Data from A Comprehensive PDX Gastric Cancer Collection Captures Cancer Cell–Intrinsic Transcriptional MSI Traits

<div>Abstract<p>Gastric cancer is the world's third leading cause of cancer mortality. In spite of significant therapeutic improvements, the clinical outcome for patients with advanced gastric cancer is poor; thus, the identification and validation of novel targets is extremely important from a clinical point of view. We generated a wide, multilevel platform of gastric cancer models, comprising 100 patient-derived xenografts (PDX), primary cell lines, and organoids. Samples were classified according to their histology, microsatellite stability, Epstein–Barr virus status, and molecular profile. This PDX platform is the widest in an academic institution, and it includes all the gastric cancer histologic and molecular types identified by The Cancer Genome Atlas. PDX histopathologic features were consistent with those of patients' primary tumors and were maintained throughout passages in mice. Factors modulating grafting rate were histology, TNM stage, copy number gain of tyrosine kinases/<i>KRAS</i> genes, and microsatellite stability status. PDX and PDX-derived cells/organoids demonstrated potential usefulness to study targeted therapy response. Finally, PDX transcriptomic analysis identified a cancer cell–intrinsic microsatellite instability (MSI) signature, which was efficiently exported to gastric cancer, allowing the identification, among microsatellite stable (MSS) patients, of a subset of MSI-like tumors with common molecular aspects and significant better prognosis. In conclusion, we generated a wide gastric cancer PDX platform, whose exploitation will help identify and validate novel “druggable” targets and optimize therapeutic strategies. Moreover, transcriptomic analysis of gastric cancer PDXs allowed the identification of a cancer cell–intrinsic MSI signature, recognizing a subset of MSS patients with MSI transcriptional traits, endowed with better prognosis.</p>Significance:<p>This study reports a multilevel platform of gastric cancer PDXs and identifies a MSI gastric signature that could contribute to the advancement of precision medicine in gastric cancer.</p></div>

Gastric cancer is the world's third leading cause of cancer mortality. In spite of significant therapeutic improvements, the clinical outcome for patients with advanced gastric cancer is poor; thus, the identification and validation of novel targets is extremely important from a clinical point of view. We generated a wide, multilevel platform of gastric cancer models, comprising 100 patient-derived xenografts (PDX), primary cell lines, and organoids. Samples were classified according to their histology, microsatellite stability, Epstein-Barr virus status, and molecular profile. This PDX platform is the widest in an academic institution, and it includes all the gastric cancer histologic and molecular types identified by The Cancer Genome Atlas. PDX histopathologic features were consistent with those of patients' primary tumors and were maintained throughout passages in mice. Factors modulating grafting rate were histology, TNM stage, copy number gain of tyrosine kinases/KRAS genes, and microsatellite stability status. PDX and PDX-derived cells/organoids demonstrated potential usefulness to study targeted therapy response. Finally, PDX transcriptomic analysis identified a cancer cell-intrinsic microsatellite instability (MSI) signature, which was efficiently exported to gastric cancer, allowing the identification, among microsatellite stable (MSS) patients, of a subset of MSI-like tumors with common molecular aspects and significant better prognosis. In conclusion, we generated a wide gastric cancer PDX platform, whose exploitation will help identify and validate novel "druggable" targets and optimize therapeutic strategies. Moreover, transcriptomic analysis of gastric cancer PDXs allowed the identification of a cancer cell-intrinsic MSI signature, recognizing a subset of MSS patients with MSI transcriptional traits, endowed with better prognosis. SIGNIFICANCE: This study reports a multilevel platform of gastric cancer PDXs and identifies a MSI gastric signature that could contribute to the advancement of precision medicine in gastric cancer.

Understanding mechanisms of primary resistance to checkpoint inhibitors will lead to precision immunotherapy of advanced gastric cancer

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.

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

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.

