Paul R. Gavine

AZD4547: An Orally Bioavailable, Potent, and Selective Inhibitor of the Fibroblast Growth Factor Receptor Tyrosine Kinase Family

The fibroblast growth factor (FGF) signaling axis is increasingly implicated in tumorigenesis and chemoresistance. Several small-molecule FGF receptor (FGFR) kinase inhibitors are currently in clinical development; however, the predominant activity of the most advanced of these agents is against the kinase insert domain receptor (KDR), which compromises the FGFR selectivity. Here, we report the pharmacologic profile of AZD4547, a novel and selective inhibitor of the FGFR1, 2, and 3 tyrosine kinases. AZD4547 inhibited recombinant FGFR kinase activity in vitro and suppressed FGFR signaling and growth in tumor cell lines with deregulated FGFR expression. In a representative FGFR-driven human tumor xenograft model, oral administration of AZD4547 was well tolerated and resulted in potent dose-dependent antitumor activity, consistent with plasma exposure and pharmacodynamic modulation of tumor FGFR. Importantly, at efficacious doses, no evidence of anti-KDR-related effects were observed, confirming the in vivo FGFR selectivity of AZD4547. Taken together, our findings show that AZD4547 is a novel selective small-molecule inhibitor of FGFR with potent antitumor activity against FGFR-deregulated tumors in preclinical models. AZD4547 is under clinical investigation for the treatment of FGFR-dependent tumors.

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

Purpose: 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. Experimental Design: 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. Results: 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. Conclusion: 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. Clin Cancer Res; 19(9); 2572–83. ©2013 AACR.

Translating the Therapeutic Potential of AZD4547 in FGFR1-Amplified Non–Small Cell Lung Cancer through the Use of Patient-Derived Tumor Xenograft Models

Purpose: To investigate the incidence of FGFR1 amplification in Chinese non–small cell lung cancer (NSCLC) and to preclinically test the hypothesis that the novel, potent, and selective fibroblast growth factor receptor (FGFR) small-molecule inhibitor AZD4547 will deliver potent antitumor activity in NSCLC FGFR1–amplified patient-derived tumor xenograft (PDTX) models. Experimental Design: A range of assays was used to assess the translational relevance of FGFR1 amplification and AZD4547 treatment including in vitro lung cell line panel screening and pharmacodynamic (PD) analysis, FGFR1 FISH tissue microarray (TMA) analysis of Chinese NSCLC (n = 127), and, importantly, antitumor efficacy testing and PD analysis of lung PDTX models using AZD4547. Results: The incidence of FGFR1 amplification within Chinese patient NSCLC tumors was 12.5% of squamous origin (6 of 48) and 7% of adenocarcinoma (5 of 76). AZD4547 displayed a highly selective profile across a lung cell line panel, potently inhibiting cell growth only in those lines harboring amplified FGFR1 (GI50 = 0.003–0.111 μmol/L). AZD4547 induced potent tumor stasis or regressive effects in four of five FGFR1-amplified squamous NSCLC PDTX models. Pharmacodynamic modulation was observed in vivo, and antitumor efficacy correlated well with FGFR1 FISH score and protein expression level. Conclusions: This study provides novel epidemiologic data through identification of FGFR1 gene amplification in Chinese NSCLC specimens (particularly squamous) and, importantly, extends the clinical significance of this finding by using multiple FGFR1-amplified squamous lung cancer PDTX models to show tumor stasis or regression effects using a specific FGFR inhibitor (AZD4547). Thus, the translational science presented here provides a strong rationale for investigation of AZD4547 as a therapeutic option for patients with squamous NSCLC tumors harboring amplification of FGFR1. Clin Cancer Res; 18(24); 6658–67. ©2012 AACR.

Establishment of patient-derived non-small cell lung cancer xenograft models with genetic aberrations within EGFR, KRAS and FGFR1: useful tools for preclinical studies of targeted therapies

