PIK3CA alterations and benefit with neratinib: analysis from the randomized, double-blind, placebo-controlled, phase III ExteNET trial

Background Neratinib is an irreversible pan-HER tyrosine kinase inhibitor that inhibits PI3K/Akt and MAPK signaling pathways after HER2 receptor activation. The ExteNET study showed that neratinib significantly improved 5-year invasive disease-free survival (iDFS) in women who completed trastuzumab-based adjuvant therapy for early breast cancer (EBC). We assessed the prognostic and predictive significance of PIK3CA alterations in patients in ExteNET. Methods Participants were women aged ≥ 18 years (≥ 20 years in Japan) with stage 1–3c (modified to stage 2–3c in February 2010) operable breast cancer, who had completed (neo)adjuvant chemotherapy plus trastuzumab ≤ 2 years before randomization, with no evidence of disease recurrence or metastatic disease at study entry. Patients were randomized to oral neratinib 240 mg/day or placebo for 1 year. Formalin-fixed, paraffin-embedded primary tumor specimens underwent polymerase chain reaction (PCR) PIK3CA testing for two hotspot mutations in exon 9, one hot-spot mutation in exon 20, and fluorescence in situ hybridization (FISH) analysis for PIK3CA amplification. The primary endpoint (iDFS) was tested with log-rank test and hazard ratios (HRs) estimated using Cox proportional-hazards models. Results Among the intent-to-treat population (n = 2840), tumor specimens were available for PCR testing (991 patients) and PIK3CA FISH (702 patients). Overall, 262 samples were PIK3CA altered: 201 were mutated (77%), 52 (20%) were amplified, and 9 (3%) were mutated and amplified. iDFS was non-significantly worse in placebo-treated patients with altered vs wild-type PIK3CA (HR 1.34; 95% CI 0.72–2.50; P = 0.357). Neratinib’s effect over placebo was significant in patients with PIK3CA-altered tumors (HR 0.41; 95% CI 0.17–0.90, P = 0.028) but not PIK3CA wild-type tumors (HR 0.72; 95% CI 0.36–1.41; P = 0.34). The interaction test was non-significant (P = 0.309). Conclusions Although there was a greater absolute risk reduction associated with neratinib treatment of patients with PIK3CA-altered tumors in ExteNET, current data do not support PIK3CA alteration as a predictive biomarker of response to neratinib in HER2-positive EBC. Trial registration ClinicalTrials.gov, NCT00878709. Trial registered April 9, 2009. Electronic supplementary material The online version of this article (10.1186/s13058-019-1115-2) contains supplementary material, which is available to authorized users.


Introduction
Randomized phase III studies have demonstrated that 12 months' treatment with the anti-HER2 antibody trastuzumab plus adjuvant chemotherapy significantly improves clinical outcomes in women with HER2-positive early-stage breast cancer (BC) [1][2][3]. Despite the initial dramatic benefits with adjuvant trastuzumab, relapses are observed on longer follow-up. At the first combined analysis of the NSABP B-31 and NCCTG N9831 trials, disease-free survival (DFS) was 87.1% in trastuzumabtreated patients after a median follow-up of 2 years [4]; however, DFS was 73.7% at the 10-year follow-up [2]. In the HERA study, 2-year DFS was 85.8% in trastuzumab-treated patients (median follow-up 1 year) [5], whereas 10-year DFS after 11 years' follow-up was estimated at 69% [1]. Longer follow-up in the BCIRG 006 trial demonstrated a similar ongoing relapse rate to earlier data in the trastuzumab-treated arms (10-year DFS 73-74.6%), particularly in patients with hormone receptor-positive and HER2-positive BC [3,6].

