BACKGROUND:Factors or signatures predicting response to chemotherapeutic agents are of great interest for breast cancer patient care. There is conflicting data regarding microtubule-associated protein tau as predictive marker of paclitaxel sensitivity. Paclitaxel plays an important role in the adjuvant and metastatic therapy of breast cancer. However, a substantial proportion of patients treated with paclitaxel do not derive benefit from this therapy. Therefore, evaluating potential predictive factors is increasingly important. The authors attempted to validate these findings in vitro utilizing the ATP tumorchemosensitivity assay (ATP-TCA).MATERIALS AND METHODS:The in vitro drug sensitivity to paclitaxel was evaluated in 48 fresh primary breast cancer specimens using the ATP-TCA. ATP-TCA results were analysed using the area under the curve (AUC) of growth inhibition. These results were correlated with the expression of tau mRNA measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Tau was also compared between patients with progesterone receptor (PgR) positive and negative and estrogen receptor (ER) positive and negative breast cancer, respectively.RESULTS:The correlation of tau with the AUC for paclitaxel was weak, Spearman Rho was -0.267 with a p-value of 0.064. As described before, multiple regression analysis confirmed T-stage (p = 0.01) and PR status (p = 0.01) as independent predictors of paclitaxel chemosensitivity. Using multiple regression analysis and defining tau mRNA expression as dependent variable estrogen receptor status as measured by immunohistochemistry was a highly significant predictor for tau mRNA expression (p < 0.001). Grade (p = 0.002) as well as PgR expression (p < 0.001) were also found to be predictors of tau mRNA expression. CON- CLUSIONS: In the present data set the authors were not able to show that MAP-tau mRNA could predict benefit from the addition of a taxane to adjuvant chemotherapy. They found that ER expression is associated with tau protein expression. Estrogen gene transcription is reported to carry weak predictive significance for endocrine sensitivity, therefore it might be worth pursuing whether, tau mRNA could possibly be a predictor for endocrine therapy response.

Zielsetzung: Wir verglichen die prognostische Bedeutung von CD8 und CD4 positiven tumorinfiltrierenden Lymphozyten sowie Immunglobulin Kappa C (IGKC) positiven tumorinfiltrierenden Plasmazellen bei unterschiedlichen molekularen Subtypen.

Hintergrund: Infiltration durch zytotoxische T Lymphozyten (CTL) ist ein häufig auftretendes Phänomen beim Mammakarzinom. Wir untersuchten die prognostische Bedeutung von CD8 positiven CTL bei nodal-negativen Mammakarzinomen

Biomarkers predictive of pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) of breast cancer are urgently needed. Using a training/validation approach for detection of predictive biomarkers in HER2-negative breast cancer, pre-therapeutic core biopsies from four independent cohorts were investigated: Gene array data were analysed in fresh frozen samples of two cohorts (n=86 and n=55). Quantitative reverse transcription polymerase chain reaction (qRT–PCR) was performed in formalin-fixed, paraffin-embedded (FFPE) samples from two neoadjuvant phase III trials (GeparTrio, n=212, and GeparQuattro, n=383). A strong predictive capacity of thymosin beta 15 (TMSB15A) gene expression was evident in both fresh frozen cohorts (P<0.0001; P<0.0042). In the GeparTrio FFPE training cohort, a significant linear correlation between TMSB15A expression and pCR was apparent in triple-negative breast cancer (TNBC) (n=61, P=0.040). A cutoff point was then defined that divided TNBC into a low and a high expression group (pCR rate 16.0% vs 47.2%). Both linear correlation of TMSB15A mRNA levels (P=0.017) and the pre-defined cutoff point were validated in 134 TNBC from GeparQuattro (pCR rate 36.8% vs 17.0%, P=0.020). No significant predictive capacity was observed in luminal carcinomas from GeparTrio and GeparQuattro. In TNBC, TMSB15A gene expression analysis might help to select patients with a high chance for pCR after NACT.

Proliferation hat eine große prognostische Bedeutung beim Mammakarzinom. Wir haben den prognostischen Einfluss der Proliferation für unterschiedliche molekulare Subtypen (luminal, basal-like, erbB2-like) in 766 nodal-negativen Patientinnen untersucht.