BackgroundPatient-derived tumor xenograft models have been established and increasingly used for preclinical studies of targeted therapies in recent years. However, patient-derived non-small cell lung cancer (NSCLC) xenograft mouse models are relatively few in number and are limited in their degree of genetic characterization and validation. In this study, we aimed to establish a variety of patient-derived NSCLC models and characterize these for common genetic aberrations to provide more informative models for preclinical drug efficacy testing.MethodsNSCLC tissues from thirty-one patients were collected and implanted into immunodeficient mice. Established xenograft models were characterized for common genetic aberrations, including detection of gene mutations within EGFR and KRAS, and genetic amplification of FGFR1 and cMET. Finally, gefitinib anti-tumor efficacy was tested in these patient-derived NSCLC xenograft models.ResultsTen passable patient-derived NSCLC xenograft models were established by implantation of NSCLC specimens of thirty-one patients into immunodeficient mice. Genetic aberrations were detected in six of the models, including one model with an EGFR activating mutation (Exon19 Del), one model with KRAS mutation, one model with both KRAS mutation and cMET gene amplification, and three models with FGFR1 amplification. Anti-tumor efficacy studies using gefitinib demonstrated that the EGFR activating mutation model had superior sensitivity and that the KRAS mutation models were resistant to gefitinib. The range of gefitinib responses in the patient-derived NSCLC xenograft models were consistent with the results reported from clinical trials. Furthermore, we observed that patient-derived NSCLC models with FGFR1 gene amplification were insensitive to gefitinib treatment.ConclusionsTen patient-derived NSCLC xenograft models were established containing a variety of genetic aberrations including EGFR activating mutation, KRAS mutation, and FGFR 1 and cMET amplification. Gefitinib anti-tumor efficacy in these patient-derived NSCLC xenografts containing EGFR and KRAS mutation was consistent with the reported results from previous clinical trials. Thus, data from our panel of patient-derived NSCLC xenograft models confirms the utility of these models in furthering our understanding of this disease and aiding the development of personalized therapies for NSCLC patients.

Volitinib, a potent and highly selective c-Met inhibitor, effectively blocks c-Met signaling and growth in c-MET amplified gastric cancer patient-derived tumor xenograft models

To investigate the incidence of cMET gene copy number changes and protein overexpression in Chinese gastric cancer (GC) and to preclinically test the hypothesis that the novel, potent and selective cMET small‐molecule inhibitor volitinib, will deliver potent anti‐tumor activity in cMET‐dysregulated GC patient‐derived tumor xenograft (PDX) models.

Targeting Fibroblast Growth Factor Receptor Signaling Inhibits Prostate Cancer Progression

Purpose: Extensive correlative studies in human prostate cancer as well as studies in vitro and in mouse models indicate that fibroblast growth factor receptor (FGFR) signaling plays an important role in prostate cancer progression. In this study, we used a probe compound for an FGFR inhibitor, which potently inhibits FGFR-1–3 and significantly inhibits FGFR-4. The purpose of this study is to determine whether targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Experimental Design: Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in prostate cancer cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. Results: AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nmol/L, which is an achievable in vivo concentration. This results in marked inhibition of extracellular signal–regulated kinase (ERK) phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all prostate cancer cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Conclusions: Targeting FGFR signaling is a promising new approach to treating aggressive prostate cancer. Clin Cancer Res; 18(14); 3880–8. ©2012 AACR.

Trastuzumab anti-tumor efficacy in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models.

BackgroundTrastuzumab is currently approved for the clinical treatment of breast and gastric cancer patients with HER-2 positive tumors, but not yet for the treatment of esophageal carcinoma patients, whose tumors typically show 5 ~ 35% HER-2 gene amplification and 0 ~ 56% HER-2 protein expression. This study aimed to investigate the therapeutic efficacy of Trastuzumab in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models.MethodsPDECX models were established by implanting patient esophageal squamous cell carcinoma (ESCC) tissues into immunodeficient (SCID/nude) mice. HER-2 gene copy number (GCN) and protein expression were determined in xenograft tissues and corresponding patient EC samples by FISH and IHC analysis. Trastuzumab anti-tumor efficacy was evaluated within these PDECX models (n = 8 animals/group). Furthermore, hotspot mutations of EGFR, K-ras, B-raf and PIK3CA genes were screened for in the PDECX models and their corresponding patient’s ESCC tissues. Similarity between the PDECX models and their corresponding patient’s ESCC tissue was confirmed by histology, morphology, HER-2 GCN and mutation.ResultsNone of the PDECX models (or their corresponding patient’s ESCC tissues) harbored HER-2 gene amplification. IHC staining showed HER-2 positivity (IHC 2+) in 2 PDECX models and negativity in 3 PDECX models. Significant tumor regression was observed in the Trastuzumab-treated EC044 HER-2 positive model (IHC 2+). A second HER-2 positive (IHC 2+) model, EC039, harbored a known PIK3CA mutation and showed strong activation of the AKT signaling pathway and was insensitive to Trastuzumab treatment, but could be resensitised using a combination of Trastuzumab and AKT inhibitor AZD5363. In summary, we established 5 PDECX mouse models and demonstrated tumor regression in response to Trastuzumab treatment in a HER-2 IHC 2+ model, but resistance in a HER-2 IHC 2+/PIK3CA mutated model.ConclusionsThis study demonstrates Trastuzumab-induced tumor regressions in HER-2 positive tumors, and highlights PIK3CA mutation as a potential resistance mechanism to Trastuzumab treatment in pre-clinical patient-derived EC xenograft models.

