbB-2 was performed on membrane extracts from 3,208 well-characterized primary breast cancers. Overall, relapse-free, distant disease-free, and local/regional-free patient survival data were available on 1,123 of these tumors. IHC scoring for ErbB-2 status (HercepTest; DAKO, Glostrup, Denmark) was performed on adjacent sections of 151 cases, and receptor functionality was measured in 230 tumors by an antibody specific for phosphorylated (Y1248-P) ErbB-2. RESULTS: Unlike nonmalignant breast tissues, breast tumors showed increased ErbB-2 levels in a bimodal distribution, with 12% constituting a distinct set of ErbB-2-overexpressing tumors. The intermodal threshold value for ErbB-2 overexpression distinguished tumors with reduced estrogen and progesterone receptor content, high IHC score for ErbB-2, and significantly increased levels of phosphorylated (Y1248-P) ErbB-2 receptor. By multivariate analysis, EIA-determined ErbB-2 overexpression predicted significantly reduced patient survival that was unaffected by tamoxifen or cyclophosphamide, methotrexate, and fluorouracil adjuvant therapy. CONCLUSION: Determination of ErbB-2 receptor expression by EIA offers a clinically valuable alternative to semiquantitative IHC assessment of breast tumor ErbB-2 overexpression and affords the opportunity to evaluate ErbB-2 phosphorylation, which may represent an important predictive parameter of receptor functionality.

Potential prognostic value of mitogen-activated protein kinase activity for disease-free survival of primary breast cancer patients.

Signaling through pathways involving mitogen-activated protein kinases (MAP kinases) has been implicated in the pathogenesis of cancer. Thus, the activity of MAP kinase is essential in the malignant potential of human breast tumors. p42/44(MAPK) was significantly higher expressed in tumor samples than in matching normal tissues adjacent to the tumor. p42/44(MAPK) protein expression correlated with enhanced MAP kinase activity only in a subset of tumors, indicating that over-expression of MAP kinases does not reflect the activation status of these enzymes. MAP kinase activity was significantly elevated in 131 tissue samples from primary breast tumors when compared to 18 normal tissues adjacent to tumors. A trend for higher MAP kinase activity in primary tumors of node-positive patients was observed when compared with tumors from node-negative patients. Similarly, higher MAP kinase activities were observed in specimens from patients who had a relapse within the follow-up time of 40 months when compared with patients with no relapse. A survival analysis demonstrated that the MAP kinase activity in primary breast tumors is potentially prognostic for relapse-free survival of patients.

A yeast-based bioassay for the determination of functional and non-functional estrogen receptors.

The response to endocrine therapy of breast cancer is not entirely predictable from hormone receptor status alone since some point mutated or splicing variants of the estrogen receptor (ER) show altered biological activities. In order to characterize the activities of all forms of ER in a heterogeneous breast tumor, a functional assay in Saccharomyces cerevisiae was developed. Total RNA isolated from breast cancer cells and one breast cancer specimen was reverse transcribed and the ER cDNA was amplified by PCR. The products were then cloned into an expression vector by in vivo homologous recombination in yeast. The yeast strain carries a reporter gene ( ADE2 ) coupled to an estrogen response element. Activation of the reporter by ER yielded white colonies whereas lack of ER activity produced red colonies. This permitted the testing for functionality of individual ER molecules and subsequent analysis by rescuing of the ER expression plasmids and complete DNA sequencing. This simple visual test allows discrimination between wild-type ER, constitutively active ER and inactive ER.

Markers of tumor angiogenesis and proteolysis independently define high- and low-risk subsets of node-negative breast cancer patients.

