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Old 05-25-2014, 10:55 AM   #2
'lizbeth
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Re: Phosphorylated p-70S6K Predicts Tamoxifen Resistance in Postmenopausal Breast Can

Methods

Patients and Materials

We have recollected primary tumor tissue blocks from stage I to III postmenopausal breast cancer patients who were randomized (2:1) between 1 year of tamoxifen (30 mg/day) versus no adjuvant therapy (IKA trial, 1982 to 1994).[18,19] Study data were part of the Oxford meta-analysis.[20] After 1989, based on two interim analyses showing a significant improvement in recurrence-free survival in lymph node-positive patients, node-positive patients in this trial skipped the first randomization and all received 1 year of tamoxifen. After 1 year a second randomization 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 received adjuvant chemotherapy. The patient characteristics and clinical outcome of the original study group (1,662 patients) have been presented elsewhere.[19]
Sufficient tumor material was available for 739 patients, who did not differ in prognostic factors from the total group (Table S1 in Additional file 1). After revision of ERα status as assessed with immunohistochemistry (IHC), a total of 563 ERα-positive tumors were used for subsequent analysis. We used a cutoff value ≥10% of positive tumor cells for ERα positivity, since this is common practice in the Netherlands and also this would avoid the potential inclusion of basal-like tumors[21] 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 participants. For this retrospective translational study, no additional consent was required according to Dutch legislation[22] 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.[23]
Immunohistochemistry

Tissue microarrays (TMAs) were constructed using formalin-fixed paraffin-embedded tumor blocks. A total of three (0.6 mm) cores per tumor were embedded in the TMAs that were stained for ERα, progesterone receptor (PgR) and HER2. ERα and PgR were considered positive when ≥10% of invasive cells showed nuclear reactivity. HER2 was considered positive when membranous staining was DAKO score 3.[24] In the case of DAKO score 2, chromogenic in situ hybridization was performed. For tumors without sufficient cores in the TMA, whole slides were cut and assessed for ERα (n = 60), PgR (n = 55) and HER2 (n = 36). The tumor grade was scored on a hematoxylin and eosin-stained slide using the modified Bloom–Richardson score.[25]
Antibodies used for immunohistochemistry of downstream phosphorylated (p) proteins are shown in Table S2 in Additional file 1. For p-AKT(Ser473), antigen retrieval was performed using citrate buffer and slides were incubated overnight with antibody (dilution 1:50). All other phospho-protein stainings (p-AKT(Thr308), p-mTOR, p-ERK1/2 and p-p70S6K) were performed using a standardized protocol on the Ventana Benchmark® Ultra system (Ventana Medical Systems, Tucson, USA). To ensure phospho-specificity of the antibodies, for each antibody a test TMA containing positive cores was dephosphorylated by λ-phosphatase before staining, resulting in disappearance of the positive staining (Figure S1 in Additional file 1).
Cytoplasmic intensity (0 to 3) was assessed for p-AKT(Ser473), p-AKT(Thr308) 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 manner by a second observer to calculate inter-observer variability. For further analyses, we used the scores produced by the first observer (MO).
Since the stability of phospho-proteins is a matter of debate,[26] we tested whether the relative age of tumor samples (divided in quartiles) 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 the inclusion center was associated 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.[27] Since a secondary contralateral 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 occurrence. The association between expression of downstream activated proteins and known prognostic factors was tested using Fisher's exact test.
We hypothesized that high expression of downstream activated proteins is associated with tamoxifen resistance. Our primary analysis was therefore to test whether tamoxifen benefit was dependent on any of the downstream activated proteins in the PI3K and/or MAPK pathway. We analyzed these markers as a binary factor, using the median level as the cutoff value. Adjusted Cox proportional hazard regression analyses were performed including an interaction variable. Covariates included age (≥65 vs. <65), grade (grade 3 vs. grade 1 to 2), tumor size (T3 to T4 vs. T1 to T2), HER2 status (positive vs. negative), and PgR status (positive vs. negative). All survival analyses were stratified for nodal status. Owing to the multiple co-primary endpoints of this study, we apply a more conservative level of significance (α = 0.01) when assessing the interactions.
Further exploratory analyses examined tamoxifen benefit when the markers were implemented as continuous linear variables. For those continuous linear variables that showed an interaction with tamoxifen treatment, we explored which level of dichotomization best predicted tamoxifen benefit, by comparing Akaike's information criteria of the Cox proportional hazards models for all possible cutoff values. In addition, based on knowledge derived from preclinical studies,[1,3] 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 tamoxifen resistance. To assess the prognostic value of the phospho-proteins, we analyzed their putative prognostic potential in the subgroup of patients who were randomized 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 ERα-positive breast cancer patients would derive similar benefit from tamoxifen. Since the phospho-protein might be associated with tamoxifen resistance, simply correcting for the assumed tamoxifen 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 (REMARK) criteria[28] as outlined in Table S3 in Additional file 1.
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