593 Background: Recently, we have shown that the Siemens Prognostic Score (SPS) based on mRNA expression of nine informative genes predicted outcome in node-positive (N+) patients with breast cancer (SABCS 2008, abstract 6044). The aim of this retrospective biomarker study was to examine the utility of the SPS in combination with TP53 expression to predict benefit from adjuvant taxane therapy. Methods: The 211 N+ patients included in this study were treated in the context of a randomized two-arm phase III study (E-T-CMF vs. E-CMF) investigating adjuvant dose-dense sequential chemotherapy with epirubicin (E) followed by CMF with or without paclitaxel (T). RNA was isolated from formalin-fixed, paraffin-embedded tissue samples, using a Siemens proprietary method, followed by kinetic one-step RT-PCR for assessment of mRNA expression of the nine SPS genes, TP53 and two normalization genes. The continuous SPS was calculated using a linear combination of expression values of the SPS genes. Patients were separated into a high- and low-risk group using a cutoff at the median of the SPS. Optimal cutoff for low or high TP53 expression was defined on the basis of a ROC curve in SPS high-risk patients. Distant metastasis-free survival (MFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared using the log-rank test. Results: For patients with high SPS or high TP53 expression, we observed a trend for a better MFS in the E-T-CMF arm (SPS: p = 0.18; HR = 0.66; TP53: p = 0.23; HR = 0.67; n = 211). Combining both parameters, patients with high SPS and high TP53 expression (n = 44) had a significantly better MFS following E-T-CMF compared to E-CMF (5-year MFS 80% vs. 40%, p = 0.003, HR = 0.21; OS: p = 0.09; HR = 0.34). On the other hand, patients with high SPS and low TP53 expression (n = 32) showed a trend for a worse outcome with E-T-CMF (MFS: p = 0.09; HR = 3.54; OS: p = 0.35, HR = 1.90). Conclusions: Our prognostic algorithm combined with TP53 mRNA expression predicts the benefit from the addition of paclitaxel to E-CMF and might be used for identification of patients who should be considered for adjuvant taxane therapy. This hypothesis needs to be confirmed in an independent clinical study. [Table: see text]

Abstract Abstract #6044 Background: Recently, we identified and validated a prognostic multigene score, which predicted outcome in early node-negative breast cancer patients that did not receive systemic therapy. The aim of this study was to examine the performance of our prognostic multigene algorithm in node-positive patients treated with adjuvant chemotherapy. Patients and Methods: Patients were treated with adjuvant anthracycline-based chemotherapy in the context of a randomized Phase III study. This was a two-arm trial (E-CMF vs. E-T-CMF) investigating postoperative dose-dense sequential chemotherapy with epirubicin (E) followed by CMF with or without paclitaxel (T). RNA was isolated from 222 formalin-fixed, paraffin-embedded tumor tissue samples, using a Siemens proprietary automated method based on silica-coated magnetic beads, followed by kinetic one-step RT-PCR for mRNA expression analysis of 9 informative genes and 2 normalization genes. For each patient a risk score was calculated using a linear combination of expression values. Patients were separated into high, intermediate and low risk by applying two thresholds of the score. As used in node-negative patients, distant metastasis-free survival (MFS) and overall survival (OS) were estimated by the Kaplan-Meier method and compared using the log-rank test. Cox analysis for MFS and OS was also performed. Results: The prognostic score could be calculated for 213 patients (102 E-T-CMF; 111 E-CMF). Looking at all patients independently of type of chemotherapy, the score could classify the patients into three distinct risk groups, which were significantly different in terms of MFS (log-rank test, p=0.004) and OS (p=0.01). The separation of the risk groups was even better when focusing on patients with more than three involved lymph nodes (MFS: p=0.0001; OS: p=0.0015; n=166). The respective analysis of the two treatment arms showed that the separation of the risk groups was only significant in E-CMF-treated patients (E-CMF: p=0.002; E-T-CMF: p=0.37). Interestingly, the subgroup of patients with more than three involved lymph nodes and classified as intermediate or high risk (n=77) had a nearly significant better MFS when treated with E-T-CMF in comparison with E-CMF (p=0.07; HR: 0.53; 95% CI: 0.2685 to 1.060). Conclusions: Our prognostic algorithm, identified and validated in node-negative patients that had not been systemically treated, predicts outcome also in node-positive chemotherapy-treated patients. Our findings suggest that the prognostic score may also be predictive of benefit from the addition of taxanes to adjuvant chemotherapy. This hypothesis needs to be confirmed in a larger cohort of samples from an independent clinical study. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6044.

