Parental cells did not have any detectable E542K mutant sequence. The mutation

After 1 year a sec ond randomization オーダー ARQ 197 was performed to receive another 2 years of tamoxifen or to stop further treatment. In total, 1,662 patients were included. None of these patients re ceived adjuvant chemotherapy. The patient characteris tics and clinical outcome of the original study group have been presented elsewhere. Sufficient tumor material was available for 739 pa tients, who did not differ in prognostic factors from the total group. After revision of ER status as assessed with immunohistochemistry, a total of 563 ER positive tumors were used for subsequent analysis. We used a cutoff value 10% of positive tumor cells for ER positivity, since this is com mon practice in the Netherlands and also this would avoid the potential inclusion of basal like tumors in our analysis.<br><br> The original trial was approved by the cen tral ethics committee of the Netherlands Cancer Insti tute and purchase AZD0530 informed consent was obtained from all study participants. For this retrospective translational study, no additional consent was required according to Dutch legislation since the use of archival pathology left over material does not interfere with patient care. Tumor tissue was handled according to the Dutch code of conduct for dealing responsibly with human tissue in the context of health research. Immunohistochemistry Tissue microarrays were constructed using formalin fixed paraffin embedded tumor blocks. A total of three cores per tumor were embedded in the TMAs that were stained for ER, progesterone re ceptor and HER2. ER and PgR were considered positive when 10% of invasive cells showed nuclear re activity.<br><br> HER2 was considered positive when membran ous staining was DAKO score 3. In the case of DAKO score 2, chromogenic in situ Alvocidib 臨床試験 hybridization was performed. For tumors without sufficient cores in the TMA, whole slides were cut and assessed for ER, PgR and HER2. The tumor grade was scored on a hematoxylin and eosin stained slide using the modified Bloom Richardson score. Antibodies used for immunohistochemistry of down stream phosphorylated proteins are shown in Table S2 in Additional file 1. For p AKT, antigen retrieval was performed using citrate buffer and slides were incu bated overnight with antibody. All other phospho protein stainings, p mTOR, p ERK1 2 and p p70S6K were performed using a stan dardized protocol on the Ventana Benchmark Ultra system.<br><br> To ensure phospho specificity of the antibodies, for each antibody a test TMA containing positive cores was dephosphorylated by phosphatase before staining, resulting in disappear ance of the positive staining. Cytoplasmic intensity was assessed for p AKT, p AKT and p p70S6K. The percentage of tumor cells with submembranous staining was scored for p mTOR, and the proportion of positive nuclei was scored for p ERK1 2. For each staining, one of the TMAs was quantified independently in a blinded man ner by a second observer to calculate inter observer variability. For further analyses, we used the scores pro duced by the first observer.