Evaluation of Trastuzumab Anti-Tumor Efficacy and its Correlation with HER-2 Status in Patient-Derived Gastric Adenocarcinoma Xenograft Models

The aim of the study was to investigate trastuzumab anti-tumor efficacy and its correlation with HER-2 status in primary xenograft models derived from Chinese patients with gastric adenocarcinoma. Patient-derived gastric adenocarcinoma xenograft (PDGAX) mouse models were firstly generated by implanting gastric adenocarcinoma tissues from patients into immune deficient mice. A high degree of histological and molecular similarity between the PDGAX mouse models and their corresponding patients’ gastric adenocarcinoma tissues was shown by pathological observation, HER-2 expression, HER-2 gene copy number, and mutation detection. Based on Hoffmann’s criteria in gastric cancer, three models (PDGAX001, PDGAX003 and PDGAX005) were defined as HER-2 positive with fluorescence in situ hybridization (FISH) amplification or immunohistochemistry (IHC) 2+/ 3+, while two models (PDGAX002, PDGAX004) were defined as HER-2 negative. Upon trastuzumab treatment, significant tumor regression (105 % TGI) was observed in model PDGAX005 (TP53 wt), while moderate sensitivity (26 % TGI) was observed in PDGAX003, and resistance was observed in PDGAX001, 002 and 004. A significant increase in HER-2 gene copy number was only observed in PDGAX005 (TP53 wt). Interestingly, trastuzumab showed no efficacy in PDGAX001 (HER2 IHC 3+ and FISH amplification, but with mutant TP53). Consistent with this finding, phosphor-HER2 modulation by trastuzumab was observed in model PDGAX005, but not in PDGAX001.


Introduction
Gastric cancer (GC) is the second most common cancer and leading cause of cancer mortality worldwide [1][2][3][4]. Early stage patients with GC usually show no specific symptoms and as a consequence, GC patients often present with advanced disease upon diagnosis, especially in countries where gastric cancer screening is not routinely performed [4,5].
Surgery remains the fundamental treatment option for resectable gastric cancer. However, even after curative surgery, local and distant recurrence rates are still high. Advancedstage gastric cancer is associated with poor prognosis and low 5-year survival rates (5-20 %) [6]. Although adjuvant chemotherapy using epirubicin, cisplatin, and 5-flurouracil (5-FU) has gradually become standard-of-care therapy for GC patients in many countries [7], most patients with inoperable or metastatic disease require palliative treatment and have a median overall survival of less than 1 year and a limited chance of long-term survival [6,8].
Due to recent genomic and proteomic advances and improved understanding of the key molecular pathways in cancer, molecular targeted therapies have emerged as additional treatment options in clinical practice. HER2 protein overexpression is increasingly recognized as a frequent molecular abnormality, known to be driven by HER-2 gene amplification in breast cancer [9]. Overexpression of the HER2 protein and amplification of the HER-2 gene in gastric cancer were first described in 1986 [10]. In recent years, HER-2 has gradually become a new prognostic factor and a novel therapeutic target in GC [9].
Trastuzumab is a recombinant humanized monoclonal antibody targeted against the HER2 extracellular domain [11]. The 'Trastuzumab in gastric cancer' (ToGA) study was the first randomized, controlled phase III trial to evaluate trastuzumab efficacy and safety in HER2-positive advanced gastric cancer [12]. In this trial, 22.1 % of the patients were identified as positive for HER-2 protein expression [8] and the median overall survival was significantly improved after trastuzumab treatment plus chemotherapy, compared to chemotherapy alone (13.8 vs 11.1 months) [13]. Importantly, trastuzumab showed no significant concomitant increase in treatment side effects [13], leading to its approval in 2010 in the EU and US for use in combination with 5-FU or capecitabine plus cisplatin for the first-line treatment of patients with HER2-positive metastatic adenocarcinoma of the stomach or GE junction [1,5]. The European Medicines Agency recommended immunohistochemistry (IHC) as the first-line test for HER-2, with patients scoring 3+ eligible for trastuzumab therapy and 2+ cases classified as equivocal and necessitating a confirmatory fluorescence in situ hybridization (FISH) result [14]. The US FDA defined positivity by utilizing the eligibility criteria of the ToGA trial: IHC 3+ or 2+ plus FISH amplification [14]. However, unlike breast cancer [15], accurate and finalized HER-2 scoring criteria for gastric cancer patient selection still remains a subject for debate [16].
Herein we describe the generation and characterization of xenograft mouse models derived directly from patients' gastric adenocarcinoma tissues. Furthermore, we use these novel clinically relevant models to evaluate the current HER-2 scoring system and to explore the opportunity for trastuzumab targeted therapy in gastric carcinoma patients.