SummaryBackgroundResponse to immunotherapy in gastric cancer is associated with microsatellite instability (or mismatch repair deficiency) and Epstein-Barr virus (EBV) positivity. We therefore aimed to develop and validate deep learning-based classifiers to detect microsatellite instability and EBV status from routine histology slides.MethodsIn this retrospective, multicentre study, we collected tissue samples from ten cohorts of patients with gastric cancer from seven countries (South Korea, Switzerland, Japan, Italy, Germany, the UK and the USA). We trained a deep learning-based classifier to detect microsatellite instability and EBV positivity from digitised, haematoxylin and eosin stained resection slides without annotating tumour containing regions. The performance of the classifier was assessed by within-cohort cross-validation in all ten cohorts and by external validation, for which we split the cohorts into a five-cohort training dataset and a five-cohort test dataset. We measured the area under the receiver operating curve (AUROC) for detection of microsatellite instability and EBV status. Microsatellite instability and EBV status were determined to be detectable if the lower bound of the 95% CI for the AUROC was above 0·5.FindingsAcross the ten cohorts, our analysis included 2823 patients with known microsatellite instability status and 2685 patients with known EBV status. In the within-cohort cross-validation, the deep learning-based classifier could detect microsatellite instability status in nine of ten cohorts, with AUROCs ranging from 0·597 (95% CI 0·522–0·737) to 0·836 (0·795–0·880) and EBV status in five of eight cohorts, with AUROCs ranging from 0·819 (0·752–0·841) to 0·897 (0·513–0·966). Training a classifier on the pooled training dataset and testing it on the five remaining cohorts resulted in high classification performance with AUROCs ranging from 0·723 (95% CI 0·676–0·794) to 0·863 (0·747–0·969) for detection of microsatellite instability and from 0·672 (0·403–0·989) to 0·859 (0·823–0·919) for detection of EBV status.InterpretationClassifiers became increasingly robust when trained on pooled cohorts. After prospective validation, this deep learning-based tissue classification system could be used as an inexpensive predictive biomarker for immunotherapy in gastric cancer.FundingGerman Cancer Aid and German Federal Ministry of Health.

Introduction: Gastric cancer is common in Asia and Eastern Asia where more than half of the world's cases arise. As is the case for Caucasian patients, amplification and overexpression of the HER2 gene is a critical event in the tumorigenesis of gastric cancer and is present in 10 to 20% of Asian patients. Trastuzumab has been approved for treatment of HER2-positive gastric cancer. However, primary and secondary resistance to Trastuzumab is a significant problem and new strategies to overcome resistance are needed. Models which recapitulate this differential response aid the development of new therapies targeting HER2. We recently enlarged our collection of gastric cancer Patient Derived Xenografts (PDX), which in the past focused on Caucasian patients, by developing PDX from Eastern Asian patients. In this study we investigated whether the newly developed PDX collection is reflective of the clinical situation and contains HER2 amplified/overexpressing tumors. We evaluated the sensitivity to Trastuzumab treatment of gastric cancer PDX which are HER2 amplified/overexpressing and searched for possible additional molecular determinants of sensitivity. Material and Methods: Gastric tumors were xenografted in nude mice and were characterized by Affymetrix SNP V6.0 array and qPCR for gene copy number variation, Sanger sequencing and Sequenom MassARRAY OncoCarta panels 1, 2 and 3 for mutations, Affymetrix HGU133 plus 2.0 arrays for gene expression and using immunohistochemistry (IHC) for protein expression. Response to HER2-targeted therapy was assessed in vivo by treating gastric PDX with Trastuzumab 10 mg/kg/day at days 7, 14, and 21. Results: A total of 29 gastric cancer PDX were established (7 PDX of Caucasian and 22 of Eastern Asian origin). Among these 29 PDX, 1 PDX from a Caucasian patient and 4 from Eastern Asian patients expressed high amounts of HER2 mRNA due to gene amplification that ranged from 10 to >50 copies. IHC in situ analyses revealed that the HER2 amplification/mRNA overexpression correlated with strong HER2 protein expression (score 3+). Ongoing analyses investigating these HER2-amplified/overexpressing PDX for sensitivity to Trastuzumab revealed one PDX sensitive and one PDX resistant to treatment. We will present analyses of key biomarkers such as NRAS/KRAS/BRAF mutations, PIK3CA/PTEN status, HER2 integrity, gene expression profiles and their correlation with sensitivity/resistance to trastuzumab. Conclusion: In agreement with what is observed in clinical practice, we identified Caucasian and Eastern Asian gastric tumors amplified and overexpressing HER2. The PDX from these tumors allowed the investigation of response to therapy targeting HER2. These PDX will aid in testing new HER2 targeted treatments and in identifying potential molecular determinants of resistance to Trastuzumab and other HER2 targeting agents. Citation Format: Vincent Vuaroqueaux, Andreas Ackermann, Jianing Guo, Anne-Lise Peille, Rebekka Krumbach, Frederic Foucault, Thomas Metz, Heinz-Herbert Fiebig. The molecular determinants of sensitivity to HER2 targeted therapy in Patient Derived Xenograft gastric tumor models from Caucasian and Eastern Asian patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2774. doi:10.1158/1538-7445.AM2013-2774

