22170 Background: Preliminary reports indicate that dutasteride can suppress progesterone transformation into 5-alpha pregnane by SRD5A1 in breast cancer, resulting in suppression of cell proliferation and detachment. The aim of this study was to investigate SRD5A1 expression according to the breast cancer subtypes and patient characteristics. Methods: SRD5A1 mRNA expression levels were investigated in three breast cancer data sets consisting of a 64 genes Qrt-PCR data set (STB, 168 patients) and two micro-arrays data sets (295 and 286 patients). Results: SRD5A1 mRNA expression was found to be significantly higher in estrogen receptor (ER) negative tumors including the Basal like and HER2-positive subtypes. Moreover, elevated SRD5A1 were found to be associated with pre-menopausal status of the patients, high tumor grade and poor clinical outcome. Conclusions: SRD5A1 mRNA expression was found to be associated with ER-negative breast cancer phenotypes mainly found in tumors obtained from pre-menopausal women. Possible effects of dutasteride as well as a better characterization of 5-alpha pregnane receptors should be further investigated in these subtypes of breast cancer. No significant financial relationships to disclose.

The assessment of ERα, 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 ERα, 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 ERα, 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.

A multiplexed fluorescence immunoassay using a novel planar waveguide technology-based microarray system, ZeptoMARK (Zeptosens), was developed to detect simultaneously urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and vascular endothelial growth factor (VEGF) in extracts of breast cancer tissues. The three analytes assay was cross-validated with single-analyte ELISA/chemiluminescence immunosorbent assay tests, revealing good correlations and enhanced assay sensitivities (LODs) of 1 pg/mL for uPA, 33 pg/mL for PAI-1, and 1 pg/mL for VEGF. Values were well within the 80-120% limits for assay recovery and within the +/-20% limits for assay precision. The uPA, PAI-1, and VEGF results obtained from 50 breast cancer cytosols using the protein array system demonstrated that the microarray-based multiplexed assay is a sensitive and robust tool to be used for the simultaneous quantification of cancer markers in small breast cancer tissue samples (core biopsies). The miniaturized, multiplexed assay format has a potential to be used for the quantitative analysis of a larger set of validated markers with significance in disease management.

ERBB2 (HER2/Neu) gene amplification and overexpression is associated with increased risk of metastases and shorter survival in breast cancer. Tyrosine 1248 is a major phosphorylation site of ERBB2 and reflects the activation status of the receptor. The aim of this study was to investigate the relationships between quantitative levels of pY1248-ERBB2 (p-ERBB2) and the expression of epidermal growth factor receptor (EGFR)-family members, and whether p-ERBB2 could provide additional prognostic value compared with established prognostic markers. For this purpose we developed a highly sensitive chemiluminescence-linked immunoassay (CLISA) and detected p-ERBB2 levels in 70 primary breast cancer biopsies. Phosphorylated ERBB2 correlated with EGFR and ERBB2, and inversely with oestrogen receptor (ER), progesterone receptor (PgR) and ERBB4 expression levels. Additionally, p-ERBB2 was associated with poor clinical outcome in univariate and multivariate Cox regression analysis. Further studies are needed to evaluate the predictive value of p-ERBB2.

20120 Background: Molecular profiling recently defined biological characteristics of several long-recognized breast cancer subtypes including ER-positive (luminal subtype), ER-negative/ERBB2-positive (ERBB2 subtype) and ER-negative/ErBB2-negative (basal-like subtype). Each of these particular subtypes has different impact on patient outcome and should be therefore taken in consideration for individual scoring calculations. Methods: The quantitative RNA expression levels of 70 relevant genes were simultaneously determined in fresh frozen samples of 317 primary breast cancer (BC) patients comprehending ER-positive (70%), ER-negative/ERBB2-positive (15%) and ERBB2-negative/ER-negative (15%) and with known follow-up data. Five years distant recurrence scoring systems were calculated by means of Cox-hazard regression models. Results: Two main prognostic scoring systems were developed: one based on genes relative to proliferation representing tumor growth and its velocity, the other based on proteases. A low proliferation score identified 30% of patients at very good prognosis (probability of distant recurrence 12%, CI: 1.5–22%) all belonging to the ER-positive subcategory as compared to cases with higher proliferation (probability of distant recurrence 31%, 32–38%). The probability to develop distant recurrence within 5 years for 30% of ERBB2-positive patients was of only 12% (CI 0–25%) when accompanied by low levels of proteases as compared to the remaining ERBB2-positive patients with a probability of recurrence of 40% (CI 22–54%). Conclusions: ER, ERBB2 and the expression levels of the few identified genes involved in tumor proliferation and invasion can be easily and precisely detected by means of QRT-PCR. This robust method allows fine tuned prognosis and gives predictive information for the treatment of individual breast cancer. [Table: see text]

