Gastric Cancer Patient-derived Xenograft Models Research Articles

Abstract 2118: SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor models

Abstract Carcinoembryonic antigen cell adhesion molecule 5, CEACAM5, is a glycosylphosphatidylinositol-anchored glycoprotein expressed on the cell surface of most of the colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) and of more than 2/3 of gastric cancer (GC) and non-small cell lung cancer (NSCLC) while normal tissue expression is limited. The high prevalence of CEACAM5 expression prompted us to develop a novel CEACAM5 topoisomerase I inhibitor (Topo1i) antibody drug conjugate (ADC) with a DAR of 8, SGN-CEACAM5C/SAR445953. The anti-CEACAM5 antibody was chosen based on its high selectivity for CEACAM5 and its potential to direct cytotoxic payloads to tumor. The Topo1i payload was optimized for potency and enhanced bystander activity. SGN-CEACAM5C/SAR445953 is rapidly internalized and demonstrates in vitro cytotoxicity with EC50 values in the sub-nM range while it induces no toxicity on CEACAM5-negative cells. The potent anti-tumor activity is mediated by direct cytotoxicity on CEACAM5-expressing tumor cells and by a strong bystander effect due to the diffusion of the payload to the neighboring CEACAM5-negative tumor cells. Accordingly, the ADC incubated in a co-culture of CEACAM5-positive and -negative cells at a ratio of 1/1 induces a ~50% growth inhibition of CEACAM5-negative cells. Interestingly, with incubated ADC, only 1% of CEACAM5-positive cells in co-culture are sufficient to provide 11% growth inhibition of CEACAM5-negative cells. In vivo, SGN-CEACAM5C/SAR445953 is stable in circulation in SCID mice with a t1/2 close to 15 days. In vivo efficacy at 1, 3 and 10 mg/kg (single administration) was evaluated in panels of 4 CRC, 3 PDAC, 4 NSCLC and 3 GC patient-derived xenograft (PDX) models. At 10 mg/kg, the ADC elicits antitumor regression in 14/14 models. At 3 mg/kg, tumor regression occurs in 12/14 models. This potent, specific and dose dependent anti-tumor activity was further confirmed in Single Mouse Trials (SMT) of 20 CRC PDX models, 31 lung cancer PDX models and 19 gastric cancer PDX models. SMT consists in use of one animal per PDX model per treatment arm and for which the evaluation of efficacy is based on RECIST (Response Evaluation Criteria In Solid Tumors) criteria used in clinic. In CRC, gastric and lung cancer SMT, disease control rates are 95%, 84% and 87%, respectively with overall response rates of 55%, 68% and 71% including 15%, 10% and 26% of complete responses, respectively. The high anti-tumor activity across panels of PDX models of several CEACAM5 positive indications supports further evaluation of SGN-CEACAM5C/SAR445953 in patients with CRC, PDAC, GC and lung cancers (NCT06131840). Citation Format: Yves Baudat, Celine Nicolazzi, Johann Petur Sigurjonsson, Celine Amara, Astrid Clarke, Ryan lyski, Dave Meyer, Valeria Fantin, Marielle Chiron, Stephanie Decary. SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2118.

First-in-Human Study with Preclinical Data of BCL-2/BCL-xL Inhibitor Pelcitoclax in Locally Advanced or Metastatic Solid Tumors.

B-cell lymphoma-extra-large (BCL-xL) regulates apoptosis and is an attractive anticancer therapeutic target. However, BCL-xL inhibition also kills mature platelets, hampering clinical development. Using an innovative prodrug strategy, we have developed pelcitoclax (APG-1252), a potent, dual BCL-2 and BCL-xL inhibitor. Aims of this study were to characterize the antitumor activity and safety of pelcitoclax and explore its underlying mechanisms of action (MOA). Cell line-derived xenograft and patient-derived xenograft (PDX) models were tested to evaluate antitumor activity and elucidate MOA. Subjects (N = 50) with metastatic small-cell lung cancer and other solid tumors received intravenous pelcitoclax once or twice weekly. Primary outcome measures were safety and tolerability; preliminary efficacy (responses every 2 cycles per RECIST version 1.1) represented a secondary endpoint. Pelcitoclax exhibited strong BAX/BAK‒dependent and caspase-mediated antiproliferative and apoptogenic activity in various cancer cell lines. Consistent with cell-based apoptogenic activity, pelcitoclax disrupted BCL-xL:BIM and BCL-xL:PUMA complexes in lung and gastric cancer PDX models. Levels of BCL-xL complexes correlated with tumor growth inhibition by pelcitoclax. Combined with taxanes, pelcitoclax enhanced antitumor activity by downregulating antiapoptotic protein myeloid cell leukemia-1 (MCL-1). Importantly, pelcitoclax was well tolerated and demonstrated preliminary therapeutic efficacy, with overall response and disease control rates of 6.5% and 30.4%, respectively. Most common treatment-related adverse events included transaminase elevations and reduced platelets that were less frequent with a once-weekly schedule. Our data demonstrate that pelcitoclax has antitumor activity and is well tolerated, supporting its further clinical development for human solid tumors, particularly combined with agents that downregulate MCL-1.

Patient-derived xenograft models for gastrointestinal tumors: A single-center retrospective study.