10566 Background: Proliferation has an important prognostic impact in breast cancer. We examined the importance of proliferation amongst different molecular subtypes (luminal, basal-like, erbB2-like) in 766 node-negative breast cancer patients without adjuvant systemic therapy. METHODS Microarray based gene-expression data of three independent and previously published cohorts of node-negative breast cancer patients (Mainz, Rotterdam, TRANSBIG) were analysed. After unsupervised hierarchical cluster analysis co-regulated genes related to proliferation were identified. Then a metagene as a surrogate for all genes within the proliferation cluster was calculated. The patients were assigned to different molecular subtypes according to the gene-expression of estrogen receptor (ESR1) and erbB2 (ERBB2). Metastasis-free survival (MFS) was compared with the Log-rank test as well as univariate and multivariate Cox regression which accounted for traditional prognostic factors like age, tumor size, estrogen receptor, histological grade and erbB2. RESULTS Patients with higher expression of the proliferation metagene showed worse MFS in the whole cohort of patients (HR 1.65, 95% CI 1.39-1.96, p<0.001). Using multivariate Cox regression, the proliferation metagene retained its independent prognostic significance (HR 1.58, 95% CI 1.16-2.27, p=0.004). Besides the proliferation metagene only tumor size (HR 1.79, 95% CI 1.18-2.72, p=0.007) showed an independent association with MFS. 521 patients (68%) belonged to the luminal subtype, 106 (14%) were erbB2-like, and 139 (18%) basal-like, respectively. Prognostic impact of IGKC was limited to the luminal subtype (HR 2.30, 95% CI 1.80-2.95, p<0.001). The proliferation metagene failed to show an association with survival both in erbB2-like (HR 0.95, 95% CI 0.50-1.78, p=0.868) as well as basal-like (HR 0.83, 95% CI 0.55-1.25, p=0.371) subtypes, respectively. CONCLUSIONS Proliferation plays a crucial and independent role in the whole cohort of node-negative patients. However, the prognostic significance of proliferation is confined to the luminal subtype and lost in basal-like and erbB2-like breast cancer, respectively.

Abstract Background: Infiltration of cytotoxic T lymphocytes (CTL) is a common feature in breast cancer. We examined the prognostic impact of CD8 positive CTL in formalin-fixed-paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) in node-negative breast cancer and validated our findings in previously published cohorts using RNA expression. Methods: CD8 was evaluated in FFPE tissue of 339 medically untreated node-negative breast cancer patients utilizing IHC. Results were validated utilizing microarray based gene-expression data of four cohorts of medically untreated node-negative breast cancer patients (n=824). Impact of CD8 on metastasis-free survival (MFS) was analyzed with univariate and multivariate Cox regression. Meta-analysis of previously published cohorts was performed using a random effects model. Prognostic significance was examined in the whole cohort and in different molecular subtypes (ER+/HER2−, ER−/HER2−, HER2). Correlation between RNA expression and IHC was analyzed according to Spearman. Results: Immunohistochemical detection of CD8+ CTL was associated with MFS in univariate (hazard ratio [HR] 0.76, 95% confidence interval [95% CI] 0.59−0.98, P=0.037) as well as in multivatiate analysis (HR 0.61, 95% CI 0.46−0.81, P=0.001). Protein levels of CD8 had a good correlation with RNA expression (r=0.488). Higher RNA expression of CD8 was related to better MFS in a meta-analysis of the whole cohort (HR 0.74, 95% CI 0.60−0.90, P=0.0023). However, prognostic significance was confined to ER+/HER2− (HR 0.71, 95% CI 0.51−0.99, P=0.0201) and HER2+ (HR 0.60, 95% CI 0.38−0.97, P=0.037) but not to ER−/HER2− molecular subtypes (HR 0.77, 95% CI 0.52−1.14, P=0.1965). Conclusion: CD8 positive CTL have independent prognostic significance in node-negative breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-13.

