Alterations of the genes involved in the PI3K and estrogen-receptor pathways influence outcome in human epidermal growth factor receptor 2-positive and hormone receptor-positive breast cancer patients treated with trastuzumab-containing neoadjuvant chemotherapy

Patients with human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer were known to have a poor prognosis in the era when trastuzumab was not available [1‐3]. After the introduction of trastuzumab, the outcome of HER2+ operable patients changed significantly, and many patients who achieved a pathological complete response (pCR) were expected to have been cured of the disease [2, 3]. However, pCR rates are 30-60%, and the 3-year relapse-free survival (RFS) is 71-78% in patients with operable breast cancer, indicating that a substantial number of patients who undergo surgical resection after the chemotherapy have recurrence [2, 3]. Patients with HER2+ breast cancer are usually treated with a combination of trastuzumab and taxanes with or without other chemotherapeutic agents [1‐3], but predictors that indicate the response to the chemotherapy and outcome are not fully investigated.

Alterations in the HER2-PI3K-AKT pathway, which include expression of an extracellular domain-truncated HER2 (p95HER2), mutation and amplification of PIK3CA, loss of PTEN or INPPB4, and mutation of AKT1, are known to result in a poor response to chemotherapy with trastuzumab or poor outcome for breast cancer patients [4, 5]. In addition, there are two types of HER2+ breast cancer; namely, hormone receptor (HR)-positive (HR+) and HR-negative (HR-), and some investigators have reported different biological characteristics including pathological responses between the two [6, 7]. Crosstalk between the estrogen receptor (ER) pathway and the PI3K or ERK/MAPK pathway is thought to be involved in the resistance to trastuzumab-containing chemotherapy in HER2+/HR+ breast cancer [8]. However, there have been few studies aiming to resolve the mechanism of chemotherapy resistance or to identify biomarkers that indicate pCR and relapse using clinical samples.

It has been reported that 0.9% - 18.5% of HER2 immunohistochemistry (IHC) 3+ tumors had a single copy of HER2[8]. The technical shortcomings of IHC that can result in false-positive and false-negative results may be one of the reasons for the discordance between IHC grades and HER2 copy numbers [9, 10], however, there may be true single-gene overexpressers although the incidence may be low. Although metastatic breast cancer patients with the discordance between IHC and HER2 copy numbers seemed to show a low probability of responding to HER2-targeted therapy [11], there has been no study to clarify that single-gene overexpressers with operable breast cancer will respond to trastuzumab, and the mechanisms for the possible resistance to the trastuzumab-containing chemotherapy.

An alternatively spliced form of the human HER2 gene, Δ16HER2, containing an in-frame deletion was found in human breast cancer [12]. Mitra et al. showed that ectopic expression of the Δ16HER2 transcript, but not wild-type HER2 transcript, promotes receptor dimerization, cell invasion, and trastuzumab resistance in NIH3T3 and MCF7 tumor cells [13]. More recently, it was reported that Δ16HER2-expressing transgenic mice, but not wild-type HER2-expressing mice, developed multiple mammary adenocarcinomas [14]. However, the clinical significance of Δ16HER2 has not been fully examined in human breast cancer.

We hypothesized that genomic alterations detectable by single-nucleotide polymorphism (SNP) arrays and HER2 copy numbers and levels of HER2 transcripts would suggest mechanisms of resistance and prognostic factors for patients treated with trastuzumab-containing chemotherapy. Thus, we studied SNP array patterns of 143 breast cancer samples, including 46 HER2+ tumors, obtained at the time of diagnosis. We found that alterations of genes involved in the estrogen-receptor (ER) and PI3K pathways indicated worse RFS rates in patients with a HR+ but not HR- tumor, who were treated with chemotherapy with trastuzumab, followed by adjuvant trastuzumab (plus endocrine therapy for patients with a HR+ tumor). We also found that patients with a tumor showing a single HER2 copy number had more difficulty in achieving pCR, and tended to have worse RFS rates than those having a tumor with HER2 amplification. These findings may help to clarify the mechanisms for resistance to the chemotherapy with trastuzumab, and improve the efficacy of chemotherapy in HER2+ breast cancer.

On the basis of routine methods, 77 tumors were classified as the HER2-/HR+ type, 28 as the HER2+/HR+ type, 18 as the HER2+/HR- type, and 20 as the HER2-/HR- type. The 46 HER2+/HR+ and HER2+/HR- tumors are the subjects of this paper. The numbers of four chromosome aberrations, including gain, loss, amplification and UPD, were examined in each tumor. Gain and amplification of oncogenes were individually described in Additional file 1: Table S1, however, they were combined and are referred to as gain in the following analyses. All the 46 tumors showed at least some chromosome aberrations.