Patient-derived xenograft (PDX) models have shown a great efficiency in preclinical and translational applications. Gastrointestinal (GI) tumors have a strong heterogeneity, and the engraftment rate of PDX models remarkably vary. However, the clinicopathological and molecular characteristics affecting the engraftment rate still remain elusive. A total of 312 fresh tumor tissue samples from patients with GI cancer were implanted into immunodeficient mice. The median follow-up time of patients was 37 months. Patients' characteristics were compared in terms of PDX growth and overall survival. PDX models of 3-6 generations were used for drug evaluation. In total, 171 (54.8%, 171/312) PDX models were established, including 85 PDX models of colorectal cancer, 21 PDX models of esophageal cancer, and 65 PDX models of gastric cancer. Other than tumor site, histology, differentiation degree, and serum alpha-fetoprotein (AFP) level, no significant differences were found between transplantation of xenografts and patients' characteristics. For patients who had undergone neoadjuvant therapy, the incidence of tumor formation was higher in those with progressive disease (PD) or stable disease (SD). In gastric cancer, the results showed a higher transplantation rate in deficient mismatch repair (dMMR) tumors, and Ki-67 could be an important factor affecting the engraftment rate. The gene mutation status of RAS and BRAF, two important molecular markers in colorectal cancer, showed a high degree of consistency between patients' tumors and PDXs. However, no significant effects of these two mutations on PDX engraftment rate were observed. More importantly, in this study although KRAS mutations were detected in two clinical cases, evident tumor inhibition was still observed after cetuximab treatment in both PDX models and patients. A large-scale PDX model including 171 cases was successfully established for GI tumors in our center. The relationship between clinicopathological and molecular features and engraftment rates were clarified. Furthermore, this resource provides us with profound insights into tumor heterogeneity, making these models valuable for PDX-guided treatment decisions, and offering the PDX model as a great tool for personalized treatment and translation research.

Abstract 1143: ATG-022, an antibody-drug conjugate targeting Claudin 18.2, demonstrated potent in vivo efficacy in gastric cancer patient-derived xenografts

Abstract Background: Antibody-drug conjugates (ADCs) have become promising antitumor agents in recent years. ADCs are comprised of a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells, cross-linked to small molecule payloads with cytotoxic activity. Human Claudin 18.2 (CLDN18.2) is overexpressed in a large proportion of gastric and pancreatic cancers, with restricted normal tissue expression. The monoclonal antibody targeting CLDN18.2 (IMAB362) has demonstrated promising clinical benefit in combination with chemotherapies for gastric cancer patients. However, the same antibody shows suboptimal efficacy in patients with low CLDN18.2 expression. Here we report the potent in vivo efficacy of ATG-022, a CLDN18.2 ADC, in multiple gastric cancer patient-derived xenograft (PDX) models, including those with low CLDN18.2 expression. Methods: The binding affinity of ATG-022 with CLDN18.2 was detected by SPR and FACs analysis. The in vitro 50% inhibition concentration (IC50) of ATG-022 was determined in CLDN18.2 positive cell lines using CellTiter-Glo luminescent cell viability assay. In vivo efficacy of ATG-022 was evaluated in a series of gastric cancer PDX models with different expression level of CLDN18.2 The expression level of CLDN18.2 was determined using IHC staining. ATG-022 was dosed twice at 1 mg/kg, 3 mg/kg or 10 mg/kg every two weeks. Results: ATG-022 binds to CLDN18.2 protein with sub-nM affinity. It induced potent in vitro cytotoxicity in CHOK1 cells overexpressing CLDN18.2, with an IC50 of 5-7nM. Bystander killing by ATG-022 was observed. ATG-022 demonstrated potent in vivo antitumor efficacy in a gastric cancer PDX model with high CLDN18.2 expression. Intravenous dosing of 1 mg/kg, 3 mg/kg, and 10 mg/kg ATG-022 induced 35.41%, 71.56% tumor growth inhibition (%TGI) and tumor regression respectively. In a PDX model with low CLDN18.2 expression, 3 mg/kg ATG-022 or 10 mg/kg clinical benchmark CLDN18.2-ADC did not inhibit tumor growth, while 10 mg/kg ATG-022 induced tumor regression. Conclusions: ATG-022 demonstrated potent in vitro and in vivo antitumor effects, with in vivo efficacy observed in CLDN18.2-low expression PDX models, suggesting a promising therapeutic strategy for gastric cancer patients with a broad range of CLDN18.2 expression levels. Citation Format: Peng Chen, Yun Liu, Min Deng, Linjie Tian, Kevin Lynch, Bo Shan, Jay Mei, Bing Hou. ATG-022, an antibody-drug conjugate targeting Claudin 18.2, demonstrated potent in vivo efficacy in gastric cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1143.

From AVATAR Mice to Patients: RC48-ADC Exerted Promising Efficacy in Advanced Gastric Cancer With HER2 Expression.

RC48-ADC is a novel humanized antibody specific for human epidermal growth factor receptor 2 (HER2)in conjugation with a microtubule inhibitor via a cleavable linker. This study was to evaluate the antitumor activity and mechanism of RC48-ADC in gastric cancer (GC) and explore the population that may benefit from RC48-ADC treatment. Four human GC cell lines and nine patient-derived xenograft (PDX) models were exploited to evaluate the antitumor effect of RC48-ADC or trastuzumab treatment in vitro and in vivo. The expression and phosphorylation of HER2 were assessed by immunohistochemistry (IHC) staining. Critical molecules of downstream PI3K/AKT and cell cycle and apoptosis signaling pathways were detected and quantified by immunoblotting. Combined with preliminary results of preclinical research, three patients with IHC3+, IHC2+/FISH+, and IHC2+/FISH- of HER2 were enrolled to verify the efficacy of RC48-ADC treatment in advanced GC. In vitro, RC48-ADC had superior antiproliferative effects in a dose-dependent manner on GC cells, especially on HER2-positive cells. In vivo, RC48-ADC exceeded trastuzumab in GC PDX models with HER2 expression, even in models with moderate to low expression of HER2. Further exploration of mechanism showed that RC48-ADC exerted the antitumor effect by inhibiting phosphorylation of HER2, inducing G2/M phase arrest and cell apoptosis in HER2-expressed PDX models. In clinical practice, RC48-ADC had satisfactory efficacy in HER2-positive and HER2 moderately expressed GC patients and demonstrated promising efficacy in HER2-positive patients who have progressed after anti-HER2 therapy. In conclusion, RC48-ADC exerted promising antitumor activity in HER2-positive as well as score of 2+ in IHC and ISH-negative AGC patients after progression of systematic treatment.