Frequency, characterization, and prognostic analysis of PIK3CA gene mutations in Chinese esophageal squamous cell carcinoma

PIK3CA gene mutations are found in numerous cancers but correlate differently with prognosis. Although the frequency of PIK3CA gene mutation in esophageal squamous cell carcinoma (ESCC) has been previously studied, a prognostic analysis has not been reported. Ninety-six surgically resected ESCC tissues were collected from Chinese patients and DNA was extracted. Gene mutations in PIK3CA (exons 9 and 20), EGFR (exons 18, 19, 20 and 21), KRAS (exons 2 and 3), and BRAF (exons 11 and 15) were screened using mutant-enriched liquid chip technology. PIK3CA gene mutations were identified in 12 of 96 ESCC cases (12.5%). No mutations were identified in EGFR, KRAS or BRAF genes in this study. Correlations between clinicopathological features and PIK3CA mutation status were analyzed and finally, patient survival information was used to determine the prognostic significance of PIK3CA mutation. Interestingly, the frequency of PIK3CA mutation was higher in female ESCC patients (31.3%, 5/16) than in males (8.8%, 7/80), and higher in patients with non-lymph node metastasis (19.6%, 10/51, P = .013) than in patients with lymph node metastasis (4.4%, 2/45, P = .025). Furthermore, patients with PIK3CA-mutated tumors showed a trend towards favorable overall survival (P = .085) but not disease-free survival (P = .238), suggesting that PIK3CA gene status may be a favorable predictive marker in ESCC patients.

PD-L1 expression and its relationship with oncogenic drivers in non-small cell lung cancer (NSCLC)

In order to explore the potential patient population who could benefit from anti PD-1/PD-L1 mono or combination therapies, this study aimed to profile a panel of immunotherapy related biomarkers (PD-1, PD-L1, CTLA-4 and CD8) and targeted therapy biomarkers (EGFR, KRAS, ALK, ROS1 and MET) in NSCLC. Tumor samples from 297 NSCLC patients, including 156 adenocarcinomas (AD) and 129 squamous cell carcinomas (SCC), were analyzed using immunohistochemistry, immunofluorescence, sequencing and fluorescence in situ hybridization. 43.1% of NSCLC patients had PD-L1 positive staining on ≥ 5% tumor cells (TC). Furthermore, dual color immunofluorescence revealed that the majority of PD-L1/CD8 dual positive tumor infiltrating lymphocytes (TIL) had infiltrated into the tumor core. Finally, combined analysis of all eight biomarkers showed that tumor PD-L1 positivity overlapped with known alterations in NSCLC oncogenic tumor drivers in 26% of SCC and 76% of AD samples. Our illustration of the eight biomarkers’ overlap provides an intuitive overview of NSCLC for personalized therapeutic strategies using anti-PD-1/PD-L1 immune therapies, either as single agents, or in combination with targeted therapies. For the first time, we also report that PD-L1 and CD8 dual positive TILs are predominantly located within the tumor core.

Assessment and prognostic analysis of EGFR, HER2, and HER3 protein expression in surgically resected gastric adenocarcinomas

Aim To investigate the significance of epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER)2, and HER3 expression on survival outcomes in Chinese gastric cancer patients. Materials and methods Formalin-fixed, paraffin-embedded specimens from 121 patients who underwent gastrectomy at Shanghai Renji Hospital from 2007–2010 were retrospectively examined. Fluorescence in situ hybridization and immunohistochemistry techniques were used to identify gene amplification and protein overexpression. Correlations between the expression or amplification of HER family genes and clinicopathological parameters were then determined using statistical analysis. Results EGFR protein overexpression, an increase in HER2 copy number and gene amplification, and HER3 protein overexpression were identified in 33.1%, 17.4%, and 62.0% of samples, respectively. Statistical analysis showed a significant association between EGFR expression and tumor invasion depth or tumor stage. HER2 was also shown to be significantly associated with the tumor grade. In addition, EGFR protein overexpression was found to be significantly associated with worse overall survival (P=0.03). Conclusion The HER family members showed a high expression in gastric cancer. EGFR protein expression was associated with overall survival.