Inhibitation Of Xenograft Models Tumour Growth By Herceptin In Patient Derived Esophageal Squamous Carcinoma

Part I Establishment of primary EC xenograft modelsObjective:Establishment of Patient Derived Esophageal Carcinoma Xenograft Model.Methods:Harvested 106 fresh EC specimen, every fresh EC speciman was separated into three parts:first part was put into medium containing antibiotics immediately after surgical resection under sterile conditions and transported to animal facility within 2 hours for implantation into immunodeficient mice; second part of tumor was snap frozen in liquid nitrogen immediately for DNA/RNA extraction; and third part of tumor was fixed in formalin and embedded into paraffin (FFPE) for H&E and IHC analysis.8-10-week-old male nude (nu/nu) mice (Vital River, Beijing, China) were used in this study. All experiments with nude mice were performed in accordance with the guidelines approved by IACUC. The human primary EC mouse models were established with fresh EC tissues harvested from EC patient during surgery. In brief, surgically removed EC tissues (FO tissue) were cut into~2 mm3 fragment and implanted via Trocar needle into nude mice subcutaneously within 2 hours after the surgery. The xenograft tissues subsequently obtained from the mice implanted with FO tissue if the patient's EC tissue could grow up in nude mice within three months, were further implanted and expanded in male nude mice. When the xenograft tumors reached size 500-1000 mm3, tumors were excised, cut into~10 mm3 fragments and implanted into nude mice for growth of next generation. Xenograft tumors in certain passage were harvested and cut into two pieces; one was snap frozen at -80℃for DNA/RNA extraction, whilst the other was fixed in 10% formalin buffer for 24 hours and embedded in paraffin (FFPE) for immunohistochemical (IHC) analysis. Larger amount of fresh tumor fragments between passage 3 and 5 were frozen in liquid nitrogen for further model recovery.Results:successed 57cases and failed 32 caess in 106 fresh EC specimen,46cases implanted F5, implanted success ration43.4%,2 model successfully established from 3 fresh tumor fragments were frozen in liquid nitrogen recovery, implanted success ration 66.7%.Conclusions:Patient Derived Esophageal Carcinoma Xenograft Model could established and continue application.PartⅡImmunohistochemistry assay to detect Her-2 expressObjective:To detect her-2 expression in esophageal carcinoma and adjacent tissue.Methods:All the EC primary xenograft tumor were harvested and fixed in 10% buffered formalin within 30min. Tissues are processed following the routine procedure after 24h fixation. Sectioned slides stained with Hematoxylin and eosin were reviewed by the pathologist to confirm EC diagnosis. squamous-celled carcinoma 97, adenocarcinoma 0, adenosquamous Carcinoma 3, Neuroendocrine carcinoma 3, squamous-celled carcinoma and part of Neuroendocrine carcinoma 3.Dual core from each case were made on tissue microarray (TMA) for immunohistochemistry (IHC) staining.IHC detect her-2 expression in esophageal carcinoma and adjacent tissue.Results:No her-2 positive staining was observed in non-tumor tissues, HER2 IHC++or+++staining was observed in 19/99 (19.2%) EC tumor samples, HER2 IHC+staining was observed in 13/99 (13.1%) EC tumor samples, HER2 IHC negative staining was observed in 67/99 (67.7%) EC tumor samples.8 and 11 EC tumor samples were tumor gradeⅡand .1,13 and 5 EC tumor samples were clinical stageⅠ,ⅡandⅢ.Conclusions:HER-2 positive expression was lower in esophageal carcinoma and was corporation with malignant of esophageal carcinoma.PartⅢFISH assay to detect Her2 amplificationObjective:To detect Her2 amplification in esophageal carcinoma tissue and to analysis the relation between Her2 amplification and her2 expressionMethods:Dual core from each case were made on tissue microarray (TMA), The tissue microarray was cut at 4μm thickness for FISH assay. FISH to detect Her2 amplification in esophageal carcinoma tissue. FISH interpretation was done using Olympus BX61 fluorescent microscope under 100x Objective and 50 tumour cells were scored for each case. HER2 FISH amplification is defined as ratio of HER2/CEP17 is>2Results:Ratio of HER2/CEP17 is>2 was observed in 6/99 (6.1%) EC tumor samples,2 and 4 EC tumor samples were tumor gradeⅡandⅢ.4 and 2 EC tumor samples were clinical stageⅡandⅢ.Conclusions:HER-2 amplification was lower in esophageal carcinoma and consistent with Her-2 over-expression and was corporation with malignant of esophageal carcinoma. Part4 Anti-tumor activity (efficacy) of Herceptin and Chemotherapy in primary EC modelsObjective:TO evaluate anti-tumor activity of Herceptin in primary EC models and correlation with her-2 expression and amplification.Methods:Based on Her2 expression data, three primary EC xenograft modelsEC004, EC016 and EC044 were selected to test efficacy of Herceptin and 5FU/Cisplatin, which was one of the standard cares for EC patients in clinic. For the efficacy study, seven tumor-bearing mice were randomized into vehicle control and Hercepin or 5FU/Cisplatin treatment groups. The treatment with tolerable and efficacious dose of Hercepin (15 mg/kg, twice a week, ip), and 5FU/Cisplatin (5FU:10 mg/kg/ip/qd,5 day-on, 2 day-off weekly; Cisplatin:5 mg/kg/iv, once a week) was initiated at tumor volume 150~250 mm3. Tumor size was measured in two dimensions twice weekly with caliper. Tumor volumes (V) calculated by the formula:V= (length+[width]2)/2 and related to the values at the first treatment day. Percentage of tumor growth inhibition. The body weight of tumor-bearing mice was measured twice weekly and the change in body weight was recorded as variable of tolerability. Percentage of tumor growth inhibition (%TGI=…) was used for the evaluation of anti-tumor efficacy.Results:EC004,44% TGI by Herceptin in this lower Her2 expression EC model, 50% TGI by SoC in the EC primary model. EC016, No efficacy of Herceptin was observed in this lower Her2 expression EC model.47% TGI by SoC in the EC primary model. EC044,123% TGI by Herceptin in this lower Her2 expression EC model,29% TGI by SoC in the EC primary model.Conclusions:The clinical use of trastuzumab for HER-2-overexpressing esophageal cancer, which is a significant fraction of the patient population.