Trastuzumab anti-tumor efficacy in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models

The incidence of esophageal carcinoma (EC) and the histological type varies widely with geographical location [1]. Notably, esophageal squamous cell carcinoma (ESCC) is the predominant histological type in Asian areas with a high risk of cancer development in the esophagus, in contrast to Western countries (Northern Europeans and Caucasians in the USA) [1]. In the United States and North and Western Europe, where esophageal adenocarcinoma is the predominant histological type, the incidence of ESCC has been decreasing in recent decades, whilst the incidence of esophageal adenocarcinomas, especially of the gastroesophageal junction, has been increasing [2, 3].

Surgery remains the mainstay of therapy for esophageal carcinoma patients. However, ESCC diagnosis is associated with a high mortality owing to its aggressive behavior and the 5-year survival for non-metastatic disease ranges from 20% to 40% [2]. Patients with metastatic disease who are treated with palliative chemotherapy using a combination of 5-Fluorouracil (5-FU) and Cisplatin have a median survival of less than one year and response rates of 25% to 45% in phase II and III trials [4, 5]. Salvage options for patients with refractory EC are very limited [6]. Unfortunately, there are no clinically approved targeted therapies for the treatment of esophageal carcinoma.

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in breast and other types of human cancers and has been successfully developed as a therapeutic target [6, 7]. Antibody-based therapy with Herceptin (Trastuzumab) has been used for the clinical treatment of HER-2-positive breast cancer [6, 7]. Recently, Trastuzumab therapeutic efficacy has also been observed in HER-2-overexpressing gastric cancer [7].

Previous studies in patients with ESCC indicated that the frequency of HER-2 protein expression ranged from 0 to 56% [8, 9], whilst the frequency of HER-2 gene amplification ranged from 5 to 35% [8, 9]. These data suggested that a proportion of ESCC patients could be candidates for Trastuzumab targeted therapy. Results from early clinical trials demonstrated a correlation between HER-2 expression and gene amplification with Trastuzumab therapeutic efficacy in patients with esophageal adenocarcinomas [10]. However, over expression and amplification of HER-2 appeared to be fundamentally different within esophageal squamous cell carcinoma, with a tendency of lower positive rates and lower level amplification, compared to adenocarcinoma [11]. Accordingly, it is not possible to imply Trastuzumab efficacy within esophageal squamous cell carcinoma, and as such, these studies aimed to address this preclinically. Therefore, we established xenograft mouse models derived from patient’s ESCC tissues and used these novel, clinically relevant ESCC models to explore the anti-tumor efficacy of Trastuzumab targeted therapy.

It is well established that patient-derived xenograft mouse models can better represent human tumors through increased diversity of molecular lesions and the preservation of three-dimensional tumor-stromal cell components and interactions [15]. Although some patient-derived xenograft models, e.g., NSCLC [16, 17], melanoma [18], colon cancer [19], breast cancer [20], HCC [21, 22], GIST [23], have been established and used for evaluation of anti-tumor compounds [24], the availability of patient-derived EC xenograft models is limited. Previous studies have however, developed xenograft EC models by implanting human EC cell lines into mice [25, 26] and the first objective of this study was to develop novel PDECX models. After successfully establishing 25 PDECX mouse models through implantation of 54 patient EC samples, 5 PDECX mouse models were selected for evaluation of Trastuzumab anti-tumor efficacy based on HER-2 expression levels. Although the five models displayed varying growth rates (Figure 1), this was not related to differentiation and HER-2 positivity (Table 1). Indeed, the varying growth rates of patient-derived models are likely related (at least in part), to the requirement to directly implant tumor fragments, rather than standardized defined numbers of tumor cells.

Trastuzumab anti-tumor efficacy correlates well with HER-2 expression in patients with breast [6, 27‐29] and gastric cancer [7, 24, 30]. Esophageal carcinoma represents another disease with a high frequency of tumor HER-2 expression and gene amplification [8, 9, 11]. In this setting, the frequency of patients with tumor HER-2 positive expression ranges from 0 to 56% [8, 9], whilst the frequency of HER-2 gene amplification varies from 5% to 35% [8, 9, 11]. In addition, the predominant histological subtype of esophageal carcinoma differs between Eastern and Western countries, and adenocarcinoma has been shown to exhibit higher rates of HER-2 protein expression positivity and gene amplification compared to squamous cell carcinoma [11]. Accordingly, most current preclinical and clinical studies are focused on esophageal adenocarcinoma and literature data supports a correlation between HER-2 gene expression (or amplification) and Trastuzumab efficacy in esophageal adenocarcinoma (preclinical and clinical phase I/II data [10, 31] however, such data is rare in esophageal squamous cell carcinoma [32]. In order to address the therapeutic efficacy of Trastuzumab in esophageal squamous cell carcinoma preclinical studies, we characterized 5 PDECX mouse models for HER-2 gene amplification and protein expression. In this study, we did not observe strong positive staining for HER-2 expression (3+) or gene amplification (FISH score ≥ 6) in the 25 established PDECX mouse models. Two PDECX mouse models (EC039 and 044) were positive (IHC 2+) for HER-2 expression and had relatively high HER-2 copy number (~3.9). HER-2 gene copy number and protein expression were highly consistent between the 5 PDECX mouse models and their corresponding patient’s EC tissues (Figure 2), suggesting that the PDECX mouse models better represent clinical EC disease, compared to standard cell line-derived xenografts.