Abstract PS5-40: CDKN2A loss can be a predictive marker of palbociclib in breast and gastric cancer

Abstract Background: Abnormal cell cycle progression is a characteristic of cancer, and targeting the cell cycle is a strategy for cancer treatment. TCGA reported that 7% and 12% of gastric cancers exhibit CCND1 or CCNE1 alterations, respectively. Besides, Cyclin D1 is overexpressed in 25% to 60% of invasive breast carcinomas, and gene amplification is observed in 10% to 30% of breast cancer cases. Furthermore, CDK4/6 and CDNK2A/B aberrations are frequently observed in gastric (39.6%) and breast (7.6%) cancers. The presence of such abnormalities in cell cycle-related molecules suggests that gastric and breast cancers are good candidates for treatment with cell cycle inhibitors. Palbociclib is a specific inhibitor of CDK4/6, a vital regulator of the G1 checkpoint, and has been approved by the FDA because it provided a significant benefit by extending PFS in a phase III trial for hormone-positive advanced breast cancer. However, the predictive marker of palbociclib is not determined. Even though CDKN2A loss has been considered as a sensitive marker of palbociclib, there is no preclinical evidence to support whether CDKN2A deficient cancer shows sensitivity to palbociclib, especially in gastric and breast cancer. Therefore, we investigated the effects of palbociclib on CDKN2A loss gastric and breast cancer cell lines as well as patient derived-xenograft (PDX) models. Methods: The cytotoxic assay, cell cycle analysis, and western blotting were conducted to determine the anti-tumor effect and action mechanisms of palbociclib on gastric and breast cancer cell lines. Moreover, modulation of CDKN2A expression was conducted by siRNA and plasmid overexpression. These in vitro data were validated in vivo model and gastric cancer PDX models which have CDKN2A loss as well. Results: There is a meaningful correlation between CDKN2A loss and palbociclib sensitivity among gastric and breast cancer cell lines. CDKN2A loss cells showed G1 cell cycle arrest by blocking Rb phosphorylation and inhibited proliferative cell signaling. Moreover, palbociclib promoted senescence rather than apoptosis. The depletion of CDKN2A expression using siRNA increased palbociclib sensitivity with G1 cell cycle arrest accompanied by senescence. In contrast, CDKN2A overexpression in sensitive cells showed insensitivity to palbociclib. The anti-tumor effects of palbociclib on CDKN2A loss breast cancer cells were validated in the xenograft model, and the two different gastric cancer PDX models have CDKN2A loss also showed a significant response to palbociclib as well. Conclusions: CDK4/6 inhibitor palbociclib showed an anti-tumor effect in vitro and in vivo xenograft model of CDKN2A loss gastric and breast cancer. Our results suggest that palbociclib has therapeutic potential for the treatment of not only breast cancer but also gastric cancer, not limited to a hormone-positive breast cancer type. Our results provide a rationale for the future clinical trials of palbociclib in the treatment of breast cancers. Citation Format: Ahrum Min, Yu Jin Kim, Miso Lee, Kyung-Hun Lee, Seock-Ah Im. CDKN2A loss can be a predictive marker of palbociclib in breast and gastric cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-40.

Abstract 74: Co-targeting BCL-xL and HER2 high expression to overcome apoptosis blockade in gastric cancer

Abstract Background: Gastric cancer is one of the most prevalent cancers in the East Asia population. The five-year survival rate is approximately 20% globally. The analysis of gene expression data suggests that anti-apoptotic protein BCL-xL may be the oncogenic driver in gastric cancer as its expression levels are much higher than BCL-2 (Pan Cancer Atlas). In this study, we investigated if a clinical stage BCL-2/BCL-xL dual inhibitor APG-1252 would elicit therapeutic activity and associated mechanisms using a panel of gastric cancer patient-derived xenograft (PDX) models. Additionally, we explored if the combination with HER2 inhibition could enhance the antitumor activity in HER2+ gastric cancer models. Methods and Experiments: Eighteen PDX models of gastric cancer that bore high levels of BCL-xL expression (BCL-xLhigh) based on the gene expression profiles were selected for a mouse trial (n=2) with a dosage regimen of APG-1252 (100 mg/kg, BIW, IV) for three weeks. In a combination study (n=5), APG-1252 (50 mg/kg, BIW, IV) and lapatinib (100 mg/kg, QD, PO) were concurrently administered for three weeks. An advanced ELISA (MSD) and Western blot analysis were performed to detect BCL-xL complex and the protein level, respectively, in tumor samples. Results: APG-1252 treatment resulted in tumor growth inhibition (TGI greater than 50%) in four PDXs (20%), including three exhibiting TGI greater than 80%. PD study confirmed that BCL-xL protein levels were high, and bound with pro-apoptotic proteins PUMA (major binding partner) and BIM in the xenograft tumors of these models. Generally, in comparison with hematologic cancer cells which exhibit dominant BCL-2 complexes (i.e., Toledo), the levels of BCL-xL complexes were consistently higher than those of BCL-2 complexes in the gastric cancer PDXs, implicating a crucial role of BCL-xL in the solid tumors. After treatment with APG-1252, BCL-xL:PUMA, were disrupted and, interestingly, such a decrease in BCL-xL complex signal correlated with TGI. Additionally, the baseline levels of BCL-xL complexes also correlated with TGI. Furthermore, a consequent increase in MCL-1 complexes after APG-1252 treatment indicated that such a compensatory mechanism among anti-apoptotic proteins may confer the drug resistance. Finally, in mouse tumor models derived from HER2+ gastric cancer cell line NCI-N87 or patient-derived xenografts (PDXs), combined treatment with AGP-1252 and HER2 inhibitor lapatinib synergistically inhibited tumor growth, suggesting that lapatinib may be able to increase mitochondrial priming, thus sensitizing cancer cells to APG-1252. Conclusion: Our results demonstrate the on-target antitumor activity of APG-1252, the potential of BCL-xLhigh as a predictive biomarker, and the resistance mechanism conferred by MCL-1. Furthermore, the data provide a scientific rationale for the combined therapy with BCL-xL and HER2 inhibitors to achieve better clinical outcomes in a subset of HER2+ gastric cancers. Citation Format: Jing Deng, Xiaojing Huang, Guoqin Zhai, Kaixiang Zhang, Jiaxing Gu, Tingting Mao, Yan Yin, Ran Tao, Douglas D. Fang, Dajun Yang, Yifan Zhai. Co-targeting BCL-xL and HER2 high expression to overcome apoptosis blockade in gastric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 74.