PURPOSE: To compare the prognostic impact of tumor angiogenesis factors (vascular endothelial growth factor [VEGF], angiogenin, and basic fibroblast growth factor [bFGF]), tumor proteolysis factors (urokinase-type plasminogen activator [uPA] and plasminogen activator inhibitor-1 [PAI-1]), and conventional tumor markers (stage, grade, and steroid receptors) in early breast cancer. PATIENTS AND METHODS: In the primary clinical study, tumor angiogenesis and other factors were detected in frozen biopsies from 305 primary breast tumors. VEGF expression was assessed by chemiluminescence immunosorbent assay (ICMA); angiogenin, bFGF, uPA, and PAI-1 by enzyme-linked immunosorbent assay (ELISA); and steroid receptors (estrogen receptor [ER] and progesterone receptor [PgR]) by enzyme immunoassay (EIA). In the validating clinical study, another set of 190 node-negative primary breast tumor samples were collected at a separate institution. RESULTS: Univariate analysis of the primary study showed that VEGF levels were positively correlated with recurrence (P < .001). Angiogenin levels were positively correlated with disease relapse (P < .005) for the overall collective group, but not within the node-negative subset. No significant correlations were found between tumor bFGF levels and patient survival. In multivariate regression analysis, the only independent predictors of relapse-free survival (RFS) were VEGF, uPA, and lymph node status. In the validation set, the distribution of VEGF and uPA values were similar to those in the primary study; low expression of both VEGF and uPA identified patients with a < or = 20% likelihood of recurrence within 7 years. CONCLUSION: Separate primary and validating clinical studies concur that tumor VEGF level is the most important prognostic parameter among several markers of tumor angiogenesis and proteolysis.

Agrin can mediate acetylcholine receptor gene expression in muscle by aggregation of muscle-derived neuregulins.

The neural isoforms of agrin can stimulate transcription of the acetylcholine receptor (AChR) epsilon subunit gene in electrically active muscle fibers, as does the motor neuron upon the formation of a neuromuscular junction. It is not clear, however, whether this induction involves neuregulins (NRGs), which stimulate AChR subunit gene transcription in vitro by activating ErbB receptors. In this study, we show that agrin- induced induction of AChR epsilon subunit gene transcription is inhibited in cultured myotubes overexpressing an inactive mutant of the ErbB2 receptor, demonstrating involvement of the NRG/ErbB pathway in agrin- induced AChR expression. Furthermore, salt extracts from the surface of cultured myotubes induce tyrosine phosphorylation of ErbB2 receptors, indicating that muscle cells express biological NRG-like activity on their surface. We further demonstrate by RT-PCR analysis that muscle NRGs have Ig-like domains required for their immobilization at heparan sulfate proteoglycans (HSPGs) of the extracellular matrix. In extrasynaptic regions of innervated muscle fibers in vivo, ectopically expressed neural agrin induces the colocalized accumulation of AChRs, muscle-derived NRGs, and HSPGs. By using overlay and radioligand-binding assays we show that the Ig domain of NRGs bind to the HSPGs agrin and perlecan. These findings show that neural agrin can induce AChR subunit gene transcription by aggregating muscle HSPGs on the muscle fiber surface that then serve as a local sink for focal binding of muscle-derived NRGs to regulate AChR gene expression at the neuromuscular junction.

Increased expression of estrogen-receptor exon-5-deletion variant in relapse tissues of human breast cancer.

A substantial percentage (30-70%) of human breast carcinomas that initially respond to endocrine therapy acquire resistance during the treatment. Many patients with tumor progression despite treatment with anti-estrogen tamoxifen show continued expression of estrogen receptors (ER) and/or progesterone receptors (PgR) in the relapse tissue. This indicates that, in these tumors, mechanisms other than loss of ER expression are responsible for treatment failure. We have investigated the occurrence and frequency of the exon-5-deletion variant (d5) of ER in human breast-cancer biopsies and in normal tissues. In all normal and tumor tissues tested, both wild-type (wt) and d5 were detected, indicating that expression of the d5 variant is a naturally occurring polymorphism. However, the primary tumors of patients who relapse within 15 months (n = 13) express higher ratios of d5 than do those of patients with no relapse during the same period (p = 0.4, n = 19), though this difference is statistically not significant. A significant increase in the expression level of d5 was determined in relapse as compared with the respective primary tumor (p = 0.02). These data indicate that increased expression of the ER exon-5-deletion variant in relapse tissues might be due to clonal selection of cells resistant to anti-estrogen treatment.

Expression profile of the putative catalytic subunit of the telomerase gene.

Telomerase, a ribonucleoprotein complex, adds hexameric repeats called telomeres to the growing ends of chromosomal DNA. The enzyme telomerase activity is present in a vast majority of tumors but is repressed in most normal tissues. Recently, two groups have reported the molecular cloning of the putative catalytic subunit (hEST2/hTRT) of the telomerase gene. We investigated the expression of this gene in diverse tumor-derived cell lines and tumors as well as in various normal tissues. The expression of hEST2/hTRT was detectable in tumor-derived cell lines, primary breast tumors, pancreatic tumors, and kidney tumors. Furthermore, the expression of hEST2/hTRT was down-regulated in response to a differentiation inducer. However, several normal tissues also expressed varying levels of hEST2/hTRT. Early passage cultures of endothelial fibroblasts and some epithelial cells also expressed the telomerase gene, albeit at low levels. In contrast, the expression of TLP1/TP1, the human homologue of Tetrahymena p80 telomerase subunit, was similar in all of these samples. Our results indicate that the differences in expression of hEST2/hTRT in tumor versus normal cells are relative and are not absolute.