In the present SNP array-based study of 46 HER2+ breast cancers, we evaluated clinical and genetic factors that indicate pCR and RFS in patients who were treated with trastuzumab-containing neoadjuvant chemotherapy. SNP array analysis has a merit to detect whole genomic aberrations at once, and therefore could find a predictive or prognostic impact of combined genomic aberrations. Such an attempt seems not to have been made before. The 46 tumors were selected based on a routine HER2 study using IHC with or without FISH in deparaffinized tissue samples. We found discordance between results of the routine analysis and the present SNP and QPCR analysis with FISH in defrosted tissue samples in 17.4% (8/46) of tumors; 4 IHC 3+ (8.7%) and 4 IHC 2+ (8.7%) tumors. Previous studies reported the IHC3+, FISH negative type in 0.9%-18.5% of tumors examined [9], and the percentage of 8.7% shown in the present study may be comparable to or a little higher than the previous results. Another discordance of 4 patients with the IHC 2+ and nonamplified HER2 type may be caused by the difficulty of FISH analysis using needle biopsied tissues embedded in paraffin. It is difficult to compare this percentage of 8.7% with those of other series, because there have been no comparable studies reported.

Overexpression of protein occurs not only by mRNA overexpression, but also by post-transcriptional, translational and protein degradation regulation [26]. HER2 is efficiently ubiquitinated and downregulated by the chaperone binding ubiquitin ligase CHIP/STUB1 [27]. Recently, Jan et al. studied the expression and correlations among TID1, CHIP, and HER2 in a total of 183 breast cancer histology sections using IHC and immunoblotting assay, and found that the immunohistochemical expression of TID1 and CHIP were positively correlated with each other but were both inversely correlated to that of HER2 [28]. These findings suggest that down-regulation of CHIP could cause overexpression of HER2 by preventing its degradation. In a study of metastatic breast cancer with the HER2 IHC2+/3+ and nonamplified HER2 type, patients treated with trastuzumab plus chemotherapy had better progression-free survival than those treated with chemotherapy alone (P=0.03), suggesting the presence of the true single-gene overexpressers in the study population [11]. Thus, some HER2-single-gene overexpressers were not the results of technical shortcomings of HER2 IHC, but could be the results of disruption of HER2 protein degradation occurring in the tumor cells. Overexpression of MYCN caused by the disruption of the protein degradation but not by the amplification indicated poor outcome in patients with neuroblastoma [29].

The present study indicated that the patients with nonamplified HER2 showed more difficulty in achieving pCR and more frequent relapse than those with HER2 amplification. Unexpectedly, we found that tumors with the lower HER2 copy numbers had more frequent alterations in the PI3K pathway genes than those with the higher copy numbers (Table 1). Alterations of the PI3K pathway genes were reported to be associated with poor response to HER2-targeted therapy in patients with HER2+ tumors [30], and this finding concurs with the present finding indicating the poor outcome of patients having a tumor with HER2 IHC 2+/3+ and nonamplified HER2. These findings support the statement that FISH analysis should be the primary HER2 testing for patients who are candidates for HER2-targeted therapies [9].

In regard to the relationship between clinical and genetic factors and RFS, patients with various genetic changes in tumors identified by SNP array had or tended to have worse RFS rates than those without (Table 3). Previous studies reported that overexpression of these genes identified by various methods was associated with a poor outcome in patients with breast cancer [31‐37]. Thus, the present study indicated that the results of the SNP array analysis detecting gain of specific genes and those of other analyses detecting overexpression of the gene products were mostly consistent.

When the prognostic implications of the genetic aberrations were separately examined in HR+ and HR- tumors, a combination of mutations and gain of PIK3CA, a combined aberration of the PI3K pathway genes, and gain of FOXA1, CDH3, BIRC5, MYBL2, and AIB1 indicated worse RFS rates only in patients with a HR+ tumor (Table 4). It is noteworthy that the incidences of the most genetic changes did not differ between the two types of tumors (Table 2). We confirmed the result of a study reporting that increased PI3K pathway activity was associated with poor outcome for patients treated with HER2-targeting therapy [30], and newly found that the prognostic significance of the alterations in the PI3K and ER pathway genes applied only to the patients with a HR+ tumor (Table 4). FOXA1, a forkhead family transcription factor, is essential for optimum expression of ER and estrogen responsive genes. Although one study reported that FOXA1 expression was correlated with the luminal A subtype and a favorable outcome [38], others depicted a complicated picture of FOXA1 as a participant in multiple signaling pathways in breast cancer, which are both oncogenic and tumor suppressive [33]. Actually, a recent study reported co-expression of ER and FOXA1 in metastatic breast cancer samples, indicating oncogenic activities of FOXA1[39]. AIB1 is a nuclear receptor coactivator that interacts with ERs in a ligand-dependent fashion and enhances estrogen-dependent transcription. Harigopal et al. reported AIB1 activation to be mediated by crosstalk with other signaling kinases such as growth factor receptors including MAPK, which can be activated by HER2 in a ligand-independent fashion [40]. AIB1 is considered as a key factor in the regulation of tumor growth and carcinogenesis. In addition, patients with AIB1-positive tumors identified by IHC showed worse RFS rates than those with AIB1-negative tumors [40]. These findings indicate that crosstalk between the PI3K and ER pathways is causally involved in resistance to the chemotherapy with trastuzumab, and thus, that hormone receptor status impacted the prognostic significance of genomic alterations in HER2+ tumors.