Abstract A005: Clinicopathological factors associated with establishment of gastric cancer PDXs and cell lines

Abstract Background: Patient-derived xenograft (PDX) models has been widely accepted as a suitable preclinical model. However, the number of PDX models as well as cell-lines from gastric cancer (GC) are limited. We started our DEF study project on May 2013 for establishing new GC PDX models and cell lines for accelerating the development of new therapeutics for GC. Methods: Two hundred and fifty (250) patients, including 233 patients underwent gastrectomy and 17 patients received cell-free and concentrated ascites reinfusion therapy (CART) in our hospitals, were enrolled. We subcutaneously engrafted 232 surgically resected gastric cancer tissues into immune-deficient NOG mice. Primary or PDX tumor tissues or ascites from CART patients were cultivated for establishing GC cell lines. Results and Discussion: From 233 surgically resected tumor tissues, we have successfully established 35 gastric cancer PDX models. PDX-establishment rate was 15.1%, and differentiated type adenocarcinomas (DAs) were more effectively established than poorly differentiated type adenocarcinomas (PDAs). The histology of PDX resembled their primary tumors, and the concordance of histological differentiation grade between primary tumors and PDXs was significant (28/35, p<0.01). Twenty-three (23) GC cell lines have been established from surgically resected primary or subsequently transplanted PDX tissues. Another 2 GC lines were established by cultivating primary GC tissues directly. Among 25 GC cell lines, 24 lines could develop subcutaneous tumors (CDXs) in SCID mice. In contrast to PDX, the concordance of histological differentiation grade between primary tumors and CDXs was not significant. In addition, we have also established 7 GC cell lines from ascites from 17 patients received CART, and six of them could develop CDXs. All of the CDXs from CART cases showed PD histology. As the results, we have established both PDX and Cell lines simultaneously from 21 patients, which allowed us to invest a direct histological and molecular comparison between primary, PDX, and CDX tumors. We would also like to discuss about lymphoproliferative lesions, most of which showed proliferation of B cell with various cytological atypia, encountered during the PDX establishment process. Citation Format: Takeshi Kuwata. Clinicopathological factors associated with establishment of gastric cancer PDXs and cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A005. doi:10.1158/1535-7163.TARG-19-A005

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

Abstract 225: TR1801-ADC, an optimized anti cMet PBD ADC with high efficacy in solid tumors of the GI tract and head & neck cancer

Abstract cMet is a well-characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and more recently, antibody drug conjugates (ADCs). However, clinical benefit from c-Met targeted therapy has been limited up to this point. We developed an optimized c-Met targeted antibody drug conjugate TR1801-ADC that utilized optimization at all steps including specificity, stability, internalization, toxin linkers, conjugation site, PK, and in vivo efficacy. The results were a highly potent c-Met ADC that was superior in comparison to a conventional MMAE (monomethylauristatin E) cMet ADC in potency, efficacy and duration of response. TR1801-ADC is site-specifically conjugated to a PBD-toxin linker and has low picomolar activity in multiple cancer cell lines derived from different solid tumors including lung cancer, colorectal cancer and gastric cancer. The potency of our cMet ADC is independent of MET gene copy number and its activity was high not only in high cMet cell lines but also in medium to low cell lines (100,000 to 40,000 cMet/cell). We identified potential Phase 1 cancer indications based on IHC and cMet H-score from tissue micro arrays (TMAs). 3 cancer indications (gastric, colorectal and head & neck cancer) were chosen based on high abundance of high cMet expression (>20% abundance of H-Score higher than 150) and were used to assess the efficacy of our cMet ADC in PDX models. For each indication 10 PDX (patient-derived xenograft) models were chosen based on their cMet expression. TR1801-ADC was administered in all models as single i.v. dose at 1, 0.5, 0.25 and 0.125 mg/kg. Control ADC was administered at 1mg/kg. Tumor growth inhibition varying from >100% to around 40% was seen in all gastric cancer PDX models with single dose injections of 1 mg/kg and 0.5 mg/kg. Complete tumor regression was seen in 70% of the 1 mg/kg group, in 50% of the 0.5 mg/kg group and in 40% of the 0.25 mg/kg group. TR1801-ADC produced statistically significant growth inhibition in comparison to untreated controls in 9 colorectal cancer PDX models. Complete tumor regression was observed in 40% (4/10) PDX models when treated with 1 mg/kg TR1801-ADC. The further 50% showed partial regression (5/10 models) and one showed no significant tumor response. The head & neck cancer PDX models were in general least sensitive to TR1801-ADC. TR1801-ADC produced statistically significant growth inhibition in comparison to untreated controls in 8/10 head & neck cancer PDX models. Complete tumor regression was observed in 30% (3/10) PDX models when treated with 1 mg/kg TR1801-ADC. 50% showed statistical significant partial regression while two models (20%) showed no significant tumor inhibition. Altogether, TR1801-ADC is a novel highly potent cMet ADC that shows very promising efficacy in PDX models of gastric, colorectal and head & neck cancer with long lasting anti-tumor effect with a single dose. Citation Format: Marco Gymnopoulos, Oscar Betancourt, Vincent Blot, Ryo Fujita, Diana Ly, Sophie Nguyen, Jeanette Snedden, Jose Villicana, Jon Wojciak, Eley Wong, Neki Patel, Francois D'Hooge, Balakumar Vijayakrishnan, Conor Barry, John A. Hartley, Phil W. Howard, Roland Newman, Julia Coronella. TR1801-ADC, an optimized anti cMet PBD ADC with high efficacy in solid tumors of the GI tract and head & neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 225.