Patients and methods
Study design, randomization, and masking Details of the multicenter, randomized, double-blind, placebo-controlled phase III ExteNET study have been described previously [26]. In brief, 2840 women with histologically confirmed stage 2-3c (1-3c in original protocol) HER2-positive BC were enrolled from academicand community-based centers in 40 countries between July 2009 and October 2011. Patients were randomized (1:1) to neratinib or placebo, given for 1 year after standard locoregional treatment, chemotherapy, and (neo)adjuvant trastuzumab. Patients, investigators, and study sponsors were masked to treatment allocation. Clinical and radiologic assessments had to be negative for recurrence or metastatic disease at study entry. (Neo)adjuvant trastuzumab was completed up to 1 year (2 years in original protocol) before randomization. All patients (intention-to-treat [ITT] population) provided written informed consent; patients in the correlative cohort signed an optional consent form relating to primary tumor collection for exploratory biomarker analyses.
Three different sponsors were involved over the course of the study, resulting in three global amendments to the study design [26]; the correlative study was developed with amendment 3 and with the initial study sponsor. The Independent Data Monitoring Committee (IDMC) remained consistent throughout the study to preserve blinding integrity; the infrastructure for study conduct and monitoring remained in place to preserve operational consistency. The IDMC reviewed the data at least twice yearly. Additional study design details are described in Additional file 1.
The aim of the present analysis was to assess the prognostic and predictive significance of PIK3CA alterations in ExteNET.

Procedures
Patients were randomized to neratinib (Puma Biotechnology, Los Angeles, CA, USA) 240 mg orally once daily continuously or matching placebo for 1 year. Concurrent adjuvant endocrine therapy for women with locally determined hormone receptor-positive disease was permitted and recommended.
Tumor blocks or freshly cut, unstained sections mounted on positively charged slides were sent to a central certified laboratory. PIK3CA gene testing by reverse transcriptase polymerase chain reaction (RT-PCR) and/ or fluorescence in situ hybridization (FISH) was performed as materials were received from August 2009 through June 2011. PIK3CA FISH was used to quantify PIK3CA gene copy number in relation to the number of copies of chromosome 3 using Vysis LSI PIK3CA Spectrum Green and Vysis CEP 3 Spectrum Orange Probes (Abbott Molecular, Abbott Park, IL, USA). Slides were deparaffinized and pretreated using Vysis Paraffin Pretreatment Reagent Kit II (Abbott Molecular). Probe mixes were hybridized at 37°C for 17 h. Hybridized slides were counterstained with DAPI II. A minimum of 50 nuclei of invasive tumor cells were scored by a board-certified pathologist using a LEICA microscope at × 100 objective. Amplification status was determined from the ratio of PIK3CA to chromosome 3 centromere probe (CEP3) signals. A ratio ≥ 2.2 was considered FISH positive and < 2.2 was considered normal. The pathologist was blinded to the clinical data, randomized arm, and outcome throughout the pre-analytical and analytical process.
The proportion of tumor nuclei present was determined by a board-certified pathologist. Macro-dissection for tumor enrichment was performed if the tumor burden was < 20%. Genomic DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tumor slides (QiaAmp DNA FFPE tissue kit, Qiagen, Valencia, CA, USA). Genomic DNA (5 ng/μL) was tested for PIK3CA mutations (DxS PI3K Mutation Test, DxS, Manchester, UK) using RT-PCR to qualitatively detect E542K, E545D/E545K, and H1047R at a minimum detection level of 1% H1047R, E542K, and E545K or 2% E545D relative to the background. E545D and E545K mutations were detected in a single reaction; both were detectable but not distinguishable. PCR was performed using an ABI 7900HT machine and analyzed using SDS version 2.2.2 software (Applied Biosystems, Foster City, CA, USA).

Statistical analysis
Biomarker results were based on 5-year iDFS data. iDFS was the time from randomization to the first occurrence of invasive ipsilateral BC recurrence, invasive contralateral BC, local/regional invasive recurrence, distant recurrence, or death from any cause. Patients without DFS events were censored at the date of last physical examination. Exploratory PIK3CA biomarker analyses were performed in the correlative cohort, which included patients with samples tested for PIK3CA mutation and/ or PIK3CA amplification.
Data from the placebo arm were used for the prognostic evaluation of PIK3CA alterations. The PIK3CAaltered group comprised patients with mutated and/or amplified PIK3CA. The PIK3CA wild-type group comprised of patients with no measurable PIK3CA mutation or amplification. Kaplan-Meier methods were used to estimate annual event-free survival rates. Log-rank tests were used to compare 5-year iDFS between the PIK3CAaltered and wild-type groups in the placebo arm. HRs were estimated using the Cox regression model.
For sensitivity analysis, a multivariate Cox model was fitted to estimate the treatment effect within the subgroups, adjusting for baseline prognostic factors including age, baseline Eastern Cooperative Oncology Group performance status, race, region, menopausal status, nodal status, hormone receptor status, histological grade, radiotherapy, prior trastuzumab, time from last trastuzumab, prior surgery, and prior neoadjuvant therapy. In addition, the interaction between PIK3CA status (altered vs wild-type) and treatment (neratinib vs placebo) was tested using a Cox regression model. All analyses were exploratory, with no adjustment for multiplicity, and were performed using SAS statistical software (version 9.3; SAS Institute Inc., Cary, NC, USA).

