After revision of ER status as assessed sellckchem with immunohistochemistry, a total of 563 ER positive tumors were used for subse quent analysis. We used a cutoff of 10% of positive tumor cells for ER positivity, because this is a common practice in The Netherlands and, in addition, this would avoid the potential inclusion of basal like tumors in our analysis. The original trial was approved by the central ethics committee of the Netherlands Cancer Institute, and informed consent was obtained from all study partici pants. For this retrospective translational study, no additional consent was required, according to Dutch legislation, because the use of archival pathology leftover 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 Inhibitors,Modulators,Libraries constructed by using formalin fixed paraffin embedded tumor blocks. A total of three cores per tumor were embed ded in the TMAs that were stained for ER, progester one receptor, and HER2. ER and PgR were considered positive when 10% of invasive cells showed nuclear reactivity. HER2 was considered positive Inhibitors,Modulators,Libraries when membranous staining was DAKO score 3. In case of a DAKO score 2, chromogenic in situ hybridization was performed. For tumors without sufficient cores in the TMA, whole slides were cut and assessed for ER, PgR, and HER2. Tumor grade was scored on a hematoxylin eosin stained slide Inhibitors,Modulators,Libraries by using the modified Bloom Richardson score. Antibodies used for immunohistochemistry are shown in Additional file 1 Table S2.

Immunohistochemistry for IGF 1R and PTEN was performed by using the Ventana Benchmark Ultra system. For IGF 1R, membranous intensity was scored from 0 to 3. For PTEN protein expression, cytoplasmic intensity was scored from 0 to 3. The specificity of both antibodies was tested on a previously described series of Inhibitors,Modulators,Libraries metastatic breast cancer patients for which we had FFPE material embedded in a TMA, as well as Agilent 44 K mRNA expression data. Results are depicted in Additional file 1 Figures S1 to S2. Immunohistochemis try for downstream phosphorylated proteins in the PI3K AKT mTOR pathway, like p AKT, p extracellular signal regulated kinase 1 2, p mTOR, and p p70S6K was performed as previously described.

The interobserver variability analyzed by using the Cohen kappa coefficient is depicted in Additional file 1 Table S3. For further analyses, we used the scores produced by the first observer. DNA isolation and PIK3CA mutation analysis Inhibitors,Modulators,Libraries DNA was isolated by using a standard DNA isolation protocol, as described in Appendix 1. PIK3CA mutation status was assessed by using Seque nom mass spectrometry based genotyping technology for PIK3CA hotspot mutations in exon 9 and exon 20. PCR primers and extension primers for the various mutations are listed in Additional file selleck Z-VAD-FMK 1 Table S4.