Abstract 2503: Predicting drug sensitivity to a novel BCL-2 and BCL-xL dual inhibitor in solid tumor PDX trials

Abstract Background: BCL-2, an anti-apoptotic protein and oncogene, has been considered one of the top 10 targets for cancer drug development. The inhibition of BCL-2 and other anti-apoptotic proteins will help to restore the apoptotic pathway. Aiming to target much diverse BCL-2 family protein dependency in solid tumors, we have developed a potent BCL-2 and BCL-xL dual inhibitor APG-1252, which is currently in phase 1 trials for solid tumors in USA, China and Australia. Mechanistically, BCL-2 and BCL-xL protect cells from cell death by sequestering pro-death BH3-only proteins like BIM. Thus, BCL-2 and BCL-xL inhibitors are designed to disrupt the protein complexes and release pro-death proteins to trigger downstream apoptotic cascade. In this study, we intended to further understand the mechanism of action of APG-1252 in vivo and factors regulating its sensitivity. To this end, we conducted trials in SCID mice carrying patient-derived xenograft (PDX) tumors, and studied target engagement. Methods and Experiments: We first selected eleven gastric and esophageal cancer PDX models that bore high levels of BCL-xL (BCL-xLhigh) but normal levels of MCL-1 (MCL-1nor) based on the gene expression profiles and the consideration of that BCL-xL may play a major role in tumorigenesis in solid tumors and MCL-1 a resistant factor for the dual inhibitor. Mice bearing PDX tumors were intravenously treated with 100 mg/kg of APG-1252 twice per week for three weeks. At the end of treatments, tumor samples were collected. An advanced ELISA system (MSD, Meco Scale Discovery) and Western blotting were performed to detect BCL-2 and BCL-xL protein complex and individual proteins. Results: 1. The MSD assays revealed dominant BCL-xL:BIM complex signals in the solid tumor PDX samples in comparison with hematological malignancy cell lines, in which BCL-2:BIM was the major complex. 2. The dual inhibitor treatment disrupted BCL-xL:BIM complex, thus BCL-xL:BIM complex may serves as a pharmacodynamic (PD) marker for target engagement. 3. BCL-2 family protein profiles showed high BCL-xL and MCL-1 levels in the solid tumor PDXs. The treatment with APG-1252 led to a significant increase in BCL-2 protein levels, while there was no major change on pro-apoptotic BH3-only proteins BIM and PUMA. 4. The more efficacious responders to the treatment exhibited a more substantial decrease in BCL-xL:BIM complex signals and low MCL-1 protein levels, whereas a much less significant decrease in BCL-xL:BIM complex and higher MCL-1 levels were found in the poor or non-responders. Conclusion: Both BCL-xL protein and BCL-xL:BIM complex are dominant in the solid tumor PDX tissues. The decrease of BCL-xL:BIM complex indicates the on-target activity after the treatment with APG-1252. Thus, the complex may be used to monitor the target engagement and, potentially, a predictive biomarker for APG-1252 efficacy. These assays may be applied to patient samples to guide the clinical trials. Citation Format: Jing Deng, Xiaojing Huang, Guoqin Zhai, Tingting Mao, Yan Yin, Douglas D. Fang, Dajun Yang, Yifan Zhai. Predicting drug sensitivity to a novel BCL-2 and BCL-xL dual inhibitor in solid tumor PDX trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2503.

Voltage-dependent calcium channel α2δ1 subunit is a specific candidate marker for identifying gastric cancer stem cells.

Purpose: Cancer stem cells (CSCs) are a subpopulation of cancer cells with self-renewal property and responsible for tumor malignancy, progression and drug resistance. Researches on CSC-specific markers in gastric cancer remain limited. Our current study explored the expression of voltage-dependent calcium channel α2δ1 subunit and the potential of using α2δ1 as a CSC marker in gastric cancer. We also compared the specificity of α2δ1 and CD44 in identifying gastric cancer stem cells (GCSCs).Materials and methods: Expression of α2δ1 was analyzed in gastric cancer cell lines, patient-derived xenograft (PDX) models and clinical samples of malignant ascites of gastric cancer patients. α2δ1+ gastric cancer cells were isolated from gastric cancer cell lines. CSC properties of α2δ1+ gastric cancer cells were then verified by subsequent tests both in vitro and in vivo.Results: The expression level of α2δ1 was found to differ drastically among gastric cancer cell lines, PDX models and clinical samples of malignant ascites. α2δ1+ gastric cancer cells sorted from HGC-27 and SGC-7901 cell lines demonstrated significant self-renewal properties, including tumorigenic capacity, sphere-formation capacity and asymmetric differentiation potential. Knockdown of α2δ1 in α2δ1+ HGC-27 significantly inhibited CSC properties and rendered HGC-27 more sensitive to chemotherapy. Flow cytometry showed that α2δ1+ gastric cancer cells accounted for a small fraction of CD44+ gastric cancer cells. Isolated CD44+α2δ1+ HGC-27 cells displayed more significant tumorigenic capacity and sphere-forming capacity compared with their CD44+α2δ1− counterparts.Conclusion: α2δ1+ gastric cancer cells possessed CSC properties. α2δ1 could be a proper marker in identifying GCSCs with superior specificity than CD44. The combination of α2δ1 and CD44 could be used to identify GCSCs with improved accuracy. Knockdown of α2δ1 combined with chemotherapy displayed higher therapeutic efficacy on gastric cancer cells, suggesting that α2δ1 could be a potential target for anticancer treatment.