10616 Background: Immunological defense mechanisms play an important prognostic role in breast cancer. We examined the prognostic impact of immunoglobulin kappa C (IGKC) in 766 node-negative breast cancer patients without adjuvant systemic therapy according to molecular subtypes. Methods: IGKC (probe set ID 211645_x_at) was analysed utilizing microarray based gene-expression data of three independent and previously published cohorts of node-negative breast cancer patients (Mainz, Rotterdam, TRANSBIG). In addition to IGKC, we examined the prognostic impact of age, histological grade, tumor size, estrogen receptor (ER) and HER2. Furthermore, we investigated the prognostic significance of IGKC in luminal (ER+/HER2-), erbB2-like (HER2 +), and basal- like (ER-/HER2-) molecular subtypes, respectively. Metastasis-free survival (MFS) was analyzed with univariate and multivariate Cox regression. Results: Patients with higher expression of IGKC showed better MFS in the whole cohort of patients (HR 0.795, 95% CI 0.711-0.890, p < 0.001). Using multivariate Cox regression, IGKC retained its independent prognostic significance (HR 0.731, 95% CI 0.621-0.861, p < 0.001). Besides IGKC histological grade (HR 2.318, 95% CI 1.522-3.532, p < 0.001), tumor size (HR 1.536, 95% CI 1.131-2.085, p = 0.006), and HER2 (HR 1.829, 95% CI 1.079-3.101, p = 0.025) showed an independent association with MFS. 521 patients (68%) belonged to the luminal subtype, 106 (14%) were erbB2-like, and 139 (18%) basal-like, respectively. Prognostic impact of IGKC was conserved across luminal (HR 0.796, 95% CI 0.688-0.923, p = 0.002), erbB2-like (HR 0.542, 95% CI 0.387-0.757, p < 0.001), and basal-like (HR 0.760, 95% CI 0.600-0.963, p = 0.023) subtypes, respectively. In multivariate analysis IGKC had independent prognostic significance additionally to molecular subtypes (HR 0.746, 95% CI 0.664-0.837, p < 0.001). Conclusions: IGKC has independent prognostic impact in breast cancer. Its prognostic significance is both conserved across and independent from molecular breast cancer subtypes. Incorporating IGKC should lead to a more appropriate assessment of outcome in node-negative breast cancer. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Siemens Healthcare Diagnostics Siemens Healthcare Diagnostics

10514 Background: Novel biomarkers for prediction of a pathological complete response (pCR) in neoadjuvant chemotherapy for breast cancer are needed. Thymosin beta 15 is a small peptide with essential functions in the regulation of actin polymerization and immune response, as shown in various cancer cell lines. Methods: TMSB15A isoform mRNA expression in fresh-frozen preoperative biopsies of patients from the neoadjuvant GeparTrio trial (TAC; n = 55) as well as in cultured tumor cells was determined by Affymetrix array analysis. Kinetic RT-PCR was used to investigate mRNA expression in formalin-fixed, paraffin-embedded (FFPE) biopsies from the GeparTrio (n = 257) and the GeparQuattro trial (neoadjuvant EC-Doc or EC-DocX; n = 392). Results: TMSB15A mRNA expression was identified as a positive predictor for pCR in frozen tumor tissue from GeparTrio by a ROC analysis (p = 0.0042, auc = 0.824). In FFPE tissue from GeparTrio, TMSB15A mRNA expression again showed significant predictive impact: OR = 7.583, 95% CI = 3.798-15.139, p < 0.0001, pCR rate 41.3% vs. 8.5%. This finding was validated in the GeparQuattro cohort: OR = 3.829, 95% CI = 2.138-6.854, p < 0.0001, pCR rate 26.2% vs. 8.5%. In multivariate analysis, TMSB15A expression was predictive independently from cT, cN, histology, grading, hormone receptor expression, and age in both FFPE cohorts. A relationship between TMSB15A mRNA expression and response was further detected in cultured triple-negative Cal51 cells that revealed a strong down-regulation of TMSB15A after induction of secondary paclitaxel resistance (p = 0.004), an effect which was not seen in luminal MCF7 and HER2-positive HCC1954 cells, which primarily showed a low-level expression of TMSB15A. Conclusions: Using a training-test approach in two well-characterized cohorts of patients with operable breast cancer as well as cell culture, we identified TMSB15A mRNA expression as a promising candidate biomarker for response to neoadjuvant chemotherapy with anthracycline/taxane. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Siemens Healthcare Diagnostics AstraZeneca Siemens Healthcare Diagnostics AstraZeneca