Clinically, approximately 20% of esophageal adenocarcinoma patients who were HER-2 IHC 2+ and FISH negative demonstrated a complete clinical response [10]. Mimura et al. have also demonstrated that HER-2 expressing esophageal SCC cells could be killed by Trastuzumab-mediated ADCC and that anti-proliferative activity reflected the degree of HER-2 expression detected by flow cytometry, but not by HercepTest nor fluorescence in situ hybridization analysis. To determine if HER-2 positive (IHC 2+) and FISH negative esophageal squamous cell carcinoma could be responsive to Trastuzumab monotherapy, we tested Trastuzumab anti-tumor efficacy in 5 established PDECX mouse models with varying degrees of HER-2 expression and gene copy number. Unsurprisingly, there was no tumor regression, and no or marginal response to Trastuzumab treatment in the HER-2 negative PDECX models. Our data demonstrated that Trastuzumab induced complete tumor regression in one of the HER-2 positive models (IHC 2+) (Figure 3 and Table 1), but generated no significant anti-tumor response in the other. Whilst we believe that this potent anti-tumor effect is a direct consequence of anti-proliferative activity, we note that Trastuzumab has been observed to exert both mild anti-angiogenic and anti-metastatic effects [33] however, we were unable to assess changes in these phenotypes within the studies documented here.

To explore the underlying mechanism behind these contrasting Trastuzumab responses, additional genetic changes were analyzed. It is well known that some key gene mutations including EGFR, K-ras, B-raf, PIK3CA, often affect the efficacy of molecular-targeted therapies [34, 35]. In clinical studies of metastatic colon cancer, patients with wild type K-ras, B-raf and PIK3CA genes gain benefit from cetuximab therapy[34], whilst lung cancer patients with mutations in exons 19 and 21 of the EGFR gene benefit from Iressa or Tarceva therapy. Furthermore, mutations in ESCC patients have been recently reported; 16% of ESCC patients (5/30 cases) harbor K-ras gene mutations [36], 14% of ESCC patients (7/50 cases) contain EGFR mutation [37], and 2.2-11.8% of ESCC patients have gene mutations in exon 9 of the PIK3CA gene [38, 39]. These findings suggest that gene mutation(s) may play a key role in resistance to Trastuzumab treatment. After screening for EGFR, K-ras, B-raf and PIK3CA gene mutations in the 5 PDECX mouse models and their corresponding primary tumors, we found that model EC039 (and the corresponding patient tissue) had a helical domain mutation (G1624A, E542K) in exon 9 of the PIK3CA gene (Figure 4 and Table 1). This mutation is known to activate downstream signaling of the AKT pathway [40, 41]. As such, this result likely explains why the HER-2 positive (IHC 2+) model EC039 was unresponsive to Trastuzumab treatment, whilst model EC044 (HER-2 positive (IHC 2+)) displayed tumor regression. PIK3CA activating mutations have been reported to associate with shorter time to progression (TTP) in Trastuzumab-treated breast cancer [42]. More recently, particular attention has been paid to the role of the PIK3CA gene in Trastuzumab resistance. Mutations in exon 9 and 20 of the PIK3CA gene have been observed in 8% to 40% of breast cancers [43‐45]. Furthermore, an association between PIK3CA mutation and resistance to Trastuzumab therapy in HER-2-amplified breast cancer cell lines has also been reported [46]. Berns et al. have also suggested that oncogenic mutations of PIK3CA may render breast cancers more resistant to treatment with Trastuzumab [47], and Eichhorn et al. have further shown that mutational activation of PIK3CA can similarly render cells more resistant to the recently approved anti-HER2 agent Lapatinib. In this situation however, Lapatinib resistance caused by the dominant activating mutations in PIK3CA can be effectively reversed by treatment with the PI3K/mTOR inhibitor, NVP-BEZ235 [48]. Moreover, the AKT inhibitor AZD5363 increases the efficacy of lapatinib and/or trastuzumab in xenografts derived from HER2+ breast cancer cell lines harboring PIK3CA mutation [14]. In our study, we observed strong AKT pathway activation in the PIK3CA mutation model EC039 (Figure 5A) compared to wild type model EC044, suggesting that activated AKT pathway signaling likely contributes to Trastuzumab resistance in our efficacy study. To our knowledge, we are the first to describe a PIK3CA gene mutation which causes resistance to Trastuzumab therapy in a preclinical PDECX mouse model, and which can be partly resensitised using a combination of Trastuzumab and AKT inhibitor (Figure 5B). The inability to achieve full tumor regression in this combination study may be a consequence of only blocking PI3K pathway signaling downstream through AKT, thus still allowing PDK1, PKC and Rac signaling to occur.

In summary, we have established novel xenograft models derived from patient esophageal squamous cell carcinoma, and have used these clinically relevant animal models to investigate Trastuzumab efficacy in HER-2 expressing ESCC. Our results demonstrate that a non-amplified, HER-2 IHC 2+ model responds completely to Trastuzumab targeted therapy, and importantly, that PIK3CA gene mutation negatively associates with Trastuzumab anti-tumor efficacy in a xenograft model of ESCC. Moreover, resensitization to Trastuzumab therapy is achieved when treatment is combined with an AKT inhibitor to suppress PIK3CA pathway signaling. Taken together, this study illustrates that Trastuzumab can induce regression in a HER-2 positive tumor, and that PIK3CA mutation (in a HER-2 positive background) may be a potential resistance mechanism to Trastuzumab treatment in pre-clinical patient-derived EC xenograft models. We believe that this pre-clinical observation and hypothesis warrants further investigation in a clinical study.