Abstract 5609: ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, shows antitumor activity in gastric cancer patient-derived xenograft models with varying glypican-3 expression

Abstract Background: ERY974 is a humanized IgG4 bispecific T cell-redirecting antibody (TRAB) currently in a Phase 1 clinical trial (NCT02748837) in patients with solid tumors that are glypican-3 (GPC3)-positive. ERY974 consists of a common light chain but has two different heavy chains that each recognize a different protein, GPC3 or CD3. ERY974 simultaneously binds to GPC3 on the cancer cell surface and to CD3 on the T cell surface to induce cellular cytotoxicity mediated by the potent effector function of T cells. The Fc portion of ERY974 is modified to eliminate FcγR binding and prevent GPC3-independent Fc-mediated effector function. However, binding to FcRn, an important factor in the PK profile of IgG, is maintained. ERY974 shows strong antitumor activity against gastric, lung, ovarian, head & neck, hepatic, and esophageal cancer-derived tumors in a non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mouse model injected with human T cells. In a cohort expansion of the Phase 1 trial, the activity of ERY974 will be examined in gastric cancer; however, individual clinical samples do not show uniform expression levels of GPC3 in tumor lesions, reflecting the heterogenous and complex structure of patients' tumors. To predict the potential efficacy of ERY974 in gastric cancer patients, we examined whether ERY974 alone or in combination with chemotherapy, could show substantial activity against heterogenous tumors with different GPC3 expression profiles using patient-derived xenografts (PDX) of gastric cancer. Method & results: We evaluated the antitumor effect of ERY974 in PDX gastric cancer tumors that have high, moderate, or low expression levels of GPC3 in a NOD-SCID mouse model injected with human T cells that were expanded in vitro using CD3/CD28 beads. The expression of GPC3 was evaluated by immunohistochemistry and quantitative RT-PCR. The efficacy of ERY974 monotherapy seemed to correlate with the GPC3 expression levels in each PDX tumor. The combination of ERY974 and chemotherapy, such as paclitaxcel, cisplatin or capecitabine, showed increased antitumor activity compared with ERY974 or chemotherapy alone. Conclusion: These preclinical data support the possibility that ERY974 alone or in combination with chemotherapy will demonstrate activity in patients with gastric cancer, and the GPC3 expression profile might be a useful predictive biomarker of ERY974 efficacy for patient selection. Citation Format: Azuma Yumiko, Yuji Sano, Toshiaki Tsunenari, Yasuko Kinoshita, Yoko Miyazaki, Junko Shinozuka, Etsuko Fujii, Atsuhiko Kato, Takahiro Ishiguro, Shohei Kishishita, Junichi Nezu, Yoshiki Kawabe, Mika Endo. ERY974, a novel T cell-redirecting bispecific antibody targeting glypican-3, shows antitumor activity in gastric cancer patient-derived xenograft models with varying glypican-3 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5609.

Abstract 1037: Comprehensive molecular profiles of gastric cancer patient derived xenograft (PDX) models and its implication in precision cancer medicine

Abstract Background: Recent cancer research is focused on precision medicine with the advent of next generation sequencing (NGS) technology and patient tumor derived model systems. Here, we present molecular characteristics of gastric cancer patient derived xenograft (PDX) models and explore the potential of molecularly defined PDX model based drug development. Materials and Methods: We generated PDX models from patient tumors with advanced gastric cancer. The genomic alterations of tumors were profiled by whole exome sequencing (WES), RNA sequencing (RNAseq), targeted sequencing, in-situ hybridization (ISH) and immunohistochemistry (IHC). Further, we developed overcoming strategy of chemotherapy resistance mechanism by combination of signaling pathway inhibitor and standard chemotherapy regimen in “N-of-1” PDX trial. Results: Thirty-five PDX models were successfully established and categorized into four subgroups of The Cancer Genome Atlas (TCGA) and Asian Cancer Research Group (ACRG) classifications: EBV (2.9%), MSI (20.6%), GS (23.5%) and CIN (52.9%) subtypes by TCGA while MSI type (11.4%), MSS/EMT type (17.1%), MSS/TP53+ (31.4%) and MSS/TP53- type (40.0%) by ACRG. In the protein levels by IHC, there were 21 cases (60.0%) of any RTK proteins overexpression; nine of HER2 (25.7%), 14 of EGFR (40.0%), and 16 of c-MET (45.7%). Five tumors (14.3%) were related to PTEN loss and 22 tumors (62.9%) showed p53 overexpression or null. Targeted sequencing identified that ERBB2 (25.7%), KRAS (11.4%), and CCND1 (11.4%) were found frequently amplified gene while PIK3CA (11.4%) and CTNNB1 (8.6%) were found most mutated genes. N-of-1 PDX trial demonstrated that the response to FOLFOX in PDX tumor was concordant with that of corresponding patient. In FOLFOX resistant tumors, multiple signaling pathways were up-regulated and inhibition of these signaling pathways was regressed tumor growth. Conclusions: The utilization of molecularly catalogued gastric cancer PDX models will guide precision medicine for cancer therapy and be a useful tool for drug development and repurposing. Citation Format: Jae Eun Lee, Yoon Young Choi, Ju Yeon Lim, Su-Jin Shin, Gunho Lee, Eun Young Kim, Taeil Son, Hyoung-Il Kim, Woo Jin Hyung, Sung Hoon Noh, Hyunki Kim, Minkyu Jung, Sangwoo Kim, Soonmyung Paik, Jae-Ho Cheong. Comprehensive molecular profiles of gastric cancer patient derived xenograft (PDX) models and its implication in precision cancer medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1037.

