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

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.

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 mm³ and implanted subcutaneously via Trocar needle into female SCID mice within 2 h after surgery. The patient tumor-engrafted mice were observed daily for 90 days. Once tumors started to grow, subcutaneous caliper measurements were taken weekly. Around 500 mm³ GA tumors was harvested from each tumor-bearing mice and further implanted in another batch of female nude mice. After three consecutive mouse-to-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.

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.

All incubations were performed at room temperature and all washing steps were performed with TBST. Tissues sectioned at 4 μm were firstly dewaxed and rehydrated. Antigen retrieval was performed using a pressure cooker at 110 °C for 5 min in retrieval buffer (S2367, DAKO). Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide (S2023, DAKO). Sections were then incubated with HER-2 antibody (Herceptest^TM, DAKO) for 30 min and phospho-HER2 (pHER2) antibody (1:600, Epitomics 2521–1) for 60 min, respectively. Finally, immunocomplexes were detected by incubation with Envision system (DAKO K5204) for 30 min and diaminobenzidine (K3468, DAKO) for 10 min.

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 + .

HER2/CEP17 FISH probes were obtained from Vysis (Cat #30-161060) and FISH assays were performed as per the manufacturer’s instructions. In brief, TMA sections were dewaxed, dehydrated, and 10 μl HER2/CEP17 probes were applied. Sections were codenaturated together with probes at 75 °C for 4 min, then incubated overnight at 37 °C. Subsequently, probes were washed off twice sequentially with 0.3 % NP40/1 × SSC at 75.5 °C for 5 min, 2 × SSC at room temperature for 2 min, and then dehydrated and air-dried. Sections were mounted in 0.3 μg/ml DAPI mounting medium (diluted from Vector, Cat #H-1200), and stored at 4 °C (avoiding light) for at least 30 min prior to scoring. In each case, 50 tumor nuclei were evaluated. Intact interphase tumor nuclei identified by DAPI staining were evaluated, and gene (red signal) and CEP17 (green signal) copy numbers in each nucleus were assessed. An absolute HER-2 gene copy number lower than four or an HER-2/CEP17 ratio of less than 1.8 was considered HER-2 negative. HER-2 copy number between 4 and 6, or a HER-2/CEP17 ratio between 1.8 and 2.0 was considered HER-2 equivocal. Cases showing a tight gene cluster of HER-2 signals, an average gene copy number of HER-2 > 6, or a HER-2/CEP17 fluorescence ratio ≥ 2 were considered positive for gene amplification. The above criteria was based on the Hoffmann criteria in GC [16] and other investigations [17, 18].

For therapeutic experiments, suitable tumor-bearing, HER-2 positive mice in the tumor volume range of 150 to 250 mm³ were sorted randomly (8 animals per group) and assigned to vehicle control or trastuzumab treatment groups (Roche, China, 15 mg/kg/twice weekly intraperitoneally). Nude mouse subcutaneous tumor volumes and body weights were measured twice per week. Tumor volume was calculated from two perpendicular diameters using the formula: V = (length + [width]²) /2. The calculated volumes were standardized to the values at the first treatment day. Percentage of tumor growth inhibition (%TGI) was calculated with the formula [1 – (change of tumor volume in treatment group/change of tumor volume in control group)] × 100, which was used for the evaluation of anti-tumor efficacy.

Formalin-fixed paraffin-embedded tumor blocks were reviewed for quality and tumor content. A single representative block, containing both PDGAX mouse model and the corresponding patient GA sample (each containing at least 70 % of neoplastic cells) was screened for gene mutations. Genomic DNA was extracted using the QIAamp Mini kit (Qiagen) as per the manufacturer’s instructions. ‘Hot spot’ mutations in AKT1 (exon 18, 19, 20, 21), FGFR4 (exon 2 and 3), PIK3CA (exon 10 and 15), PTCH (exon 1, 2), PTEN (exon 1,2) and TP53 genes (exon 5 and 7) were screened using amplification refractory mutation system (ARMS, Amoy diagnostics, Fujian, China) and mutant-enriched liquid chip PCR method (SurExam Bio-Tech, Guangzhou, China) [19].

Statistical significance was evaluated using a one-tailed, two-sample Student’s t test. P ≤ 0.05 was considered statistically significant.