Longitudinal high-dimensional analysis identifies immune features associating with response to anti-PD-1 immunotherapy.

Response to immunotherapy widely varies among cancer patients and identification of parameters associating with favourable outcome is of great interest. Here we show longitudinal monitoring of peripheral blood samples of non-small cell lung cancer (NSCLC) patients undergoing anti-PD1 therapy by high-dimensional cytometry by time of flight (CyTOF) and Meso Scale Discovery (MSD) multi-cytokines measurements. We find that higher proportions of circulating CD8+ and of CD8+CD101hiTIM3+ (CCT T) subsets significantly correlate with poor clinical response to immune therapy. Consistently, CD8+ T cells and CCT T cell frequencies remain low in most responders during the entire multi-cycle treatment regimen; and higher killer cell lectin-like receptor subfamily G, member 1 (KLRG1) expression in CCT T cells at baseline associates with prolonged progression free survival. Upon in vitro stimulation, CCT T cells of responders produce significantly higher levels of cytokines, including IL-1ß, IL-2, IL-8, IL-22 and MCP-1, than of non-responders. Overall, our results provide insights into the longitudinal immunological landscape underpinning favourable response to immune checkpoint blockade therapy in lung cancer patients.

Discovery of Highly Potent and Selective IRAK1 Degraders to Probe Scaffolding Functions of IRAK1 in ABC DLBCL.

MyD88 gene mutation has been identified as one of the most prevalent driver mutations in the activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL). The published literature suggests that interleukin-1 receptor-associated kinase 1 (IRAK1) is an essential gene for ABC DLBCL harboring MyD88 mutation. Importantly, the scaffolding function of IRAK1, rather than its kinase activity, is required for tumor cell survival. Herein, we present our design, synthesis, and biological evaluation of a novel series of potent and selective IRAK1 degraders. One of the most potent compounds, Degrader-3 (JNJ-1013), effectively degraded cellular IRAK1 protein with a DC50 of 3 nM in HBL-1 cells. Furthermore, JNJ-1013 potently inhibited IRAK1 downstream signaling pathways and demonstrated strong anti-proliferative effects in ABC DLBCL cells with MyD88 mutation. This work suggests that IRAK1 degraders have the potential for treating cancers that are dependent on the IRAK1 scaffolding function.

Assessing IRAK4 Functions in ABC DLBCL by IRAK4 Kinase Inhibition and Protein Degradation.

The interleukin-1 receptor-activated kinase 4 (IRAK4) belongs to the IRAK family of serine/threonine kinases and plays a central role in the innate immune response. However, the function of IRAK4 in tumor growth and progression remains elusive. Here we sought to determine the enzymatic and scaffolding functions of IRAK4 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). We chose a highly selective IRAK4 kinase inhibitor to probe the biological effects of kinase inhibition and developed a series of IRAK4 degraders to evaluate the effects of protein degradation in ABC DLBCL cells. Interestingly, the results demonstrated that neither IRAK4 kinase inhibition nor protein degradation led to cell death or growth inhibition, suggesting a redundant role for IRAK4 in ABC DLBCL cell survival. IRAK4 degraders characterized in this study provide useful tools for understanding IRAK4 protein scaffolding function, which was previously unachievable using pharmacological perturbation.

Pharmacology of the ATM Inhibitor AZD0156: Potentiation of Irradiation and Olaparib Responses Preclinically.

AZD0156 is a potent and selective, bioavailable inhibitor of ataxia-telangiectasia mutated (ATM) protein, a signaling kinase involved in the DNA damage response. We present preclinical data demonstrating abrogation of irradiation-induced ATM signaling by low doses of AZD0156, as measured by phosphorylation of ATM substrates. AZD0156 is a strong radiosensitizer in vitro, and using a lung xenograft model, we show that systemic delivery of AZD0156 enhances the tumor growth inhibitory effects of radiation treatment in vivo Because ATM deficiency contributes to PARP inhibitor sensitivity, preclinically, we evaluated the effect of combining AZD0156 with the PARP inhibitor olaparib. Using ATM isogenic FaDu cells, we demonstrate that AZD0156 impedes the repair of olaparib-induced DNA damage, resulting in elevated DNA double-strand break signaling, cell-cycle arrest, and apoptosis. Preclinically, AZD0156 potentiated the effects of olaparib across a panel of lung, gastric, and breast cancer cell lines in vitro, and improved the efficacy of olaparib in two patient-derived triple-negative breast cancer xenograft models. AZD0156 is currently being evaluated in phase I studies (NCT02588105).

