Background
Luminal breast cancer is the most frequent intrinsic subtype, accounting for about 70% of breast cancers [
1]. Patients with luminal breast cancer have treatment options including chemotherapy and endocrine therapy; however, methods for optimizing remain unclear. Comprehensive genetic analysis reveals genetic heterogeneity of breast cancer [
1,
2]. Several genetic alterations are frequently observed among breast cancers. These are correlated with intrinsic subtype. Recently, these genetic characteristics were found to be associated with therapeutic efficacy and prognosis [
3,
4]
. PIK3CA gene encoding the p110 catalytic subunit of PI3K is most frequently mutated in luminal breast cancer [
1].
PIK3CA mutations cause activation of PI3K. PI3K converts phosphatidylinositol bisphosphate (PIP2) into phosphatidylinositol triphosphate (PIP3) and activates Akt. Phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 to PIP2 and regulates Akt signaling [
5]. PTEN is often deregulated in patients with breast cancer and can activate PI3K/Akt signaling [
1]. Akt acts downstream of PI3K to regulate many biological processes, such as cell proliferation, metabolism, differentiation, apoptosis and tumorigenesis [
6]. In patients with human epidermal growth factor receptor 2 (HER2) overexpressed breast cancer,
PIK3CA mutations and PTEN loss have been reported to cause activation of the downstream PI3K / Akt pathway associated with drug resistance [
5,
7]. Moreover, activation of the PI3K / Akt signaling is associated with endocrine therapy resistance [
8,
9]. It would be important to investigate how
PIK3CA mutations and PTEN loss, key genetic alterations in this drug resistance-associated pathway, relate to response to preoperative therapy in early breast cancer.
There is pressing needs to find the biomarker in the selection of neoadjuvant therapy in postmenopausal luminal breast cancer patients. Immunochemical markers such as Ki67, morphological markers such as tumor-infiltrating lymphocytes (TIL), and genomic profile markers such as the Oncotype DX Recurrence Score have been used in studies, but there is insufficient evidence to support their use to guide clinical decisions for neoadjuvant therapy [
10]. Several retrospective studies have reported that patients with
PIK3CA mutation or PTEN loss are less responsive to preoperative chemotherapy in all subtypes, as well as in HER2-positive and triple-negative breast cancer [
11‐
13]. However, this has not been well investigated in luminal breast cancer. We examined the hypothesis that
PIK3CA mutations and low PTEN expression affect the response to neoadjuvant therapy and prognosis in postmenopausal luminal breast cancer patients. First, we investigated the correlation between the pathological effect of neoadjuvant therapy and
PIK3CA mutations or low PTEN expression. Second, we assessed these alterations on prognosis using the same cohort.
Methods
Patients and clinicopathological data
We selected patients with Estrogen receptor (ER)-positive HER2-negative breast cancer who underwent neoadjuvant therapy at Chiba University Hospital (Chiba, Japan) and Chiba Cancer Center (Chiba, Japan) from 2003 to 2015. We limited the inclusion criteria to postmenopausal patients with up to stage II. Patients with histories of systemic therapy for other cancers, including metachronous breast cancer, were excluded. Patients provided written informed consent for the use of their biological material for future research purposes before biopsy. The local ethics committee approved this study (Chiba University Hospital on 22 March 2017 No. 113. Chiba Cancer Center on 3 March 2017 No. 2285).
Traditional prognostic factors for early breast cancer including age at diagnosis, tumor size, lymph node (LN) involvement, and tumor grade were obtained from the electronic databases in each Facilities. The expression of ER, Progesterone receptor (PR), HER2, and Ki-67 was evaluated by immunohistochemistry in accordance with the ASCO/CAP guidelines [
14,
15]. The cut off point for ER and PR positivity was 10% and that for Ki-67 positivity was 14%. HER2 was defined as positive with score 3 + , or demonstration of HER2 amplification on fluorescence in situ hybridization of ≧ 2.0.
The clinical response was assessed according to the response evaluation criteria in solid tumors (RECIST) guidelines [
16]. For evaluating the pathological effect of neoadjuvant therapy, the response criteria of the Japanese Breast Cancer Society [
17] was used. In order to evaluate the relationship between therapeutic effect and biological features, those who were pathological therapeutic effect grade 2 or 3 were defined as sensitive, and grade 0 or 1 as resistant.
The effects on prognosis were evaluated by recurrence-free survival (RFS) and overall survival (OS). RFS was defined as from the day systemic therapy started to the day of recurrence event (including locoregional recurrence, distant metastasis, and contralateral breast cancer). OS events were death from all causes.
Formalin fixed, paraffin embedded (FFPE) tissue samples of pretreatment core needle biopsy and surgical specimen were collected. The tumors were histologically evaluated on hematoxylin & eosin sections and we selected the area that contained more than 70% of the cancer cells. Four slices of 4-μm-thick section were made and then the tumor area of each section was manually dissected using a disposable scalpel. Total DNA was extracted from samples using the QIAamp DNA FFPE kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.
PIK3CA exons 9 and 20 were screened for mutations using high resolution melting (HRM) analysis on a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) according to the protocol described before [
18]. We used a plasmid containing mutation sequence for positive control. The primers used for analysis were shown in Supplementary Table S1. The M13 chimeric primers were used for subsequent Sanger sequencing.
PIK3CA (exons 9 and 20) polymerase chain reaction (PCR) products shown to be positive by HRM analysis were sequenced to confirm the presence of mutations using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, USA) according to the manufacturer's protocol. The M13 primers were used for sequencing. The sequencing products were analyzed on an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).