Abstract 2935: Preclinical efficacy of the p70S6K/AKT dual inhibitor M2698 in combination with trastuzumab in models of gastric cancer

Abstract M2698 is a potent and selective, ATP-competitive dual inhibitor of p70S6K and AKT1/3 that is being evaluated in a phase I clinical trial in cancer patients. Dual pathway inhibition by M2698 may result in improved clinical efficacy by inhibiting downstream pS6 and blocking the increased AKT activity that results from a compensatory feedback loop induced by PI3K/AKT/mTOR (PAM) pathway inhibition. In earlier studies, combination efficacy was observed with M2698 and trastuzumab in HER2+ breast cancer patient derived xenograft (PDX) models, including complete tumor regressions (Huck, BR. et.al. 105th AACR; 2014: Abstract 4516). To explore the potential combination effects in gastric cancer (GC), we evaluated the effect in in vitro and in vivo GC models. Ten human GC cell lines, which included two HER2+ lines, were treated with M2698 and trastuzumab over various concentrations and cellular proliferation was examined with a WST-8 assay. Combination effects were evaluated by Bliss independence scores and highest single agent (HSA) models. The OE-19 HER2+ cell line had the greatest Bliss and HSA sum values, whereas these measures in the other HER2+ cell line, NCI-N87, were among the lowest in the set of 10 lines, indicating that the combination is synergistic in some HER2+ GC cell lines, such as OE-19. The effects of this combination on downstream markers were analysed by Western blotting. Expression of pS6 was reduced in OE-19 cells by treatment with M2698, but pAKT and pERK were both increased. Upregulated pAKT is a known compensatory feedback mechanism that is inhibited by the dual nature of M2698. Upregulated pERK is a candidate resistance marker for PI3K pathway inhibition. However, the combination of trastuzumab with M2698 blocked this increase in pERK, suggesting that dual inhibition of the MAPK and PAM pathways may contribute to the synergistic anti-proliferative effects. The combination was then tested in the OE-19 xenograft model in vivo and significantly inhibited tumor growth compared to vehicle treatment and monotherapies (p<0.05), whereas both monotherapies were not different from vehicle (p>0.05). In an in vivo pharmacodynamic study, the combination of M2698 and trastuzumab inhibited pS6 and pERK showing effective PI3K and MAPK pathway inhibition by the combination. The agents were also tested alone and in combination (n=3/treatment group) in 27 PDX models of GC in mice (ChemPartner, Shanghai, China). The tumor control rate (tumor stasis or regression) was 11% (3/27) of models treated with M2698, 15% (4/27) with trastuzumab treatment and 22% (6/27) with the combination. The PDX models are currently being evaluated for HER2 status to determine the correlation with the efficacy of the treatments. Based on the data from the OE-19 model, pERK is a candidate marker of combination efficacy which can be further explored in additional GC models. Citation Format: Shota Fukuoka, Takashi Kojima, Yoshikatsu Koga, Mayumi Yamauchi, Masahiro Yasunaga, Yasuhiro Matsumura, Kohei Shitara, Toshihiko Doi, Takayuki Yoshino, Toshio Kuronita, Anderson Clark, Brian Elenbaas, Hong Zhang, Atsushi Ohtsu. Preclinical efficacy of the p70S6K/AKT dual inhibitor M2698 in combination with trastuzumab in models of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2935.

Abstract A008: Establishment of novel gastric cancer PDX models and cell lines for developing new therapeutics

Abstract It is essential to have appropriate preclinical models for developing new drugs for gastric cancer (GC). Cell lines have been the most commonly used model for this purpose. However, the numbers of gastric cancer cell lines established and available for this purpose are limited and not enough to deal with variety of gastric cancer molecular subtypes such as recently proposed by The Cancer Genome Atlas (TCGA). In addition, patient-derived xenograft (PDX) models have been considered more appropriate models for evaluating the efficacy of new drugs in vivo. For contributing to develop new drugs as well as to seek more effective biomarkers, we have been conducting an intramural project, called DEF study, for establishing new GC PDXs as well as new GC cell lines. So far, 30 multi-passage-competent new PDXs have been successfully established from surgically removed gastric cancer tissues or ascites. Twenty-two (22) new GC cell lines have also been established from PDX tissue (16 lines) or directly from ascites (6 lines). All GC PDXs have been examined by immunohistochemistry for HER2, EGFR, and Met expressions, and we identified 5 cases overexpressing HER2 (corresponding to Score3+) and 1 case overexpressing both EGFR and MET. Comprehensive gene mutation analysis has been performed in 21 PDX tissues, and repetitive mutations (SNV/Indel) have been observed in ERBB2, ERBB3, BRAF, APC, ARID1A, and p53. Copy number alternations have also been identified; examples include KRAS and ERBB2 for amplification and PTEN and CDKN2A for deletion. Citation Format: Takeshi Kuwata, Kazuyoshi Yanagihara. Establishment of novel gastric cancer PDX models and cell lines for developing new therapeutics [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A008.

Abstract B035: Evaluation of a novel monoclonal antibody targeting ASC amino acid transporter 2 using patient-derived xenograft mouse models of gastric cancer