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.

Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

Relationships between Chromosome 7 Gain, MET Gene Copy Number Increase and MET Protein Overexpression in Chinese Papillary Renal Cell Carcinoma Patients.

To investigate the relationships between Chromosome 7 gain, mesenchymal-epithelial transition factor (MET) gene copy number increase and MET protein overexpression in Chinese patients with papillary renal cell carcinoma (PRCC), immunohistochemistry (IHC), immunofluorescence (IF) and fluorescence in situ hybridization (FISH) were performed on 98 formalin-fixed, paraffin-embedded (FFPE) PRCC samples. Correlations between MET gene copy number increase, Chromosome 7 gain and MET protein overexpression were analyzed statistically. A highly significant correlation was observed between the percentage of tumor cells with MET gene copy number ≥3 and CEP7 copy number ≥3 (R2 = 0.90, p<0.001) across two subtypes of PRCC. In addition, the percentage of tumor cells with MET gene copy number ≥3 was found to increase along with increases in MET IHC score. This correlation was further confirmed in those PRCC tumor cells with average MET gene copy number >5 using combined IF and FISH methodology. Overall, this study provides evidence that Chromosome 7 gain drives MET gene copy number increase in PRCC tumors, and appears to subsequently lead to an increase in MET protein overexpression in these tumor cells. This supports MET activation as a potential therapeutic target in sporadic PRCC.

Intra-Tumoral Heterogeneity of HER2, FGFR2, cMET and ATM in Gastric Cancer: Optimizing Personalized Healthcare through Innovative Pathological and Statistical Analysis.

Current drug development efforts on gastric cancer are directed against several molecular targets driving the growth of this neoplasm. Intra-tumoral biomarker heterogeneity however, commonly observed in gastric cancer, could lead to biased selection of patients. MET, ATM, FGFR2, and HER2 were profiled on gastric cancer biopsy samples. An innovative pathological assessment was performed through scoring of individual biopsies against whole biopsies from a single patient to enable heterogeneity evaluation. Following this, false negative risks for each biomarker were estimated in silico. 166 gastric cancer cases with multiple biopsies from single patients were collected from Shanghai Renji Hospital. Following pre-set criteria, 56 ~ 78% cases showed low, 15 ~ 35% showed medium and 0 ~ 11% showed high heterogeneity within the biomarkers profiled. If 3 biopsies were collected from a single patient, the false negative risk for detection of the biomarkers was close to 5% (exception for FGFR2: 12.2%). When 6 biopsies were collected, the false negative risk approached 0%. Our study demonstrates the benefit of multiple biopsy sampling when considering personalized healthcare biomarker strategy, and provides an example to address the challenge of intra-tumoral biomarker heterogeneity using alternative pathological assessment and statistical methods.

Patient-Derived Gastric Carcinoma Xenograft Mouse Models Faithfully Represent Human Tumor Molecular Diversity.

Patient-derived cancer xenografts (PDCX) generally represent more reliable models of human disease in which to evaluate a potential drugs preclinical efficacy. However to date, only a few patient-derived gastric cancer xenograft (PDGCX) models have been reported. In this study, we aimed to establish additional PDGCX models and to evaluate whether these models accurately reflected the histological and genetic diversities of the corresponding patient tumors. By engrafting fresh patient gastric cancer (GC) tissues into immune-compromised mice (SCID and/or nude mice), thirty two PDGCX models were established. Histological features were assessed by a qualified pathologist based on H&E staining. Genomic comparison was performed for several biomarkers including ERBB1, ERBB2, ERBB3, FGFR2, MET and PTEN. These biomarkers were profiled to assess gene copy number by fluorescent in situ hybridization (FISH) and/or protein expression by immunohistochemistry (IHC). All 32 PDGCX models retained the histological features of the corresponding human tumors. Furthermore, among the 32 models, 78% (25/32) highly expressed ERBB1 (EGFR), 22% (7/32) were ERBB2 (HER2) positive, 78% (25/32) showed ERBB3 (HER3) high expression, 66% (21/32) lost PTEN expression, 3% (1/32) harbored FGFR2 amplification, 41% (13/32) were positive for MET expression and 16% (5/32) were MET gene amplified. Between the PDGCX models and their parental tumors, a high degree of similarity was observed for FGFR2 and MET gene amplification, and also for ERBB2 status (agreement rate = 94~100%; kappa value = 0.81~1). Protein expression of PTEN and MET also showed moderate agreement (agreement rate = 78%; kappa value = 0.46~0.56), while ERBB1 and ERBB3 expression showed slight agreement (agreement rate = 59~75%; kappa value = 0.18~0.19). ERBB2 positivity, FGFR2 or MET gene amplification was all maintained until passage 12 in mice. The stability of the molecular profiles observed across subsequent passages within the individual models provides confidence in the utility and translational significance of these models for in vivo testing of personalized therapies.