14659 Background: Tumor size, nodal status, the Gleason grading system and serum PSA values are today’s available prognostic tools of localized prostate cancer and the only help for adjuvant therapy. Based on the results of a feasibility study we continued the evaluation of the recently developed prognostic molecular signature. Methods: Of 60 CaP patients, who underwent primary prostatectomy in 2003 to 2005, fresh frozen samples of the tumor were asserved. The RNA extracted from cryocuts was tested. The quantitative RNA expression levels of 90 relevant genes involved in the different tumor hallmarks were simultaneously assessed. Results: Unsupervised agglomerative clustering of the obtained molecular profiles revealed different signatures. Correlations between these groups and the known TNM staging as well as Gleason scores were strongly present. Of interest was that all recurrences observed within this short period of time were found in a single cluster expressing higher levels of proliferation markers. Conclusions: The molecular profile of primary prostate cancer by quantitative RT-PCR is a powerful tool describing the biology of an individual tumor. Gene expression profiling can be precisely quantified and seems to be better reproducible than pathological judgments of the Gleason scores. Moreover, the gene panel is partially based on drug target genes and therefore of predictive value. Finally the method could be applied also in core biopsies. [Table: see text]

Activation of nuclear factor-κB (NFκB) has been linked to the development of hormone-independent, estrogen receptor (ER)-negative human breast cancers. To explore the possibility that activated NFκB marks a subset of clinically more aggressive ER-positive breast cancers, NFκB DNA-binding was measured in ER-positive breast cancer cell lines and primary breast cancer extracts by electrophoretic mobility shift assay and ELISA-based quantification of specific NFκB p50 and p65 DNA-binding subunits. Oxidant (menadione 100μM×30min) activation of NFκB was prevented by pretreatment with various NFκB inhibitors, including the specific IκB kinase (IKK) inhibitor, parthenolide (PA), which was found to sensitize MCF-7/HER2 and BT474 but not MCF-7 cells to the antiestrogen tamoxifen. Early stage primary breast cancers selected a priori for lower ER content (21–87fmol/mg; n=59) and known clinical outcome showed two- to four-fold increased p50 and p65 NFκB DNA-binding over a second set of primary breast cancers with higher ER content (>100fmol/mg; n=22). Breast cancers destined to relapse (13/59) showed significantly higher NFκB p50 (but not p65) DNA-binding over those not destined to relapse (46/59; p=0.04). NFκB p50 DNA-binding correlated positively with several prognostic biomarkers; however, only NFκB p50 DNA-binding (p=0.04), Activator Protein-1 DNA-binding (AP-1; p≤0.01) and urokinase-type plasminogen activator expression (uPA; p=0.0014) showed significant associations with metastatic relapse and disease-free patient survival. These clinical findings indicate that high-risk ER-positive breast cancers may be prognostically identified by increased NFκB p50 DNA-binding, and support preclinical models suggesting that therapeutic inhibition of NFκB activation may improve the endocrine responsiveness of high-risk ER-positive breast cancers.