Zielsetzung: Immunologische Abwehrmechanismen spielen eine wichtige Rolle beim Mammakarzinom. Wir haben den prognostischen Einfluss von Immunglobulin Kappa C (IGKC) bei nodal-negativen Mammakarzinomen, die keine medikamentöse adjuvante Therapie erhalten hatten, untersucht.

Authors' University Mainz, Ge Cologne, Environme TU Dortm Dortmund, West Germ Germany Note: Supp Research O

Abstract Background: Aurora kinase A (AURKA) is important for cell cycle progression. Inhibitors that target AURKA are currently in clinical development. We examined the prognostic impact of AURKA in three cohorts of node-negative breast cancer patients without adjuvant systemic therapy (n=766). Methods: AURKA (probe set ID 204092_s_at) was analysed utilizing microarray based gene-expression data of three independent and previously published cohorts of node-negative breast cancer patients (Mainz, Rotterdam, TRANSBIG). In addition to AURKA, we examined the prognostic impact of age, histological grade, tumor size, estrogen receptor (ER) and HER2. Metastasis-free survival (MFS) was analyzed with univariate and multivariate Cox regression. Results: AURKA displayed a strong positive correlation with grade (P<0.001), tumor size (P<0.001), HER2 (P<0.001), and a negative correlation with ER (P<0.001), respectively. Patients with higher expression of IGKC showed shorter MFS in the Mainz cohort (HR 1.629, 95% CI 1.243-2.134, P<0.001), Rotterdam cohort (HR 1.567, 95% CI 1.271-1.933, P<0.001), and TRANSBIG cohort (HR 1.331, 95% CI 1.081-1.640, p=0.007), respectively. Accordingly, AURKA was significant in univariate analysis in the whole cohort of patients (HR 1.523, 95% CI 1.343-1.726, P<0.001). Using multivariate Cox regression, only tumor size retained its independent prognostic significance (HR 1.818, 95% CI 1.196-2.765, p=0.005). AURKA (HR 1.221, 95% CI 0.987-1.511, p=0.065) and histological grade (HR 1.551, 95% CI 0.955-2.520, p=0.076) showed only borderline significance for MFS. Conclusion: AURKA has prognostic relevance in three independent cohorts of node-negative breast cancer patients making it a suitable target for therapy with Aurora kinase inhibitors. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-22.

There are two major questions regarding systemic therapy of breast cancer: Firstly, which patients should be treated, and secondly, how should these patients be treated? Prognostic factors aim to foresee the outcome of patients irrespective of treatment while predictive factors intend to assess the outcome of patients receiving a certain systemic therapy and thus are intimately associated with sensitivity or resistance to therapy. Ideally, a predictive factor is also a therapeutic target as it is the case with estrogen receptor (ER) or HER-2. In order to avoid over- as well as under-treatment, it is advisable to select the appropriate treatment strategy on the basis of a careful risk assessment for each individual patient. Additionally to time-honoured clinicopathological factors additional prognostic factors like urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitor 1 (PAI-1) or multiparameter gene-expression analyses have shown promising results especially in node-negative breast cancer. These multigene profiles offer new insights in breast cancer biology, like the important role of the tumor-associated immune system. ER, HER-2 and potentially newer prognostic factors like epithelial cell adhesion molecule (Ep-CAM) bridge the gap from prognosis to prediction and serve as therapeutic targets. This should allow us to quantify the risk of progression in each individual patient and tailor treatment accordingly, leading to a more personalized treatment recommendation.