Prognostic effects
The prognostic effect of PIK3CA alterations was assessed in 312 patients in the placebo arm of the correlative cohort who had sufficient tumor material for the testing of PIK3CA mutations and PIK3CA amplification. There was no significant association in iDFS observed in patients harboring PIK3CA alteration (HR 1.34; 95% CI 0.72-2.50) (P = 0.357). Estimated 5-year iDFS was 88.8% in the PIK3CA wild-type group vs 84.5% in the PIK3CAaltered group (Additional file 1: Figure S2).
A multivariate analysis adjusting for clinical prognostic covariates revealed a statistically significant benefit for neratinib vs placebo in patients with PIK3CA-altered tumors (HR 0.40, 95% CI 0.15-0.92; P = 0.041) (Additional file 1: Table S1). The proportion of patients with hormone receptor-positive tumors was similar in PIK3CA-altered (58.4%) and wild-type subgroups (59.4%). The observed benefit for neratinib in patients with PIK3CA-altered tumors was independent of hormone receptor status (Fig. 3).

Discussion
Studies have demonstrated that PIK3CA mutations and amplifications contribute to the hyperactivation of the PI3K/Akt pathways in breast and other human cancers [30][31][32][33][34][35][36]. The contribution of PIK3CA gene amplifications in breast cancer tumorigenesis is not well studied, and gene amplifications are relatively infrequent in breast cancers compared to somatic mutations. In the present analysis of data from the ExteNET study, we evaluated PIK3CA amplifications and somatic mutations to obtain a comprehensive assessment of the prognostic and predictive significance of PIK3CA alterations in patients with early-stage HER2-positive breast cancer. The ExteNET study demonstrated that 1 year of neratinib, given after standard trastuzumab-based adjuvant therapy, significantly improved iDFS in women with early-stage HER2-positive BC, with a 27% relative reduction in the risk of an iDFS event (stratified HR 0.73, 95% CI 0.57-0.92; P = 0.008), corresponding to an absolute improvement of 2.5% vs placebo [27]. In patients with hormone receptor-positive disease, most of whom received concurrent adjuvant hormonal therapy with neratinib/placebo, the magnitude of benefit was more pronounced (HR 0.60, 95% CI 0.43-0.83; P = 0.002) with an absolute improvement of 4.4%. The present correlative  receptor-negative PIK3CA-altered tumors; however, as the interaction test between PIK3CA status and neratinib benefit was not statistically significant, we cannot definitively conclude that PIK3CA status is predictive in nature for selection of neratinib therapy. PIK3CA alterations are among the most common genomic aberrations seen in patients with HER2-positive BC; data suggest that PIK3CA is altered in 36% of patients [10], with mutations in 30.7%, amplifications in 3.1%, and multiple alterations in 2.1% of cases [37]. Similar rates of PIK3CA alterations (35.9%) were reported in another cohort study, primarily comprising advanced-stage BC [38]. In their recent large pooled analysis of outcomes in patients with early-stage HER2-positive BC, Zavardas and colleagues reported an overall PIK3CA mutation rate of 22% [11], similar to the 21% mutation rate we report. We observed a higher amplification rate (8.7%) than previously reported [10], which may be related to discrepancies in gene amplification measurement methods (FISH vs copy number alterations by next-generation sequencing). Within The Cancer Genome Atlas dataset, 70% of PIK3CA mutations were at hotspots E542, E545, or H1047 [10]. Although available techniques at the time of measurement (2009-2011) only allowed us to profile two hotspot mutations in exon 9 (E542K, E545K/D) and one hotspot mutation in exon 20 (H1047R), these appear to account for over two thirds of the reported mutations across the PIK3CA gene [10].
The prognostic and predictive significance of PIK3CA alterations, particularly mutations, have been extensively studied in BC. In metastatic BC, PIK3CA mutation status showed the greatest prognostic effect of those analyzed in the CLEOPATRA study [19]. PIK3CA status was an independent poor prognostic factor, with 8.6 months' median progression-free survival (PFS) for mutated PIK3CA vs 13.8 months for wild-type PIK3CA in the control arm, and 12.5 months for mutated PIK3CA vs 21.8 months for wild-type PIK3CA in the pertuzumab arm. In the EMILIA study, PIK3CA mutations were associated with shorter PFS and OS in capecitabine plus lapatinib-treated patients, but interestingly not in T-DM1-treated patients [20].