9570 Background: In spite of complete resection of localized prostate cancer (CaP), some will recur and metastasize. New prognostic and predictive tools are needed to improve clinical management of adjuvant therapy. For this purpose we investigated 90 relevant genes in tumor and non-malignant adjacent tissue (NAT). Methods: Of 32 CaP patients, who underwent primary prostatectomy in 2003, fresh frozen samples of the tumor and respective NAT were asserved. The RNA extracted from cryocuts was tested on the Bioanalyzer 2100. The quantitative RNA expression levels were simultaneously determined on ABI 7000. Results: Unsupervised agglomerative clustering of the obtained molecular profiles clearly revealed different signatures in tumor versus NAT. Genes involved in proliferation as well as tumor markers such as PSA and prostate stem cell antigen were found at elevated levels in tumors, whereas high expression levels of other genes (e.g. cytokeratins, Glutathione S-transferase) were found in NAT. Interestingly, more expressed in NAT were certain markers of angiogenesis and cell matrix-remodeling. Conclusions: The molecular profile by quantitative RT-PCR is a powerful tool describing the biology of an individual tumor and its surrounding tissue, which in correlation with the Gleason score, pTNM staging and circulating PSA will help to predict clinical outcome and to identify at time patients who should receive hormone-deprivation. No significant financial relationships to disclose.

The aim of this study was to compare cytosol estrogen (ER) and progesterone (PgR) receptor levels measured in normal and pathological tissue specimens (n = 395) of endometrium (ED), endocervical mucosa (EM) and cervical vaginal portion (VP) from 108 patients (age 27–79). The highest ER levels were found in ED which decreased to 25% in VP. The PgR decline was more pronounced in VP since the majority of samples were PgR-negative. In addition pathological tissue from ED exhibited marked differences in steroid receptor levels: The highest ER and PgR concentrations were detected in the proliferative phase, whereas the secretory phase exhibited significantly reduced ER and PgR levels. The lowest ER and PgR amounts were measured in atrophic and quiescent tissues; in proliferative tissue ER and PgR levels were comparable to hyperplastic tissue. Cervical ER and PgR concentrations were significantly lower as compared with ED (25–30%), and no cyclic variations were detectable. Also ER and PgR levels were different between EM and VP. 96% of ER were positive in the cervix, whereas 73% were PgR-negative in VP. Furthermore an inverse correlation between ER and progesterone was found in ED and a positive correlation between estradiol and PgR. These results suggest that the cytosol ER and PgR levels vary markedly in the uterine corpus and may explain in part the different response to endocrine therapy.

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.

This study demonstrates a requirement for NF-κB activation in cis-diamminedichloroplatinum (cisplatin)-induced apoptosis in human head and neck squamous cell carcinoma (HNSCC) cell lines. This conclusion was supported by the following observations: cisplatin induced IκBα degradation and NF-κB-dependent transcriptional activation prior to cell death; pyrrolidine dithiocarbamate (PDTC), a chemical inhibitor of NF-κB activation, prevented apoptosis; lactacystin, an inhibitor of IκBα degradation, also prevented apoptosis; and finally, the expression of a super-repressor mutant IκBα blocked apoptosis. The expression of tumor necrosis factor α (TNFα) was promoted by cisplatin treatment and was suppressed by PDTC treatment. In addition, a neutralizing antibody against TNFα protected cells from cisplatin-induced apoptosis. These findings suggest that NF-κB activation is required for cisplatin-induced apoptosis and TNFα may play an important role in NF-κB-mediated apoptosis in cisplatin-treated HNSCC cell lines.

Let D be a subset of the set P of prime numbers not containing any twin primes. Kemnitz and Kolberg raised the following question. For any given natural number n, are there only finitely many such minimal sets D, of the size n, such that the induced prime distance graph has chromatic number 4? In this paper, a conditional answer to this question based on a well-known conjecture from the prime number theory is given.

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.

In this report we present an extension of the pooled analysis of the prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-I in breast cancer patients. We analyzed a different endpoint, metastasis-free survival (MFS). We checked the consistency of the estimates for uPA and PAI-1 for relapse-free survival (RFS) and MFS exploring possible sources of heterogeneity. Nodal status, the most important prognostic factor for breast cancer, introduced heterogeneity in the uPA/PAI-1 survival analyses, reflecting the interaction between nodal status and uPA/PAI-1. The estimates for uPA and PAI-1 were found to be consistent, even when a different transformation of their values was used. The heterogeneity of the separate data sets decreased if the levels of uPA and PAI-1 were ranked, data sets were pooled, and the analyses corrected for the base model that included all traditional prognostic factors, and stratified by data set. We conclude that uPA and PAI-1 are ready to be used in the clinic to help classify breast cancer patients into high and low risk groups.