Abstract Background: Molecular tests predicting outcome of breast cancer patients may be useful for treatment decisions in addition to standard clinicopathologic features. Methods: Using human genome HG-U133A array and qRT-PCR datasets, we developed and validated a gene-expression signature predicting the likelihood of distant recurrence in postmenopausal, early-stage breast cancer patients with estrogen receptor-positive, HER2-negative tumors treated with adjuvant endocrine therapy. RNA levels assessed by qRT-PCR in formalin-fixed paraffin-embedded tumor specimens were used to calculate a risk score (T5) and to determine a risk group (low or high) for each patient. The prospectively defined T5 risk score was then validated independently in patients from two large randomized phase III trials. Distant recurrence-free survival and overall survival were analyzed with Cox models adjusted for clinicopathological factors. The primary endpoint was time to distant recurrence. Results: In a training set of 964 tumors, we identified a gene-expression signature consisting of three proliferation-related genes (BIRC5, UBE2C, DHCR7), five estrogen-regulated genes (RBBP8, IL6ST, AZGP1, MGP, STC2), and three reference genes (CALM2, OAZ1, RPL37A). For the validation, RNA analysis was possible in 1702 of 1725 (99%) tumors of both validation sets. Women were classified as having low risk (n=832; 49%) or high risk (n=870; 51%) by the T5 risk score. The T5 risk score provided prognostic information independent from clinicopathologic risk as estimated by Adjuvant!Online or Ki67 labeling index. Patients with a higher T5 risk score had a significantly shorter time to distant recurrence (adjusted hazard ratio, 1.24; 95% confidence interval [CI], 1.15 to 1.33; P<0.001) and overall survival (adjusted hazard ratio, 1.13; 95% CI, 1.06 to 1.19; P<0.001) compared to patients with a lower T5 risk score. The addition of the risk characterized by the T5 risk score to the clinicopathological risk resulted in 10-year distant recurrence-free survival rates of 95% in combined low risk patients and 82% in combined high risk patients (P<0.001). Conclusions: Using formalin-fixed paraffin-embedded tumor specimens, the multigene T5 risk score provides prognostic information independent of Adjuvant!Online or Ki67 labeling index. By combining the T5 risk score with clinicopathological risk, we were able to accurately identify breast cancer patients with low risk or high risk for distant recurrence. Using this new easy-to-use multigene tool in clinical practice will assist in optimizing adjuvant therapy by reducing both undertreatment and overtreatment and thus improves outcome and quality of life of patients with early-stage breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-07.

AbstractPurpose: Members of the Bcl-2 family act as master regulators of mitochondrial homeostasis and apoptosis. We analyzed whether ERBB2 influences the prognosis of breast cancer by influencing the proapoptotic versus antiapoptotic balance of Bcl-2 family members.Experimental Design: ERBB2-regulated Bcl-2 family members were identified by inducible expression of ERBB2 in MCF-7 breast cancer cells and by correlation analysis with ERBB2 expression in breast carcinomas. The prognostic relevance of ERBB2-regulated and all additional Bcl-2 family members was determined in 782 patients with untreated node-negative breast cancer. The biological relevance of ERBB2-induced inhibition of apoptosis was validated in a murine tumor model allowing conditional ERBB2 expression.Results: ERBB2 caused an antiapoptotic phenotype by upregulation of MCL-1, TEGT, BAG1, BNIP1, and BECN1 as well as downregulation of BAX, BMF, BNIPL, CLU, and BCL2L13. Upregulation of the antiapoptotic MCL-1 [P = 0.001, hazard ratio (HR) 1.5] and BNIP3 (P = 0.024; HR, 1.4) was associated with worse prognosis considering metastasis-free interval, whereas clusterin (P = 0.008; HR, 0.88) and the proapoptotic BCL2L13 (P = 0.019; HR, 0.45) were associated with better prognosis. This indicates that ERBB2 alters the expression of Bcl-2 family members in a way that leads to adverse prognosis. Analysis of apoptosis and tumor remission in a murine tumor model confirmed that the prototypic Bcl-2 family member Bcl-xL could partially substitute for ERBB2 to antagonize tumor remission.Conclusions: Our results support the concept that ERBB2 influences the expression of Bcl-2 family members to induce an antiapoptotic phenotype. Antagonization of antiapoptotic Bcl-2 family members might improve breast cancer therapy, whereby MCL-1 and BNIP3 represent promising targets. Clin Cancer Res; 16(2); 451–60