The prognostic effect of PIK3CA mutations is less clear in early-stage disease, with seeming variability between short-and long-term outcome and across different clinical trials [11-13, 17, 18]. In our correlative analysis of ExteNET, patients with PIK3CA-mutant tumors did not have a statistically significant difference in survival compared with patients with PIK3CA wild-type tumors.
The presence of PIK3CA alterations and a statistically significant differential benefit from HER2-directed therapies has not been demonstrated in prior studies. In the pooled analysis of five neoadjuvant studies, the interaction test was not significant for PIK3CA status and benefit from combination vs single-agent HER2-directed treatments [12]. Results from FinHER [17] and NSABP B-31 [18] also failed to demonstrate a significant interaction between PIK3CA mutation and the degree of benefit from adjuvant trastuzumab relative to control. Although we observed a greater absolute improvement in iDFS for neratinib in PIK3CA-altered vs wild-type tumors in the present study, the interaction test was not statistically significant (P = 0.1842), and thus, we cannot conclude that PIK3CA status is predictive of benefit for neratinib following 1 year of trastuzumab in patients with early-stage HER2-positive BC.
One limitation of this study is that only three PIK3CA mutation hotspots were assessed, although the PCR methodology used provided good sensitivity for detecting mutations even at low allele frequencies. Today, however, a more comprehensive, whole-exome, or next-generation sequencing might be performed to detect the full spectrum of genomic aberrations across the PIK3CA gene. It is unclear if other known PIK3CA variants would trend toward a similar benefit for neratinib as seen for the E542K, E545K/D, and H1047R variants. A second limitation is that validated guidelines for a definition for PI3K amplification to be associated with biological relevance have not yet been established. Since guidelines for PIK3CA amplification have not been established, we used the same cutoff that was defined for HER2 FISH amplification by ASCO/CAP at the time of sample testing. Another limitation is that FFPE tumor tissue was only collected from 42% of the ITT population, and only 41% of these samples were tested for both PIK3CA amplification and mutation. These tissues were archival and the primary resected breast specimen, and thus reflect the state of the tumor prior to receiving any systemic therapy. Although the study results were essentially unchanged after adjusting for important prognostic factors, the possibility of bias cannot be excluded. A larger sample size would have improved the likelihood of demonstrating a statistically significant interaction test, if one truly exists. A final limitation was the fact that patients with early relapse during adjuvant trastuzumab-based therapy were excluded from ExteNET as patients had to be clinically disease-free at the study entry, with a median time from diagnosis to randomization of 22-24 months. Thus, the results and hypotheses associated with our study may not be relevant in those tumors with primary resistance to adjuvant trastuzumab.

Conclusions
The use of biomarkers to identify patient populations likely to benefit from targeted therapies is a key challenge in the treatment of patients with breast cancer. The clinical significance of PIK3CA mutation as a biomarker of resistance to HER2-targeted therapies has not been clearly established. This exploratory correlative study reports a trend toward the absolute risk reduction for neratinib treatment being greater in patients with PIK3CA-altered vs wild-type HER2-positive BC following standard adjuvant chemotherapy and trastuzumab; however, this observation was not statistically significant. Therefore, these results do not support PIK3CA alterations as a predictive biomarker for benefit from neratinib treatment. Consideration of PIK3CA status as a definitive factor for the use of neratinib in the adjuvant setting would require a prospective clinical study with sufficient power to assess predictive significance for the biomarker.

Additional file
Additional file 1: Supplementary methods. Table S1. Multivariate analyses adjusting for clinical prognostic covariates. Figure S1. ExteNET: CONSORT diagram of the correlative cohort. Figure S2. Kaplan-Meier plots of 5-year invasive disease-free survival for PIK3CA-altered vs wild-type tumors in the placebo arm of the correlative cohort, for assessment of prognostic effect. Figure S3