Abstract Introduction: Gastric cancer is a leading cause of global cancer mortality with an overall 5-year survival rate of approximately 20% and is particularly prevalent in many Asian countries. ASC amino acid transporter 2 (ASCT2), also known as SLC1A5, is a Na+-dependent glutamine/neutral amino acid transporter. ASCT2 acts as a high-affinity transporter of L-glutamine (Gln) and has been reported to be upregulated in a variety of cancerous tissues, including stomach. Several studies have shown that glutamine is a major nutrient contributing to cancer cell growth, so the glutamine metabolism pathway is an attractive target for gastric cancer treatment. In this study, we evaluated the efficacy of a novel anti-ASCT2 humanized monoclonal antibody, KM8094, as a therapeutic agent against gastric cancer and explored predictive biomarker candidates using patient-derived xenograft (PDX) mouse models. Materials and Methods: The tumor tissues from which the PDX models were generated were provided by Dr. Chan Shing Leng (NUS, Singapore). For the glutamine uptake assay, PDX-tumor cells were incubated with 3H-glutamine in the presence of KM8094. Intracellular 3H-glutamine was measured by liquid scintillation spectrophotometry. In vitro ADCC activity against human gastric cancer cells (SNU-16) was evaluated using human peripheral blood mononuclear cells (PBMC) from either gastric cancer patients or healthy controls as effector cells. In vivo antitumor activity of KM8094 was examined using PDX models. Mice were treated with KM8094 at 10 mg/kg i.v. or phosphate-buffered saline once weekly for 2-3 weeks. Finally, omics analyses of PDX tumor tissues were performed via a gene expression array analysis, DNA methylation array analysis, and metabolomics analysis. Results: All five gastric cancer PDX models histologically expressed ASCT2. KM8094 significantly inhibited the 3H-glutamine uptake in vitro and showed in vivo antitumor efficacy in the gastric cancer PDX models. In addition, KM8094 showed in vitro ADCC activity against gastric cancer cells using PBMCs from gastric cancer patients. These results indicated that KM8094 is a potential new therapeutic agent for gastric cancer treatment. Finally, we explored predictive biomarker candidates of KM8094 by a multi-omics analysis on the PDX models, and a few candidates were selected by genomic analyses. In addition, a metabolomics analysis revealed clear differences in the intracellular energy and redox status between responsive and nonresponsive PDX models. Conclusions: These results demonstrated the therapeutic potential of KM8094 for the treatment of gastric cancer and provide some insight into predictive biomarker candidates for antitumor efficacy. Citation Format: Noriyuki Kasai, Aya Sasakawa, Kenta Hosomi, Tze Wei Poh, Bernadette Lynn Chua, Wei Peng Yong, Jimmy So, Shing Leng Chan, Richie Soong, Koji Kono, Toshihiko Ishii, Kazuya Yamano. Evaluation of a novel monoclonal antibody targeting ASC amino acid transporter 2 using patient-derived xenograft mouse models of gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B035.

1639P - Tucatinib, a HER2 selective kinase inhibitor, is active in patient derived xenograft (PDX) models of HER2-amplified colorectal, esophageal and gastric cancers

Background: Tucatinib, an orally bioavailable and HER2 selective small molecule tyrosine kinase inhibitor, is currently being developed for HER2+ metastatic breast cancer in combination with capecitabine + trastuzumab (HER2CLIMB study). In addition to breast cancer, HER2 is amplified in subsets of patients with other malignancies, including gastrointestinal cancers (colorectal, esophageal and gastric cancers). To test whether tucatinib might have utility in treating HER2-amplified cancers originating from the gastrointestinal tract, tucatinib was tested alone, and in combination with trastuzumab, in cell line derived and PDX models of colorectal, esophageal and gastric cancer. Methods:In vitro assays were performed to evaluate the combination of tucatinib and trastuzumab in HER2-amplified cell lines by measuring changes in signal transduction (pHER2, pHER3, pAKT) and cell survival. The in vivo activity of tucatinib (50 mg/kg BID) and trastuzumab (20 mg/kg Q3D) was evaluated alone, or in combination, in established HER2-amplified tumor models, including PDX models of colorectal, esophageal and gastric cancers. Results: As a single agent, or in combination with trastuzumab, tucatinib demonstrated significant anti-tumor activity, including tumor regressions, in the N87 gastric cancer cell line xenograft model and in PDX models of HER2 amplified colorectal, esophageal and gastric cancers. The combination of tucatinib and trastuzumab was consistently more active than either single agent alone, and resulted in tumor growth inhibition from 85-139%, including complete tumor regressions in HER2+ gastric PDX models. Conclusions: The activity of tucatinib in HER2-amplified colorectal, esophageal and gastric tumor xenograft models supports the exploration of using tucatinib to treat HER2+ gastrointestinal cancers in the clinical setting. To this end, an open label phase II clinical study combining tucatinib with trastuzumab in HER2+/RAS wild type metastatic colorectal cancer (MOUNTAINEER) has recently been initiated. Legal entity responsible for the study: Cascadian Therapeutics Funding: Cascadian Therapeutics Disclosure: S. Peterson: Employee and shareholder of Cascadian Therapeutics, corporate officer of Cascadian Therapeutics. P. de Vries, J. Piasecki, R. Rosler: Employee and shareholder of Cascadian Therapeutics

EGFR gene status predicts response and survival benefit in a preclinical gastric cancer trial treating patient‑derived xenografts with cetuximab.

Cetuximab has been evaluated as a first-line treatment with conflicting results. The aim of the present study was to investigate the relationship between epidermal growth factor receptor (EGFR) status, and response and survival benefit following cetuximab treatment in gastric cancer (GC). Using 20patient-derived GC xenograft (PDX) models, the mice (10mice/model) were randomly assigned into two groups. The control group and treatment group were treated with PBS and cetuximab, respectively. The drug response was evaluated by monitoring tumor growth. Survival benefit was evaluated by comparing the survival curves corresponding to the time for the tumors to reach 600mm3. Our results revealed that the PDX models treated with cetuximab had better survival than that noted for the non-treated group (P<0.05). The EGFR status was measured by FISH, qPCR, RNAish and immunohistochemistry, respectively. Four cases in the treated group were identified as responsive to cetuximab. EGFR mRNA and protein overexpression were associated with the response to cetuximab (P<0.05). EGFR amplification, mRNA and protein overexpression were associated with prolonged survival in the cetuximab-treated PDX models. Moreover, in the PDX models with EGFR amplification, mRNA or protein overexpression, cetuximab treatment was associated with a better survival compared with that noted in the untreated group in the PDX models (P<0.05), while the survival was not statistically different in the other cases (P>0.05). In conclusion, cetuximab provided survival benefit in the trial. The level of EGFR amplification and overexpression significantly predicted response and survival benefit, particularly the mRNA and protein expression level. A combination of mRNA and protein expression may predict efficacy of cetuximab more efficiently.