Selective modulation of protein kinase A and protein kinase C activities in epidermal growth factor (EGF)-stimulated MCF-7 breast cancer cells.

In human MCF-7 breast cancer cells, both protein kinase A (PKA) and different members of the protein kinase C (PKC) family are stimulated upon binding of epidermal growth factor (EGF) to cell surface receptors. Selective stimulation of calcium-dependent PKCs with 10(-6) to 10(-9) M Thymeleatoxin significantly increased the proliferation rate of MCF-7 cells over 5 days in culture. This stimulation was blocked by the PKC antagonist Chelerythrine. In contrast, selective activation of PKA by addition of 1 mM dibutyryl cyclic AMP (dBcAMP) did not affect the proliferation rate of MCF-7 cells. Similarly, activation of the adenylate cyclase by 1 microM Forskolin and inhibition of PKA by the cyclic AMP analogue Rp-cAMPS did not modulate the proliferation rate of these cells. Activation of PKC stimulated the expression of the immediate early gene c-fos but c-myc expression was not significantly enhanced. On the other hand, PKA activation increased both c-myc and c-fos expression in MCF-7 cells. These results suggest that PKA activation and c-myc expression are not obligatory for proliferation of MCF-7 cells.

Tumor-necrosis factor-alpha modulates mitogen-activated protein kinase activity of epidermal-growth-factor-stimulated MCF-7 breast cancer cells.

Tumor-necrosis factor(TNF)-alpha inhibited in a dose-dependent fashion the proliferation of epidermal-growth-factor(EGF)-stimulated MCF-7 breast cancer cells with an IC50 of 0.25 nM. A comparable TNF-alpha-mediated inhibition of p42/44 mitogen-activated protein (MAP) kinase activity was observed in 10 nM EGF-stimulated cells. The MAP kinase activity dropped 50% within 3 min of TNF-alpha (1 nM) addition to EGF-stimulated MCF-7 cells. EGF and TNF-alpha, when added independently, led to a transient stimulation of MAP kinase activity with maximal activations within 6-8 min and 1-2 min, respectively. These observations suggest that MAP kinase activity in EGF-stimulated MCF-7 cells is modulated by the growth-inhibitory receptor pathways of TNF-alpha. Phosphorylation measurements on western blots determined the involvement of several individual MAP kinases, namely p42/44 MAP kinases, p38 MAP kinase and c-Jun N2-terminal kinase 1 (JNK1), in EGF and TNF-alpha-induced signalling. Phosphorylation of p42 and p38 MAP kinases only was observed after treatment with either TNF-alpha or EGF. A combination of both ligands inhibited p42 and p38 MAP kinase phosphorylation in MCF-7 cells. In contrast, no JNK1 phosphorylation was detected in these cells. Simultaneous addition of okadaic acid, a potent inhibitor of phosphatases 1 and 2A, blocked the decay of EGF-stimulated MAP kinase activity over 40 min. TNF-alpha added to EGF-stimulated and okadaic-acid-treated cells increased the MAP kinase activity twofold within 1 min. Similarly, okadaic acid treatment partly reverted the TNF-alpha-inhibited growth of MCF-7 cells. These experiments suggest that phosphatases are involved in the rapid shut-down by TNF-alpha of p42 MAP kinase activity.

[Molecular factors determine primary and secondary therapy of breast carcinoma]. / Molekulare Faktoren bestimmen die primäre und sekundäre Therapie des Mammakarzinoms.

The clinical oncology realizes that the classical approach with systemic adjuvant chemo- and hormonal therapies is not sufficient and will be challenged by cellular and molecular structures which reflect the targets for new therapeutic approaches. These targets are key proteins involved in the signal transduction cascade. In human tumors these proteins have either lost their biological functionality by oncogenic mutations or are constitutively activated. The molecular classification of primary breast cancer was performed by assessing the following factors: estrogen- and progesterone receptors, ERbB-2 mutated p53, uPA, PAI-I, VEGF, DNA-Index and S-Phase. These factors are of prognostic and predictive value.