Gastric cancer is the world third leading cause of cancer mortality. In spite of the significant therapeutic advances, the overall clinical outcome for patients with advanced gastric cancer is poor, with 5-20% 5-year survival. The only targeted therapy approved so far are trastuzumab, and Ramucirumab which have given unsatisfactory results. Around 50% of gastric tumors bear genetic alterations affecting tyrosine kinase pathways (mainly EGFR, HER3, FGFR2 and MET pathways, besides HER2) but their clinical validation as tumor drivers is missing. The need for new therapeutic options and the possible presence of ‘druggable’ targets prompted us to investigate potential targeted therapies for this disease. Our project aims at identifying and validating targeted therapeutic strategies in gastric cancer, through the generation of a platform of gastric tumor patient-derived xenografts (PDXs), animal models in which tumor surgical specimens are directly transferred into mice. Upon engraftment, the tumor is split and re-implanted in a cohort of mice, allowing the simultaneous testing of different drugs on the same tumor. Thanks to the establishment of a network of 15 Italian centers for samples collection, we generated around 80 gastric PDXs and successfully derived cell lines and organoids from engrafted tumors. Among the tumors collected so far, we found HER2, EGFR, FGFR2, MET and KRAS amplifications. This gastric PDX platform will be exploited for: 1) Validation of candidate oncogenes as relevant targets and identification of efficient therapeutic strategies 2) Identification of novel molecular targets; 3) identification of genetic predictors of response/resistance. In the PDX platform we identified one tumor bearing a high level of MET gene amplification (26 gene copies). We thus performed a preclinical study on a cohort of patient-derived xenografts generated from the MET-amplified gastroesophageal tumor. Despite the high amplification level, MET inhibitors induced only a partial response, while the combined anti-MET/EGFR treatment led to complete tumor regression. Most important, the combo treatment also prevented resistance onset. This data represent the proof of concept that a combined anti-MET/EGFR therapy can be more effective than anti-MET treatment alone in MET-amplified gastroesophageal tumors, in the absence of EGFR genetic lesions. Citation Format: Silvia Menegon, Maria Apicella, Cristina Migliore, Tania Capeloa, Marilisa Cargnelutti, Maurizio Degiuli, Anna Sapino, Paola Cassoni, Michele De Simone, Paolo M. Comoglio, Silvia Marsoni, Simona Corso, Silvia Giordano. Gastric cancer in the age of targeted agents: identification and validation of novel therapeutic strategies through the generation of a patient-derived xenografts platform. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 622.

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.

Varlitinib is a potent, reversible pan-HER inhibitor of the ATP-binding site of EGFR (IC50 = 7 nM), HER2 (IC50 = 2 nM) and HER4 (IC50 = 4 nM). Gastric Cancer is the fifth most common cancer worldwide, with 952,000 cases diagnosed in 2012, constituting 6.8% of total cancer diagnosis. Prognosis for gastric cancer is poor, with the overall 5-year survival rate for gastric cancer in the US at ∼29%. Current clinical guidelines recommend use of trastuzumab combined with chemotherapy for HER2+ (amplified) patients with advanced gastric cancer. However the majority of patients (∼80%) do not have HER2 amplification. Studies have implicated dysregulation of HER signaling in gastric cancer and there is strong scientific rationale for testing of pan-HER therapies in Gastric Cancer. We tested the anti-tumour activity of Varlitinib as a single agent in patient-derived gastric cancer xenograft in SCID mice (GC22-0808 & GC11-0414) with over-expression of HER proteins. Varlitinib is very active in tumour growth inhibition studies, causing dose-dependent inhibition with tumour stasis observed at dosing of 50 mg/kg BID and tumour regression observed at dosing of 100 mg/kg BID. Tumour protein lysates after two-day treatment with Varlitinib and vehicle were also analysed to elucidate the mechanism of action. Multiple proliferation and anti-apoptosis pathways were inhibited by Varlitinib, including AKT, PI3K and Survivin pathways. In view of the robust anti-tumour activity inhibiting multiple survival and proliferation pathways, as well as the safe tolerabilty profile of Varlitinib, ASLAN has initiated Phase 2 clinical trials of Varlitinib in gastric cancer. Citation Format: A. G. Lisa Ooi, Bertil E. Lindmark, Mark McHale, Huynh T. Hung, Richard Ong. Varlitinib demonstrates potent antitumor efficacy in patient-derived gastric cancer xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4719.

