Gastric Cancer Patient-derived Xenograft Research Articles

HER2, MET and FGFR2 oncogenic driver alterations define distinct molecular segments for targeted therapies in gastric carcinoma

Background:Gastric cancer (GC) is a leading cause of cancer deaths worldwide. Since the approval of trastuzumab, targeted therapies are emerging as promising treatment options for the disease. This study aimed to explore the molecular segmentation of several known therapeutics targets, human epidermal growth factor receptor 2 (HER2), MET and fibroblast growth factor receptor 2 (FGFR2), within GC using clinically approved or investigational kits and scoring criteria. Knowledge of how these markers are segmented in the same cohort of GC patients could improve future clinical trial designs.Methods:Using immunohistochemistry (IHC) and FISH methods, overexpression and amplification of HER2, FGFR2 and MET were profiled in a cohort of Chinese GC samples. The correlations between anti-tumour sensitivity and the molecular segments of HER2, MET and FGFR2 alterations were further tested in a panel of GC cell lines and the patient-derived GC xenograft (PDGCX) model using the targeted inhibitors.Results:Of 172 GC patients, positivity for HER2, MET and FGFR2 alternations was found in 23 (13.4%), 21 (12.2%) and 9 (5.2%) patients, respectively. Positivity for MET was found in 3 of 23 HER2-positive GC patients. Co-positivity for FGFR2 and MET was found in 1 GC patient, and amplification of the two genes was found in different tumour cells. Our study in a panel of GC cell lines showed that in most cell lines, amplification or high expression of a particular molecular marker was mutually exclusive and in vitro sensitivity to the targeted agents lapatinib, PD173074 and crizotinib was only observed in cell lines with the corresponding high expression of the drugs' target protein. SGC031, an MET-positive PDGCX mouse model, responded to crizotinib but not to lapatinib or PD173074.Conclusions:Human epidermal growth factor receptor 2, MET and FGFR2 oncogenic driver alterations (gene amplification and overexpression) occur in three largely distinct molecular segments in GC. A significant proportion of HER2-negative patients may potentially benefit from MET- or FGFR2-targeted therapies.

A subset of gastric cancers with EGFR amplification and overexpression respond to cetuximab therapy

A preclinical trial identified 4 of 20 (20%) gastric cancer (GC) patient-derived xenografts responded to cetuximab. Genome-wide profiling and additional investigations revealed that high EGFR mRNA expression and immunohistochemistry score (3+) are associated with tumor growth inhibition. Furthermore, EGFR amplification were observed in 2/4 (50%) responders with average copy number 5.8 and >15 respectively. Our data suggest that a GC subtype with EGFR amplification and overexpression benefit from cetuximab treatment.

The AKT inhibitor AZD5363 is selectively active in PI3KCA mutant gastric cancer, and sensitizes a patient-derived gastric cancer xenograft model with PTEN loss to Taxotere

IntroductionActivation of the PI3K/AKT pathway is a common phenomenon in cancer due to multiple mechanisms, including mutation of PI3KCA, loss or mutation of PTEN, or over-expression of receptor tyrosine kinases. We recently developed a novel AKT kinase inhibitor, AZD5363, and demonstrated that HGC27, a cell line harboring both PI3KCA mutation and PTEN loss, displayed the greatest sensitivity to this AKT inhibitor in vitro and in vivo.Case preparationTo further elucidate the correlation between AZD5363 response and genetic alterations in gastric cancer (GC) and identify GC patients with both PI3KCA mutations and PTEN loss, we investigated the effects of pharmacological inhibition of AKT on a panel of 20 GC cell lines and genetic aberrations in tumor samples from a cohort of Chinese GC patients. We demonstrated that GC cells with PI3KCA mutations were selectively sensitive to AZD5363. Disease linkage studies showed that PI3KCA activating mutations or PTEN loss were found in 2.7% (4/150) and 23% (14/61) of Chinese GC patients respectively. To further dissect the role of PI3KCA mutation and PTEN loss in response to AKT inhibition, we tested the antitumor activity of AZD5363 in two patient-derived GC xenograft (PDGCX) models harboring either PI3KCA mutation or PTEN loss. Our data indicated that AZD5363 monotherapy treatment led to a moderate response in the PI3KCA mutant PDGCX model. Whilst monotherapy AZD5363 or Taxotere were ineffective in the PTEN negative PDGCX model, significant anti-tumor activity was observed when AZD5363 was combined with Taxotere.ConclusionOur results indicated that PI3KCA mutation is an important determinant of response to AKT inhibition in GC and combination with AZD5363 can overcome innate resistance to Taxotere in a PTEN loss PDGCX model. It is suggested that AKT inhibitor is an attractive option for treatment of a new segment of GC patients with aberrant PI3K/AKT signaling.

