Antibodies from the following sources were used for analysis, pHER2 Y1248, Y877

Since the stability of phospho proteins is a matter of de bate, we tested whether the relative age of tumor sam ples was associated with quantitative phospho protein staining for each of these markers. In addition, to test for a possible effect of different fixation procedures, we tested whether buy ARQ 197 the inclusion center was as sociated with differences in quantitative phospho protein expression. Statistical analysis The recurrence free interval was defined as the time from the date of first randomization until the occurrence of a local, regional or distant recurrence or breast cancer specific death. Since a secondary contralat eral breast tumor cannot be inferred from the molecular make up of the primary tumor, while the other type of events can be inferred in relation to tamoxifen resistance of the primary tumor, this was not considered an event and these patients were censored at the date of this oc currence.

The association between expression of down stream activated proteins and known prognostic factors was tested using Fishers exact test. We hypothesized that high expression of downstream activated proteins is associated with tamoxifen resistance. Our primary analysis was therefore to test whether tam oxifen benefit was dependent on any of the downstream activated AZD0530 379231-04-6 proteins in the PI3K and or MAPK pathway. We analyzed these markers as a binary factor, using the me dian level as the cutoff value. Adjusted Cox proportional hazard regression analyses were performed including an interaction variable. Covariates included age, grade, tumor size, HER2 status, and PgR sta tus.

All survival analyses were strati fied for nodal status. Owing to the multiple co primary endpoints of this study, we apply a more conservative level of significance when assessing the interactions. Further exploratory analyses examined tamoxifen bene fit when the markers were implemented as continuous lin supplier Alvocidib ear variables. For those continuous linear variables that showed an interaction with tamoxifen treatment, we ex plored which level of dichotomization best predicted tam oxifen benefit, by comparing Akaikes information criteria of the Cox proportional hazards models for all possible cutoff values. In addition, based on knowledge derived from preclinical studies, we explored whether a composed variable of either high p ERK1 2 or high p mTOR indicating the activation of either the MAPK pathway or the PI3K pathway was associated with tam oxifen resistance.

To assess the prognostic value of the phospho proteins, we analyzed their putative prognostic potential in the subgroup of patients who were random ized to the control arm. The reason why we did not use all patients and corrected for tamoxifen treatment is that this correction would assume that all ER positive breast can cer patients would derive similar benefit from tamoxifen. Since the phospho protein might be associated with tam oxifen resistance, simply correcting for the assumed tam oxifen benefit without a correction for a potential interaction between treatment and phospho protein could bias the analysis for prognostic potential. Survival curves were constructed using the Kaplan Meier method. This study complied with reporting recommendations for tumor marker prognostic studies criteria as outlined in Table S3 in Additional file 1.