Patients and Tissue Samples
Five treatment-naïve gastric adenocarcinoma patient samples were obtained intraoperatively during gastrectomy resection at the Renji Hospital, Shanghai, China. Freshly harvested gastric adenocarcinoma specimens from patient tumors were separated into three parts: the first part was transferred to medium-containing antibiotics immediately after surgical resection under sterile conditions, then transported to an animal facility within 2 h for implantation into immune deficient mice; the second part was snap frozen immediately in liquid nitrogen for DNA/ RNA extraction; and the third part was fixed in formalin and embedded in paraffin for pathological and IHC analysis. All the samples were firstly evaluated by pathologist for quality control purposes. Informed consent was obtained from all patients. This study was approved by the ethics board of Renji Hospital, Shanghai Jiaotong University.

Establishment of Patient Derived Gastric Adenocarcinoma Xenograft (PDGAX) Mouse Models
Eight to 10 week old female nude (nu/nu) mice and severe combined immune deficient (SCID) mice (Vital River, Beijing, China) were used for the generation of xenograft models in this study. Animals were kept in a controlled light-dark cycle (12 h-12 h). The PDGAX mouse models were established from fresh patient gastric adenocarcinoma (GA) tissues surgically resected from GC patients. Patient's GA tissues (F0 tissue) were cut into fragments of approximately 15 mm 3 and implanted subcutaneously via Trocar needle into female SCID mice within 2 h after surgery. The patient tumorengrafted mice were observed daily for 90 days. Once tumors started to grow, subcutaneous caliper measurements were taken weekly. Around 500 mm 3 GA tumors was harvested from each tumor-bearing mice and further implanted in another batch of female nude mice. After three consecutive mouseto-mouse passages, the xenograft was considered to be stable and submitted for full characterization, including histopathological analysis, HER-2 expression by FISH and IHC assays, and mutation detection of AKT1, FGFR4, PIK3CA, PTCH, PTEN and TP53. The tumor specimens in each passage of the tumor-bearing mice were harvested and divided into three parts. The first part was implanted into immune deficient mice for the next generation of the xenograft model; the second part was snap frozen in liquid nitrogen for DNA/RNA extraction; and the third part was fixed in 10 % buffered formalin for 24 h and embedded in paraffin for IHC analysis. Surplus fresh tumor tissues at passage 3-5 were frozen with 20 % FCS in liquid nitrogen for future model recovery. The PDGAX mouse models were maintained in nude mice within ten passages for efficacy studies. All animal experiments were performed in accordance with IACUC-approved guidelines.

H&E Staining
All the xenograft and primary tissues were fixed in 10 % buffered formalin within 30 min after resection. Tissues were processed using a routine procedure after 24 h fixation. Sectioned slides were stained with hematoxylin and eosin, and then reviewed by pathologist to confirm the GA diagnosis.
In the present study, the IHC scoring criteria on human gastric cancer followed Hoffman's criteria [16]: no staining or <10 % tumor cell positive staining was defined as 0/negative; faintly or barely perceptible staining on >10 % tumor cell membrane was defined as 1+; weak to moderate positive staining on >10 % tumor cells was defined as 2+; and cohesive moderate to strong staining on the membrane was defined as 3+.

Statistical Analysis
Statistical significance was evaluated using a one-tailed, twosample Student's t test. P≤0.05 was considered statistically significant.

Establishment of PDGAX Mouse Models
Age, gender and tumor characteristics from five GA patients were collated (Table 1). All five patients' GA samples were viable and led to growth in SCID mice after subcutaneous implantation, and continued to grow in nude mice after the second generation. The growth curves of the five established PDGAX mouse models are shown in Fig. 1. Autopsy examination of all PDGAX tumor-bearing mice at 2-3 months post-implantation revealed no evidence of metastases in the stomach, brain, lung, liver or kidney.

Histology and Tumor Grade
All five PDGAX mouse models and their corresponding patient's GA tissues were classified as gastric adenocarcinoma by pathologist histological evaluation. Similar histological features were observed between each xenograft model and its corresponding patient GA tissue (Fig. 2).

Characterization of HER-2 Protein and Gene Expression in PDGAX Models
Following Hoffman's HER-2 scoring criteria [16], three PDGAX mouse models (PDGAX001, PDGAX003 and PDGAX005) were scored as strongly positive for HER-2 membrane staining (3+), one model (PDGAX002) was scored as moderately positive (2+), and one model (PDGAX004) was scored as HER-2 negative (Fig. 3). According to Hoffman's criteria, the scoring of HER-2 gene copy number determined by FISH assay was 'score 6' (gene amplification) for models PDGAX001 and PDGAX005, 'score 5' for model PDGAX002, and 'score 2' for models PDGAX003 and PDGAX004. However, only model PDGAX005 showed a gene copy number increase with an average copy number of more than 20, whereas the average gene copy numbers of the other models were less than 6.0. In addition, all five PDGAX mouse models and their corresponding patient GA tissues showed a highly similar pattern of HER-2 protein expression and gene copy number (Fig. 3).   (Fig. 5), indicating that pharmacodynamic modulation of pHER2 correlates to trastuzumab response in model PDGAX005.