FGFR2 Gene Amplification in Gastric Cancer Predicts Sensitivity to the Selective FGFR Inhibitor AZD4547

FGFR gene aberrations are associated with tumor growth and survival. We explored the role of FGFR2 amplification in gastric cancer and the therapeutic potential of AZD4547, a potent and selective ATP-competitive receptor tyrosine kinase inhibitor of fibroblast growth factor receptor (FGFR)1-3, in patients with FGFR2-amplified gastric cancer. Array-comparative genomic hybridization and FISH were used to identify FGFR2 amplification in gastric cancer patient tumor samples. The effects of FGFR2 modulation were investigated in gastric cancer cells with FGFR2 amplification and in patient-derived gastric cancer xenograft (PDGCX) models using two approaches: inhibition with AZD4547 and short hairpin RNA (shRNA) knockdown of FGFR2. Amplification of the FGFR2 gene was identified in a subset of Chinese and Caucasian patients with gastric cancer. Gastric cancer cell lines SNU-16 and KATOIII, carrying the amplified FGFR2 gene, were extremely sensitive to AZD4547 in vitro with GI50 values of 3 and 5 nmol/L, respectively. AZD4547 effectively inhibited phosphorylation of FGFR2 and its downstream signaling molecules and induced apoptosis in SNU-16 cells. Furthermore, inhibition of FGFR2 signaling by AZD4547 resulted in significant dose-dependent tumor growth inhibition in FGFR2-amplified xenograft (SNU-16) and PDGCX models (SGC083) but not in nonamplified models. shRNA knockdown of FGFR2 similarly inhibited tumor growth in vitro and in vivo. Finally, compared with monotherapy, we showed enhancement of in vivo antitumor efficacy using AZD4547 in combination with chemotherapeutic agents. FGFR2 pathway activation is required for driving growth and survival of gastric cancer carrying FGFR2 gene amplification both in vitro and in vivo. Our data support therapeutic intervention with FGFR inhibitors, such as AZD4547, in patients with gastric cancer carrying FGFR2 gene amplification.

Abstract 2774: The molecular determinants of sensitivity to HER2 targeted therapy in Patient Derived Xenograft gastric tumor models from Caucasian and Eastern Asian patients.

Abstract 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

Abstract 1052: Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of gastric cancer.