BackgroundGastric cancer is the sixth most common cancer and second-leading cause of cancer-related mortality worldwide.1 The heterogenous and genetically complex nature of this disease underlies the challenges in developing effective...

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.

Patient-derived xenograft (PDX) models have been recognized as being more suitable for predicting therapeutic efficacy than cell-culture models. However, there are several limitations in applying PDX models in preclinical studies, including their availability—especially for cancers such as gastric cancer—that are not frequently encountered in Western countries. In addition, the differences in morphology between primary, PDX, and tumor cell line-derived xenograft (CDX) models have not been well established. In this study, we aimed to establish a series of gastric cancer PDXs and cell-lines from a relatively large number of gastric cancer patients. We also investigated the clinicopathological factors associated with the establishment of PDX and CDX models, and compared the histology between the primary tumor, PDX, and CDX that originated from the same patient. We engrafted 232 gastric cancer tissues into immune-deficient mice subcutaneously and successfully established 35 gastric cancer PDX models (15.1% success rate). Differentiated type adenocarcinomas (DAs, 19.4%) were more effectively established than poorly differentiated type adenocarcinomas (PDAs, 10.8%). For establishing CDXs, the success rate was less influenced by histological differentiation grade (DA vs. PDA, 12.1% vs. 9.8%). In addition, concordance of histological differentiation grade between primary tumors and PDXs was significant (p < 0.01), while concordance between primary tumors and CDXs was not. Among clinicopathological factors investigated, pathological nodal metastasis status (pN) was significantly associated with the success rate of PDX establishment. Although establishing cell lines from ascites fluid was more efficient (41.2%, 7/17) than resected tissues, it should be noted that all CDXs from ascites fluid had the PDA phenotype. In conclusion, we established 35 PDX and 32 CDX models from 249 gastric cancer patients; among them, 21 PDX/CDX models were established from the same patients. Our findings may provide helpful insights for establishing PDX and CDX models not only from gastric but from other cancer types, as well as select preclinical models for developing new therapeutics.

Objective: The optimal preclinical models in new targeted drug development are patient-derived tumor xenografts (PDXs) models with definite molecular signatures, which will be excavated and validated in this study. Methods: The genomic profiles of 50 PDXs from advanced gastric cancer (AGC) were analyzed by targeted next-generation sequencing. The Epstein-Barr virus (EBV) infection, microsatellite instability (MSI) status, and expressions of EGFR, HER3, c-Met, PTEN and c-Myc were determined by in situ hybridization and immunohistochemisty, respectively. Results: A total of 581 non-synonymous single nucleotide variations (SNV), 513 Indels, and 225 amplifications were identified in 50 PDXs. Pathway analysis revealed recurrent alterations in the MAPK signaling, ErbB signaling, VEGF signaling, mTOR signaling and cell cycle pathway. The ErbB family amplified in 8% (EGFR), 10% (ERBB2), 2% (ERBB3) and 2% (ERBB4) of the 50 PDXs respectively. Meanwhile, several activating mutations in EGFR (L858R and G719S), ERBB3 (P1212S) and ERBB4 (R1174Q) were also detected in our study. Moreover, we detected 7 PDXs harboring PIK3CA activating mutations (E545K, Q546K, H1047R) and 2 PDXs with PTEN loss. Whether PI3K/Akt/mTOR inhibitors could turn over the primary or acquired resistance of anti-HER2 therapies remains further researches. Genes in the cell cycle pathway altered in 70% of the 50 PDXs. The mutation rates of TP53, BRCA1 and BRCA2 were 28%, 8% and 14%. We also detected amplifications of CCNE1, CCND1 and MYC in 16%, 6% and 20% of the xenografts. In this study, 9 PDXs (18%) were EBV positive, with frequent alterations of ARID1A but rare mutations of TP53, and a robust expression of PD-L1. However, we didn’t observe any loss of MMR protein expressions. Conclusions: Several potential therapeutic targets were identified based on the molecular signature of PDXs in advanced gastric cancer, which will provide evidences for drug development. Citation Format: Zuhua Chen, Jing Gao, Lin Shen. Excavation of therapeutic targets based on the molecular signature of patient-derived tumor xenografts in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3855. doi:10.1158/1538-7445.AM2017-3855