Abstract Abstract #6066 Background: Herstatin is a naturally occurring product of the HER2 gene generated by alternative mRNA splicing. It functions as a secreted inhibitor that binds to the extracellular domains of EGFR and HER2, disrupting multiple receptor combinations in response to a variety of ligands. This mode of action supports the potential utility of herstatin in the development of novel therapeutics against breast cancer. The aim of the present study was to evaluate the association of herstatin with HER family expression at the m-RNA level, as well as with other clinicopathological characteristics of patients with high-risk operable breast cancer enrolled in a randomized phase III trial. The effect of herstatin on disease-free survival (DFS) and overall survival (OS) was also investigated. Patients and Methods: 595 high-risk breast cancer patients were treated with anthracycline-based chemotherapy in the adjuvant setting (HE10/97 trial). This was a two-arm trial (E-CMF vs. E-T-CMF) investigating postoperative dose-dense sequential chemotherapy with epirubicin (E) followed by CMF with or without paclitaxel (T). RNA was extracted from 279 formalin-fixed paraffin-embedded tumor tissue samples followed by kinetic one-step RT-PCR for assessment of mRNA expression of herstatin, EGFR, HER2, HER3, and HER4. Values of RNA above the median were considered as positive expression, except for EGFR, where the 75th percentile was used as a cut-off. DFS and OS were estimated by the Kaplan-Meier method and compared using the log-rank test. Cox analysis for DFS and OS was also performed. Results: Positive herstatin expression was found to be significantly associated with ductal histology (χ2 test, p=0.004), and marginally associated with infiltrated lymph nodes (p=0.062). Furthermore, herstatin was found to be strongly correlated with HER2 expression (r=0.783, p<0.001) and weakly correlated with HER3 expression (r=0.144, p=0.02). Positive herstatin expression was associated with poor DFS and OS (log-rank, p=0.004 and 0.008, respectively). This remained unchanged when adjusted for treatment group, whereas no interaction between herstatin and treatment was identified. Interaction of HER2 overexpression (>75th percentile) with herstatin was found to be significant for OS (Wald, p=0.03), thus implying a possible prophylactic effect of herstatin in the subgroup of patients with HER2 overexpression. Conclusions: Herstatin was shown to be associated with a poor prognosis. Given the high correlation between herstatin and HER2, this finding may reflect the effect of HER2 on patient's prognosis. Our results suggest that the effect of herstatin may be altered in case of HER2 overexpression. This hypothesis should be investigated in future studies, focusing in patients with HER2 overexpression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6066.

Abstract Abstract #1071 Background: A number of molecular signatures have been published to aid breast cancer prognosis and therapy response prediction. However, most signatures rely on measurements of dozens of genes on an array platform. Here we report the identification and validation of a prognostic algorithm based on a small number of expressed genes reflecting dominant prognostic motifs like steroid hormone receptor expression, proliferation, matrix degradation and B-cell infiltration. The test is designed for mRNA extracted from formalin fixed paraffin embedded tumour tissue (FFPE). Materials and Methods: 200 fresh frozen tumours from node negative patients, who did not receive systemic therapy after surgery, were profiled on HG-U133a arrays. Primer and probes were designed for genes identified as prognostic relevant and used to profile the corresponding paired FFPE tumour tissue of our identification cohort by kinetic PCR. A genomic algorithm was constructed and tested in FFPE tumor tissue of an independent validation cohort of 240 node negative patients that remained untreated in the adjuvant setting. Results: Based on the Prognostic-score of our algorithm we classified the patients of the validation cohort into three classes: low risk (25% of patients), intermediate risk (25%) and high risk (50%). The probability to die after having a recurrence within 10 years was 4.7% for the low risk group, 11.9% for the intermediate risk group and 33.1% for the high risk group. Kaplan Meier analysis revealed that the trend for survival (Logrank test for trend P = 0.0002) was highly significant. Adjuvant! Online calculated probabilities to die of cancer were also used to classify patients into three risk groups of comparable size (25/25/50 percent of patients). The observed risk was 9.1% in the low risk, 26.7% in the intermediate and 23.6% in the high risk group. The area under the ROC curve for Adjuvant! Online was 0.619 (95% CI, 0.52 to 0.718; P = 0.031) while for our algorithm it was 0.755 (95% CI, 0.6628 to 0.8473; P < 0.0001). Discussion: We identified a prognostic algorithm for node negative breast cancer based on kinetic PCR analysis of FFPE tumor tissue. Validation in an independent cohort showed the ability of the algorithm to classify patients into clinically relevant risk classes. Preliminary analysis shows a better prognostic performance than current clinical guidelines. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1071.