Gene Mutation Screening
A panel of genes including; AKT1, FGFR4, PIK3CA, PTCH, PTEN, TP53 were screened in all of the PDGAX mouse models and corresponding patient GA tissues. Interestingly, the TP53 mutation was identified in exon 5 from both the PDGAX001 model and the corresponding patient GC tissue. This was subsequently confirmed by direct sequencing (Fig. 6). A mutation (G ->A) was identified which causes an amino acid change from cysteine to tyrosine at the 176th peptide of TP53 protein. The functional meaning of this mutation is unclear. No other mutations were detected in the PDGAX models or their corresponding patient GA tissue.

Discussion
Considering the current economic and ethical issues within modern drug discovery, relevant and predictive animal models of human cancer are immensely important in the search for new therapeutics. Subcutaneous tumor implantation has been the standard methodology in establishing animal models for human cancer research [20,21]. However, standard xenografts make use of permanent tumor cell lines instead of primary tumors, and often have poor predictive value in determining human drug efficacy [22]. PDGAX models more closely resemble primary human tumors in terms of tissue architecture and molecular heterogeneity and thus offer the potential to better predict drug efficacy in humans [22]. To date, few PDGAX mouse models have been successfully established from GA patient tissues. Herein, we have successfully created five PDGAX mouse models by directly implanting fresh GA patient tissues into immune deficient mice. All five PDGAX mouse models and their corresponding . This mutation led to an amino acid change from cysteine to tyrosine at the 176th peptide of TP53 protein patients' gastric cancer tissues displayed a highly similar pattern in HER-2 protein expression and gene copy number. This finding gives further support to the hypothesis that PDGAX mouse models can more accurately represent clinical disease. Furthermore, we have used these PDGAX mouse models to successfully evaluate the efficacy of trastuzumab in gastric adenocarcinoma. An excellent correlation between trastuzumab efficacy and HER-2 expression/ amplification has been shown in advanced gastric and gastroesophageal junction (GEJ) adenocarcinoma patients by the ToGA trials [1] and other previous studies [23]. According to the latest clinical practice guidelines [1,14,24], trastuzumab, in combination with cisplatin and a fluoropyrimidine, is recommended for advanced HER-2 positive GC. For clinicians, accurate and reliable identification of patients eligible for HER-2 targeted therapy is of crucial importance. The algorithm proposed by Hoffmann [16] incorporates a combination of IHC and FISH analyses and is the most appropriate HER-2 scoring system currently in use for the identification of trastuzumab-eligible gastric cancer patients. HER-2 positivity is defined as either IHC positive (3+) or FISH positive (HER-2/CEN-17≥2.2 or HER-2 gene copy number≥6). However, according to the results of the ToGA trial, there is little difference in the proposed algorithm for HER-2 testing in gastric cancer between the FDA in the US and the European Medicines Agency [14]. Furthermore, there is no apparent benefit in patients with IHC 0 to 1+/FISH positive disease, suggesting that an IHC assay may be better suited to the selection of patients for treatment with trastuzumab [14]. In our study, two models (PDGAX001 and PDGAX005) were identified as HER-2 positive by IHC3+ and FISH AMP. Our data demonstrated significant tumor regression after treatment with trastuzumab in model PDGAX005 model (with a higher average gene copy number), but not in PDGAX001 model (with a lower average gene copy number).
To explore the mechanism underlying the difference in trastuzumab anti-tumor efficacy between the two HER-2 IHC 3+ and FISH AMP PDGAX models (PDGAX005 and PDGAX001), molecular genetic studies were performed. After screening gene mutations in AKT1, FGFR4, PIK3CA, PTCH, PTEN and TP53 in the 5 PDGAX mouse models and their corresponding primary tumors, mutation of TP53 was identified in model PDGAX001 and the corresponding patient tissue (Fig. 4 and Table 1). This result suggested that TP53 mutation might represent a mechanism through which trastuzumab resistance may be conferred. Further work is ongoing to explore the mechanism underneath.
In summary, we have established novel xenograft models derived from patient gastric adenocarcinoma tissues and used these clinically relevant animal models to investigate the correlation of trastuzumab anti-tumor efficacy with HER-2 expression. Our results showed that PDGAX models could accurately recapitulate trastuzumab clinical data in the preclinical setting, underscoring their predictive power for future drug discovery.