BRCA1 mutations found in archived early onset breast tumours.

Inherited mutations in the BRCA1 gene are thought to account for approximately 5% of breast cancers in women under the age of 45 years. In order to determine whether mutations could be found at the expected frequency, 60% of the protein coding region of BRCA1 was screened in 75 archived early-onset breast tumours, taken from women under 45 years of age. Two of the 75 tumours (2.7%) had detectable mutations, in close agreement to that predicted. Since BRCA1 mutations found in breast tumours are invariably germline, two immediate consequences are apparent. Firstly, family members of affected patients are likely to carry mutations as well, and should be considered for BRCA1 screening; and secondly, persons harbouring a germline BRCA1 mutation should be examined frequently and indefinitely for new primary tumours in remaining breast tissue.

Tumour necrosis factor and interferon are selectively cytostatic in vitro for hormone-dependent and hormone-independent human breast cancer cells.

Since experimental studies have shown that tumour necrosis factor-alpha (TNF-alpha) has potent anti-tumour activity that can be potentiated with cytokines, we tested the efficacy of TNF-alpha with interferon-gamma (IFN-gamma) on different human breast cancer cell lines, particularly comparing hormone-dependent and -independent phenotypes. TNF-alpha inhibited the growth of hormone-dependent human MCF-7, ZR-75-1 and T47-D breast cancer cells with a half maximal concentration of 0.25 nM. In contrast, the growth of hormone-independent cells MDA-MB-231 and HS578T was not affected by TNF-alpha alone, but a synergistic inhibition was observed when using IFN-gamma and TNF-alpha together. The mRNA for the proto-oncogene C-MYC, as an intracellular indicator of cell activation, was significantly increased in MCF-7 cells in the presence of TNF-alpha. In MDA-MB-231 cells this mRNA was increased only in the presence of both TNF-alpha and IFN-gamma, without a change in the number of surface TNF receptors. These findings indicate that TNF-alpha treatment in combination with IFN-gamma may provide a successful approach to overcome the cellular heterogeneity of advanced breast tumours.

Chemiluminescence immunoassay for vascular endothelial growth factor (vascular permeability factor) in tumor-tissue homogenates.

We developed a two-site chemiluminescence immunoassay for human vascular endothelial growth factor (VEGF). The assay recognized both VEGF121 and VEGF165 isoforms, but had no detectable cross-reactivity with platelet-derived growth factor or placenta growth factor. The range of detection was between 30 ng/L and 30 micrograms/L VEGF. Inter- and intraassay variations were 8.2-8.3% and 7.2-7.6%, respectively. VEGF concentrations were measured in the cytosolic extracts of 45 ovarian and 142 primary breast tumors. The amount of VEGF in the ovarian tumors (median = 0.46 ng/mg total protein, range 0-15.8 ng/mg) was significantly (P = 0.03) higher compared with the breast tumors (median = 0.24 ng/mg total protein, range 0-12.3 ng/mg). In 32 and 7 extracts of normal breast tissues adjacent and distant to the tumors, respectively, VEGF concentrations were significantly much lower (P < 0.0001). The detection of substantial amounts of VEGF in two invasive tumors (compared with normal tissues) suggests that the assay should be a useful tool for investigating the prognostic value of VEGF in breast and ovarian carcinomas and for selecting patients for future anti-VEGF therapy.

The dual role of mutant p53 protein in chemosensitivity of human cancers.

Mutational loss of p53 tumor suppressor functions has been observed in a wide range of neoplasms and was associated with either enhanced or decreased chemosensitivity of affected tumors. The dual role of wild-type p53 as a DNA repair initiator and a trigger for apoptosis raises the possibility that appropriately designed chemotherapy could be selectively applied against p53-defective tumor cells. The cytotoxic effects of DNA-crosslinking chemotherapeutica such as cisplatin could be enhanced by mutated p53 which is no longer able to repair drug-induced DNA damage. In contrast, DNA synthesis blockers such as fluorouracil can induce apoptosis through p53-dependent mechanisms. Thus, loss of p53 functions results in decreased sensitivity to this type of drugs. Clinical studies will reveal the role of abberant p53 in the efficacy of chemotherapy for individual patients.