PIK3CA mutations, phosphatase and tensin homolog, human epidermal growth factor receptor 2, and insulin-like growth factor 1 receptor and adjuvant tamoxifen resistance in postmenopausal breast cancer patients

Introduction Inhibitors of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway can overcome endocrine resistance in estrogen receptor (ER) α-positive breast cancer, but companion diagnostics indicating PI3K/AKT/mTOR activation and consequently endocrine resistance are lacking. PIK3CA mutations frequently occur in ERα-positive breast cancer and result in PI3K/AKT/mTOR activation in vitro. Nevertheless, the prognostic and treatment-predictive value of these mutations in ERα-positive breast cancer is contradictive. We tested the clinical validity of PIK3CA mutations and other canonic pathway drivers to predict intrinsic resistance to adjuvant tamoxifen. In addition, we tested the association between these drivers and downstream activated proteins. Methods Primary tumors from 563 ERα-positive postmenopausal patients, randomized between adjuvant tamoxifen (1 to 3 years) versus observation were recollected. PIK3CA hotspot mutations in exon 9 and exon 20 were assessed with Sequenom Mass Spectometry. Immunohistochemistry was performed for human epidermal growth factor receptor 2 (HER2), phosphatase and tensin homolog (PTEN), and insulin-like growth factor 1 receptor (IGF-1R). We tested the association between these molecular alterations and downstream activated proteins (like phospho-protein kinase B (p-AKT), phospho-mammalian target of rapamycin (p-mTOR), p-ERK1/2, and p-p70S6K). Recurrence-free interval improvement with tamoxifen versus control was assessed according to the presence or absence of canonic pathway drivers, by using Cox proportional hazard models, including a test for interaction. Results PIK3CA mutations (both exon 9 and exon 20) were associated with low tumor grade. An enrichment of PIK3CA exon 20 mutations was observed in progesterone receptor- positive tumors. PIK3CA exon 20 mutations were not associated with downstream-activated proteins. No significant interaction between PIK3CA mutations or any of the other canonic pathway drivers and tamoxifen-treatment benefit was found. Conclusion PIK3CA mutations do not have clinical validity to predict intrinsic resistance to adjuvant tamoxifen and may therefore be unsuitable as companion diagnostic for PI3K/AKT/mTOR inhibitors in ERα- positive, postmenopausal, early breast cancer patients.

To evaluate the predictive and prognostic capacity of the different molecular alterations in PI3K/AKT/mTOR pathway in postmenopausal breast cancer randomized between adjuvant tamoxifen versus control.

Hypothesis
The presence of these molecular alterations have been shown to result in in vitro activation of the PI3K/AKT/mTOR pathway, which results in endocrine resistance. We hypothesize that the presence of a molecular alteration in the PI3K/AKT/mTOR pathway is associated with clinical tamoxifen resistance.

Characteristics
From 1982 until 1994 a randomized clinical trial was conducted in the Netherlands, studying the benefit of adjuvant tamoxifen (IKA-trial) in postmenopausal breast cancer patients.

Inclusion criteria
In the original study, 1662 breast cancer patients were included who were were post-menopausal, less than 76 years of age and had a T 1-Exclusion criteria Mastitis or palpable supra-or infraclavicular lymph nodes Treatment Patients were randomized in a 2:1 distribution between 1 year tamoxifen (30 mg per day) versus no adjuvant therapy. After 1 year a second randomization was performed to receive another 2 years of tamoxifen or to stop further treatment. From 1989, based on two interim analyses showing a significant improvement in recurrence free-free survival in lymph node positive patients, these node positive patients were all allocated to the tamoxifen treatment arm (ie skipped the first randomization).

Methods (2) Specimen characteristics
Material used Formalin-fixed paraffin-embedded (FFPE) breast tumor tissue of the primary tumor. DNA was isolated from FFPE material.
Preservation/storage Formalin fixation and paraffin embedding. Storage at room temperature. Tumor DNA was stored at 4ºC.

Assay
Genotyping for PIK3CA exon 9 (E542K and E545K) and exon 20 mutations (H1047L and H1047R) was performed on genomic DNA using Sequenom mass spectrometry-based genotyping technology. HER2 status was assessed with a standard immunohistochemistry protocol and considered positive when membranous staining was DAKO score 3. In case of a DAKO score 2, Silver in situ Hybridization was performed using ultraView SISH Detection Kit (Ventana ®) according to the manufacturer's instructions and amplified cases were considered as HER2 positive. Immunohistochemistry for IGF-1R (Ventana anti-IGF-1R rabbit monoclonal antibody) and PTEN (Cell Signaling # 9559) was performed using the Ventana Benchmark ® Ultra system.
Protocol PCR primers and extension primers for the various PIK3CA mutations are listed in Table S8. Immunohistochemical stainings were performed using a standardized protocol on the Ventana Benchmark ® Ultra system. Staining protocols can be downloaded from our website: http://research.nki.nl/linnlab/index.htm