Immunohistochemical analysis
Immunocytochemistry (IHC) was performed using ER (1D5; Dako, Tokyo, Japan) and PgR (PgR636; Dako), Ki-67(MIB-1; Dako), PTEN (6H2.1; Dako) antibodies and HercepTest II (Dako) following the manufacturer’s instructions. The stained slides were reviewed by two observers without knowledge of the clinical data. Staining as scored semi quantitatively using a histoscore (H-score), which was calculated as staining intensity (scored as 0–3) x percentage of stained cells (0–100%) as previously described [
19]. Because the scoring system for PTEN expression by using immunohistochemical methods are not standardized, PTEN H-scores less than 50 were considered as low in this analysis,[
20].
Statistics
The association between various clinicopathological features and PIK3CA mutations or PTEN expression was compared using Fisher’s exact test or Wilcoxon’s test if appropriate. The probability of a pCR was estimated using a logistic regression model which was fitted to the pathological effects of neoadjuvant therapy, clinicopathological features, PIK3CA mutation status, and PTEN expression. In order to avoid overfitting, factors were carried in subsequent multivariate analyses if the statistical significance was p < 0.10 in univariate analyses. Odds ratios, 95% confidence intervals, and p-values were estimated. RFS and OS rates were calculated with the Kaplan–Meier method and evaluated with the log-rank test. Multivariate analyses of the predictors of pathological effects, recurrence, and death were conducted using the Cox proportional hazards model. All analyses were conducted with the JMP® software program (version 13.0, SAS Institute Inc., Cary, USA). p-values ≤ 0.05 were considered to indicate statistically significant differences.
Discussion
To our knowledge, this is the first report showing the effects of PIK3CA mutations and PTEN expression on pathological therapeutic effect of neoadjuvant therapy and the prognosis in postmenopausal Asian patients with ER-positive HER2-negative breast cancer. There are several treatment options for patients with early-stage luminal breast cancer, including surgery, chemotherapy, and endocrine therapy. Our study showed that in clinical practice, the examination of PIK3CA mutations and PTEN expression may provide useful information to determine the indication for neoadjuvant therapy.
Tumors with
PIK3CA mutations had poorer response to NAC than tumors with
PIK3CA wild-type. To confirm the effect of
PIK3CA mutations on pathological therapeutic effect of NAC, multivariate analysis was performed. Only
PIK3CA mutation status was an independent predictor for pathological therapeutic effect of NAC in postmenopausal luminal breast cancer patients. Although Ki-67 was reported as a predict marker of response to NAC even in luminal breast cancer [
21], there is no relationship between them in our cohort. If postmenopausal patients with high Ki-67 luminal breast cancer plan NAC especially in expectation of tumor shrinkage in order to carry out breast conserving surgery, it would be better to confirm that tumor does not have
PIK3CA mutations.
It has been reported that
PIK3CA mutations are not related to the response to NAE in ER positive breast cancer [
22,
23]. However, these studies included HER2-positive patients and some evaluation of the response was performed after short term treatment. Our cohort including only ER-positive, HER2-negative breast cancer patients also showed that
PIK3CA mutations did not affect the response to 6 months NAE.
When response to NAC and prognosis were compared among breast cancer-intrinsic subtypes, patients with luminal tumors had a lower pathologic complete response rate but showed better outcomes compared with triple negative type and HER2 type [
24]. Likewise, luminal tumors with
PIK3CA mutations showed chemoresitance compared with
PIK3CA wild-type but
PIK3CA mutation status was not a prognostic marker for postmenopausal patients with luminal tumors. The relationship between
PIK3CA mutations and prognosis in breast cancer patients is controversial. Kalinsky et al. [
25] reported that patients with tumors harboring a
PIK3CA mutation had significant improvement in OS and breast cancer-specific survival in 590 patients. A meta-analysis of eight retrospective cohort studies also showed that the group with
PIK3CA mutation had superior clinical outcomes [
26]. On the other hand, our study and a prospective clinical trial on adjuvant endocrine therapy [
27] showed that
PIK3CA mutations were not associated with prognosis. It is speculated that the impact of
PIK3CA mutations on the prognosis varies with subtype and therapy, and there is no effect in postmenopausal luminal breast cancer patient.
Loss of PTEN expression had associations with poor prognosis and triple negative type in breast cancer patients as previously reported [
28]. We confirmed that low PTEN expression before treatment is a poor prognostic factor even in luminal breast cancer. Expression changes in PTEN were seen after treatment (Table
2) but were not significantly associated with therapeutic effects in both NAC and NAE group (data not shown). There are various causes of loss of PTEN function, such as somatic mutation, epigenetic silencing, and protein interaction [
29], and there are many ways to assess it. However, IHC may be a simple and useful test in the clinic to evaluate protein expression that reflects actual function of PTEN.
The frequency of
PIK3CA mutations in patients with luminal breast cancer is 30–50% using digital PCR or next-generation sequencing (NGS) [
1,
22,
23]. The Sanger sequence is less sensitive than PCR and NGS [
30]. However, our study showed that the HRM analysis and Sanger sequencing method was useful for identifying clinically significant mutations. This method, which can be performed easily and inexpensively, has economic benefits given that
PIK3CA mutations are clinically measured in over 1 million women with newly-diagnosed breast cancer, per year.
Our study had several limitations. First, DNA analysis using FFPE specimens is subject to DNA degradation that could affect the variability of the data. Second, this was a retrospective study; therefore, it is impossible to eliminate selection bias. Third, it is possible that our results were due to the small number of patients. Finally, our study only includes patients with ER-positive, HER2-negative breast cancer up to stage II, and it is unclear whether the findings would apply to patients with other subtypes or stages of breast cancer. The study only evaluates the relationship between PIK3CA mutations and PTEN expression with the response to neoadjuvant therapy and survival outcomes, and it is unclear whether these biomarkers have any therapeutic implications. Further prospective studies are needed to confirm our results.
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