In den zurückliegenden Jahren sind zahlreiche prognostische und prädiktive Multiparameteralgorithmen, basierend auf der Analyse von messenger-Ribonukleinsäure (mRNA), veröffentlich worden. Viele dieser Algorithmen erfordern RNA, die aus Frischgewebe bzw. frisch eingefrorenem Gewebe isoliert werden muss. Für die klinische Routine ist formalinfixiertes Gewebe jedoch ein weitaus geeigneteres Ausgangsmaterial. Siemens Healthcare Diagnostics entwickelt deswegen eine robotergestützte, vollautomatische Methode für die Extraktion von mRNA und Desoxyribonukleinsäure (DNA) aus formalinfixiertem sowie paraffineingebettetem Gewebe für das Pathologielabor. Die Methode soll erstmals als Teil eines Prognosetests für das Metastase- und Todesrisiko von Patientinnen mit nodal-negativem Brustkrebs Anwendung finden, der sich ebenfalls bei Siemens Healthcare Diagnostics in der Produktentwicklung befindet.

A multitude of prognostic and predictive multiparameter algorithms based on the analysis of mRNA have been published in recent years. Many of the algorithms require fresh or fresh frozen tissue as a source of the mRNA. However, practical considerations suggest formalin-fixed paraffin-embedded tissue (FFPE tissue) to be a more suitable starting material for routine diagnostic applications. Therefore, Siemens Healthcare Diagnostics is developing a fully automated method to extract mRNA and DNA from FFPE tissue for use in pathology laboratories. Initially, the method will be used as part of a prognosis assay to predict the likelihood of distant metastasis and death for node-negative breast cancer patients.

INTRODUCTION:Glycodelin and survivin are key polypeptide regulators of cellular proliferation, apoptosis and angiogenesis. In view of contradictory reports on their functional role in tumors, we studied their transcriptional levels in localized breast cancer.PATIENTS AND METHODS:Glycodelin and survivin messenger ribonucleic acid (mRNA) was isolated and amplified by quantitative reverse-trancription PCR from paraffin-embedded breast carcinomas of 275 women. A normalized score was calculated by the use of GAPDH, RPL37A reference genes and was correlated with clinicopathologic/molecular parameters and patient outcome.RESULTS:A total of 272 patients were eligible, most harbored stage III node-positive breast carcinomas larger than 2 cm. Glycodelin mRNA was expressed in 68 patients (25%), more frequently in premenopausal women (P = 0.01) and those with HER2 mRNA-positive tumors (P = 0.02). Survivin mRNA was present in 263 tumors (97%) and its levels correlated significantly with high nuclear grade, VEGF mRNA and p53 mRNA presence (P < 0.05). At a median follow-up of 64 months, neither glycodelin nor survivin mRNA expression demonstrated prognostic utility for overall or disease-free survival at univariate and multivariate analysis.CONCLUSIONS:Glycodelin and survivin transcriptional activity are associated with adverse clinicopathologic and molecular characteristics of node-positive primary breast cancer but do not predict patient outcome. Further study is needed for illumination of their functional roles in tumorigenesis.