Control experiments
The specificity of the PTEN and IGF-1R antibodies was tested on a previously described series of metastatic breast cancer patients 21 for which we had FFPE material embedded in a TMA as well as Agilent 44K mRNA expression data. Results are depicted in Figure S1-2. For genotyping for PIK3CA mutation status control experiments were performed using PCR and capillary sequencing. For exon 9, a total of 349 samples could be compared between the two techniques (Sequenom mass spectrometry-based genotyping technology versus PCR and capillary sequencing) resulting in 91 % concordant results. In 8% of the cases a mutation was found with Sequenom mass spectrometry-based genotyping which was not detected with PCR and capillary sequencing, while in 1% of the cases a a mutation was found with PCR and capillary sequencing which was not detected with Sequenom mass spectrometry-based genotyping. For exon 20, a total of 408 samples could be compared between the two techniques (Sequenom mass spectrometry-based genotyping technology versus PCR and capillary sequencing) resulting in 96 % concordant results. In 3% of the cases a mutation was found with Sequenom mass spectrometry-based genotyping which was not detected with PCR and capillary sequencing, while in 1% of the cases a mutation was found with PCR and capillary sequencing which was not detected with Sequenom mass spectrometry-based genotyping.

Reproducibility
For each immunohistochemical staining, one of the TMAs was quantified independently in a blinded manner by a second observer to calculate inter-observer variability. The inter-observer variability analyzed using the (weighted) Cohen's kappa coefficient is depicted in Table S3 Quantification Quantification of immunohistochemical staining was performed as described in the method section for immunohistochemistry.

Blinding
Scoring of the immunohistochemical stainings was done without knowledge regarding both the recurrence-free-interval survival as well as the treatment arm at the time of scoring.

Case selection
A randomized controlled trial. The translational study presented her was performed retrospectively. The median duration of follow-up for patients without a recurrence event was 7.8 years. Patient records were re-evaluated for recurrence until 2000.

Clinical endpoints
The improvement of recurrence free interval (RFI) with tamoxifen versus nil was assessed according to the different levels of the tested drivers and downstream activated proteins as specified below. RFI included local, regional, distant recurrences and breast cancerspecific death, but not contra-lateral breast cancer, as the primary event.
Rational for sample size The sample size of the translational study is based on the amount of available tumor blocks containing invasive, ERα positive tumor cells, that could be recollected and a power calculation based on events in this group assuring that meaningful results could be deduced.

Methods (5) Statistical analysis
Statistical methods and variable selection procedure 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. A secondary contra-lateral breast tumor was not considered as an event and these patients were censored at the date of this occurrence. The association of PIK3CA mutations, PTEN, HER2 and IGF-1R protein expression with expression of downstream activated proteins in the PI3K/AKT/mTORpathway (like p-AKT, p-mTOR and p-p70S6K) was evaluated using linear by linear tests. Survival curves were constructed using the Kaplan-Meier method. All survival analyses were stratified for nodal status.All p-values are based on a two-sided test. All calculations were made with Statistical Package for the Social Sciences (SPSS) 15.0 Inc., IL, USA.
Missing data Cases with a missing value for one of the variables were excluded from the multivariate analysis, with the exception of missing HER2 and PgR data for which a separate level was created

Marker handling in analysis
In our primary analysis we tested the clinical validity of these molecular alterations as single markers, analyzed as binary factor. Covariate adjusted Cox proportional hazard regression analyses were performed including an interaction variable. The following molecular alterations were tested: PIK3CA mutation status (exon 9 mutant versus exon 9 wild type and exon 20 mutant versus exon 20 wild type imputed as separate factors in one model), HER2 (positive versus negative), PTEN (negative versus positive) and IGF-1R (score 3 versus score 0-2). In addition, we tested the interaction with tamoxifen for a composed variable defined as any of these molecular alterations present versus no molecular alteration. Covariates included age (≥ 65 versus < 65), grade (grade 3 versus grade 1-2), tumor size (T3-4 versus T1-T2), HER2 status (positive versus negative), and PgR status (positive versus negative). Due to multiple co-primary endpoints, the level of significance was set at 0.01. In addition we tested the prognostic effects of these molecular alterations in control patients using covariate adjusted Cox proportional hazard regression analyses.

Data
Flow of patients See Figure S3 for description of patients excluded for this translational study. See Table S1 for characteristics of total study patients versus the 739 patients with sufficient tumor material included in TMA .

Results (2) Analysis and presentation
Relation to standard prognostic variables See  Tables 2 and 3 and Supplementary Table S9. Estimated effects with CIs for marker and all other variables in the model.

Interpretation, limitations and implication
See discussion section 4 out of these had both exon 9 and exon 20 mutation Figure S1: Membranous IGF-1R protein expression according to immunohistochemical staining of TMA cores from primary breast cancers compared to mRNA levels that were available from hybridization to a 44K oligoarray (Agilent Technologies). In total 40 cases out of 69 patients were evaluable for IHC. In total 6 IGF-1R probes were available, showing all similar results. The figure shows the data for the first IGF-1R probe (A_23_P205986). Linear by linear test was performed using IGF-1R mRNA levels split by quartiles. Figure S2: Cytoplasmic PTEN protein expression according to immunohistochemical staining of TMA cores from primary breast cancers compared to mRNA levels that were available from hybridization to a 44K oligoarray (Agilent Technologies). In total 36 cases out of 69 patients were evaluable for IHC. In total 3 PTEN probes were available, showing all similar results. The figure shows the data for the first PTEN probe (A_23_P98085)