Abstract Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the Ras/Raf/MEK/ERK signaling cascade plays a critical role in signal transduction pathways that regulate growth factor response, angiogenesis, tumor cell proliferation, and apoptosis, blocking this signaling pathway could thus have therapeutic efficacy. This study aims to target this pathway in preclinical models of GC with refametinib (BAY 86-9766, RDEA119), an orally available, allosteric inhibitor of MEK1/2. Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with refametinib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or refametinib-treated xenografts were analyzed by immunohistochemistry for cell proliferation, apoptosis and microvessel area, using antibodies against p-Histone H3Ser10, cleaved PARP and CD31, respectively. Tumor lysates were analyzed by Western Blotting with antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis.Results: Refametinib demonstrated significant tumor growth inhibition ranging from 66-87% in eight GC models at a dose of 15 mg/kg/d. Refametinib inhibited cell proliferation by 2-4 fold as observed by reduced staining for p-Histone H3Ser10. Increased staining for caspase-cleaved p85 PARP by 1.5 to 9 fold was detected, indicating that refametinib induced apoptosis. Furthermore the compound significantly inhibited angiogenesis in 2 of 8 models by 3-fold compared to the vehicle-treated group as indicated by reduced CD31 staining. Reduced cell proliferation and induction of apoptosis were associated with decreased levels of p-ERK1/2, p-cdk2Thr14/Tyr15, p-p90RSKSer359/363, p-p70S6KThr421/Ser424, p-4EBP1Thr70, p-S6RSer235/236 and p-eIF4ESer209 and elevation of p-AktSer473, Bim and cleaved PARP. Conclusion: We show that single-agent refametinib has significant anti-tumor activity in patient-derived gastric cancer preclinical models. Our findings support further investigation of refametinib either in monotherapy or in combination with inhibitors of the PI3K-AKT-mTOR pathway in the treatment of GC. Citation Format: Huynh T. Hung, Richard Ong, Khee Chee Soo, Florian Puehler, Arne Scholz, Dominik Mumberg, Dieter Zopf, Karl Ziegelbauer. Refametinib (BAY 86-9766): An allosteric inhibitor of MEK1/2, potently inhibits tumor growth in patient-derived xenograft models of gastric cancer. [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 1052. doi:10.1158/1538-7445.AM2013-1052

Abstract B2: Regorafenib, an oral multi-kinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases, potently inhibits tumor growth and angiogenesis in patient-derived xenograft models of gastric cancer.

Abstract Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the VEGFR and PDGFR signaling cascade plays a critical role in the development and progression of gastric cancer and VEGF expression is correlated with angiogenesis, tumor progression and poor prognosis in patients with GC, blocking these signaling pathways could thus have therapeutic efficacy. This study aims to investigate the potential of a combined therapeutic approach to target these pathways in preclinical models of GC with regorafenib an oral multi-kinase inhibitor targeting VEGFR1–3, PDGFR, FGFR, TIE-2, RET and KIT. Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with regorafenib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or regorafenib-treated xenografts were analyzed by immunohistochemistry for apoptosis, microvessel area, and cell proliferation using antibodies against cleaved PARP, CD31 and phospho-Histone3Ser10, respectively. Tumor lysates were analyzed by Western Blotting with respective antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis. Results: Regorafenib demonstrated significant tumor growth inhibition of 81%, 85% and 88% in the GC09-0109 model after oral administration of 5, 10 and 15 mg/kg/d, respectively. In addition potent tumor growth inhibition ranging from 72–96% was observed in seven other GC models at a dose of 10 mg/kg/d. Regorafenib significantly inhibited angiogenesis in all models by 4–12 fold compared to the vehicle-treated group, independent of their degree of vascularization, as indicated by reduced CD31 staining. Furthermore inhibition of cell proliferation by 2–4 fold was observed by reduced staining for p-histone 3Ser10. Increased staining for caspase-cleaved p85 PARP by 2 to 20 fold was detected, indicating that regorafenib induced apoptosis. Finally, regorafenib also induced tumor necrosis as detected by histological staining. Inhibition of angiogenesis, reduced cell proliferation and induction of apoptosis and necrosis was associated with decreased levels of p-ERK1/2, p-cdk2Thr14/Tyr15, p-VEGFR2Tyr951, p-p70S6KThr421/Ser424, p-S6RSer235/236 and p-eIF4ESer209 and elevation of cleaved caspase-3 and cleaved PARP. Conclusion: We show that regorafenib is an efficacious anti-tumor targeted agent in patient-derived gastric cancer preclinical models. Our findings support further investigation of regorafenib in the treatment of GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B2.