The present study also showed that gain of DEK and BIRC5 genes, which seems not to be involved in the PI3K or ER pathway, had clear prognostic significance in patients with breast cancer (Table 3), suggesting presence of other pathways exhibiting crosstalk with the PI3K or ER pathway. Lu et al. reported that HER2-mediated up-regulation of survivin expression contributed to Taxol resistance through a survivin-mediated faster exit from mitosis [41]. Xia et al. showed that acquired resistance to lapatinib in the BT474 cell line was associated with a switch in the regulation of survivin from HER2 to ER [42]. Thus, various roles of survivin in chemotherapy resistance may contribute to the prognostic significance of BIRC5 gain in the present series of patients with HR+ and HR- breast cancers.

A splice variant of the human HER2 transcript lacking exon 16 (Δ16HER2) has been detected in human breast cancers [12]. Mitra and colleagues showed that ectopic expression of Δ16HER2, but not wild-type HER2 mRNA, promoted receptor dimerization, cell invasion, and trastuzumab resistance in the NIH3T3 and MCF7 cell lines [13]. Recently, a mouse line that transgenically expresses the Δ16HER2 transcript has been generated and all the transgenic females developed multifocal mammary tumors with a rapid onset [14]. This oncogenic isoform has been associated with trastuzumab resistance in various in vitro and transgenic mouse studies [12, 14]. The present study showed that levels of the wild-type HER2 transcript and those of the Δ16HER2 transcript were correlated, and that there was no difference in the RFS rate between patients with higher levels of Δ16HER2 mRNA and those without. In addition, we also found that patients with higher percentages of the Δ16HER2 mRNA (≥ 2.4%) and lower levels of the wild-type HER2 mRNA (< 400) were likely to have recurrence. The decreased RFS rates for patients with lower levels of wild-type HER2 mRNA may be explained by the decreased susceptibility to trastuzumab in patients with such tumors. The patients with the higher percentage of Δ16HER2 mRNA also had the lower level of wild-type HER2 mRNA, and this lower level might have resulted in a lower denominator and hence the higher percentages of Δ16HER2 mRNA. Thus, the poor prognosis of patients with the higher percentage of Δ16HER2 mRNA may be related to the lower levels of the wild-type HER2 mRNA, or to presently unknown mechanisms.

The present study showed no difference in RFS between patients who attained pCR and those who did not. Previous studies indicated better overall survivals for patients with pCR than those with no pCR [2, 3]. von Minckwits and colleagues studied pCR and its prognostic impact on survival in intrinsic breast cancer subtypes and concluded that pCR is a suitable surrogate end point for patients with HER2+ (nonluminal) but not luminal B/HER2+ breast cancer [43]. More recently, results of a meta-analysis showed that patients with a HR-/HER2+ or HR+/HER2+ tumor who achieved pCR had greater event-free survival than those with the respective subtype of tumor who did not [44]. The contradictory finding of the present study might be caused by the small sample size and inclusion of HR+/HER2+ and HR-/HER2+ breast cancer samples.

Several clinical trials have been carried out to improve the outcome of patients with HER2+ tumors. Two trials reported that the combination of an aromatase inhibitor with trastuzumab or lapatinib improved outcome for patients with HER2+/HR+ metastatic breast cancer compared with an aromatase inhibitor alone [45, 46]. Both trials showed better progression-free survival for the patients treated with the combination. These studies were undertaken because the crosstalk between the PI3K and ER pathways could cause resistance to trastuzumab and anti-hormone agents. In addition, one phase 2 and another phase 3 clinical trials were carried out using a combination of lapatinib and trastuzumab with or without cytotoxic chemotherapy for patients with a HER2+/HR+ or HER2+/HR- early stage tumor, and both studies showed improved pCR rates [47, 48]. These studies were undertaken because primary and acquired resistance to both agents could be overcome, their partly non-overlapping mechanisms of action, and the well-characterized synergistic interaction between them could be expected in patients with HER2+ breast cancer. The present study showed that the PI3K and ER pathway genes were specifically altered in HER2+/HR+ tumors of patients who subsequently relapsed after receiving neoadjuvant chemotherapy with trastuzumab. Lapatinib targets the intracellular ATP domain of HER2, preventing self-phosphorylation and subsequent activation of the PI3K and MAPK signal pathways [5]. It may be reasonable to add lapatinib to the present trastuzumab-containing chemotherapy for patients with a HER2+/HR+ tumor to overcome the resistance due to the altered PI3K pathway genes and to obtain a better outcome.

MT, TH, KT, MH, and JW participated in data collection, interpretation and molecular analysis. FK and MK carried out molecular cytogenetic and pathological studies. ASO and OS were responsible for SNP array analysis. MT, HT, KI, MM, and YK contributed to concept design and drafted the manuscript. All authors have read and approved the manuscript.