9574 Objectives: In an effort to identify predictive markers that may have the potential to identify patients who are likely to respond to paclitaxel in a dose-dense sequential chemotherapy regimen we analyzed DNA alterations in fixed tumor tissues obtained from the adjuvant breast cancer trial HE 10/97. This is a two-arm trial (E-CMF vs. E-T-CMF) investigating postoperative dose-dense sequential chemotherapy with epirubicin (E) followed by CMF with or without paclitaxel (T). Disease -free survival (DFS) was the primary study endpoint. Methods: DNA from fixed tumor tissues was isolated employing an experimental method based on magnetic beads from Bayer HealthCare Diagnostics. The amplification of genes with putative relevance to breast cancer was verified by TaqMan™ PCR analyses. The amplification status of candidate genes was then used to identify differences in DFS in subgroups of patients. Results: By analyzing fixed tumor tissues from 270 patients, we found that amplification of genomic DNA frequently occurred as an overexpression of multiple (10–180) neighboring genes. We therefore named these genomic regions ARCHEONs (“Amplified Regions of Chromosomal Expression Observed in Neoplasia”). Notably, patients with ARCHEONs on chromosome 2q 33–34 or 4q34.1–3 that were treated with paclitaxel had a DFS rate of 84% at 48 months, which was significantly higher (p=0,006) than the 70% DFS rate observed in patients that had the ARCHEONs but were not treated with paclitaxel, or a rate of 68% seen in patients that were treated with paclitaxel but did not have the ARCHEONs. Conclusion: The described ARCHEONs appear therefore to influence the response of high-risk breast cancer patients to dose-dense sequential chemotherapy and to have predictive value in identifying patients that can benefit from paclitaxel. Identification of such ARCHEONs prior to the initiation of chemotherapy could help physicians determine whether to add paclitaxel to an adjuvant anthracyclin-based treatment. No significant financial relationships to disclose.

New PNA monomers containing aminoprolines or pyrrolidines as backbones have been synthesized. Oligomerisation was carried out on a solid support. Resistance to enzymatic degradation was tested.

Ehrlich ascites cells were cultured under different O2 partial pressures from less than 0.1 ppm to 2 x 10(5) ppm. During the artificial hypoxia and following reoxygenation the DNA synthesis rate was measured and the relative frequency of replicon initiations was examined by analyzing the length distributions of replicative daughter strand DNA. These studies were complemented by evaluation of growth and cycling of the cells and by biochemical analyses. It was demonstrated that the reversible shut-down of clusters of replication units already described before (Probst, H., Gekeler, V., and Helftenbein, E. Exp. Cell Res., 154: 327-341, 1984) occurred between 0.25 and about 2.5 microM dissolved O2. Above 2.5 microM, a transition range to aerobiosis extended to about 16 microM O2. Below 0.25 microM O2, the cells suffered damage impairing the reversibility of the shutdown. The observed changes of growth and cycling correlated well with the respective changes of replication. Analogous oxygenation dependent regulatory events in replication were also observed during growth of the cells as an in vivo ascites tumor. Obviously, the particular oxygenation conditions in the peritoneal cavity strongly influence tumor growth via the oxygen dependent regulation of replication.

Cultured Ehrlich ascites cells were subjected to hypoxic conditions for about 2 h, then reaerated or allowed to remain hypoxic. The newly formed DNA of hypoxic or reaerated cells was labeled with [3H]thymidine using different pulse and pulse/pulse-chase protocols. The chain length distribution of the labeled DNA molecules was analysed by sedimentation after lysing the cells on the top of alkaline sucrose gradients. The results indicated that the hypoxia effectively and reversibly suppressed the initiation of new replication units. Initiation, growth and integration of Okazaki pieces into active replicons was not noticeably affected. In marked contrast to aerobic cells, the use of hypoxic cells allows the separation of Okazaki pieces as a distinct class of pulse labeled short DNA chains. Short daughter DNA of very recently initiated replicons did not interfere at pulse times shorter than 4 min. For examination of the newly initiated replicons it seems favourable to trigger a burst of initiations by reaeration.