Identification and validation of dysregulated MAPK7 (ERK5) as a novel oncogenic target in squamous cell lung and esophageal carcinoma.

BACKGROUND: MAPK7/ERK5 (extracellular-signal-regulated kinase 5) functions within a canonical three-tiered MAPK (mitogen activated protein kinase) signaling cascade comprising MEK (MAPK/ERK kinase) 5, MEKK(MEK kinase) 2/3 and ERK5 itself. Despite being the least well studied of the MAPK-modules, evidence supports a role for MAPK7-signaling in the pathology of several cancer types. METHODS AND RESULTS: Fluorescence in situ hybridization (FISH) analysis identified MAPK7 gene amplification in 4% (3/74) of non-small cell lung cancers (NSCLC) (enriched to 6% (3/49) in squamous cell carcinoma) and 2% (2/95) of squamous esophageal cancers (sqEC). Immunohistochemical (IHC) analysis revealed a good correlation between MAPK7 gene amplification and protein expression. MAPK7 was validated as a proliferative oncogenic driver by performing in vitro siRNA knockdown of MAPK7 in tumor cell lines. Finally, a novel MEK5/MAPK7 co-transfected HEK293 cell line was developed and used for routine cell-based pharmacodynamic screening. Phosphorylation antibody microarray analysis also identified novel downstream pharmacodynamic (PD) biomarkers of MAPK7 kinase inhibition in tumor cells (pMEF2A and pMEF2D). CONCLUSIONS: Together, these data highlight a broader role for dysregulated MAPK7 in driving tumorigenesis within niche populations of highly prevalent tumor types, and describe current efforts in establishing a robust drug discovery screening cascade.

Evaluation of Trastuzumab Anti-Tumor Efficacy and its Correlation with HER-2 Status in Patient-Derived Gastric Adenocarcinoma Xenograft Models.

The aim of the study was to investigate trastuzumab anti-tumor efficacy and its correlation with HER-2 status in primary xenograft models derived from Chinese patients with gastric adenocarcinoma. Patient-derived gastric adenocarcinoma xenograft (PDGAX) mouse models were firstly generated by implanting gastric adenocarcinoma tissues from patients into immune deficient mice. A high degree of histological and molecular similarity between the PDGAX mouse models and their corresponding patients' gastric adenocarcinoma tissues was shown by pathological observation, HER-2 expression, HER-2 gene copy number, and mutation detection. Based on Hoffmann's criteria in gastric cancer, three models (PDGAX001, PDGAX003 and PDGAX005) were defined as HER-2 positive with fluorescence in situ hybridization (FISH) amplification or immunohistochemistry (IHC) 2+/ 3+, while two models (PDGAX002, PDGAX004) were defined as HER-2 negative. Upon trastuzumab treatment, significant tumor regression (105 % TGI) was observed in model PDGAX005 (TP53 wt), while moderate sensitivity (26 % TGI) was observed in PDGAX003, and resistance was observed in PDGAX001, 002 and 004. A significant increase in HER-2 gene copy number was only observed in PDGAX005 (TP53 wt). Interestingly, trastuzumab showed no efficacy in PDGAX001 (HER2 IHC 3+ and FISH amplification, but with mutant TP53). Consistent with this finding, phosphor-HER2 modulation by trastuzumab was observed in model PDGAX005, but not in PDGAX001.

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.

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.

PURPOSE: 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. EXPERIMENTAL DESIGN: A range of assays were used and included; in vitro cell line panel screening and pharmacodynamic (PD) analysis, cMET fluorescence in-situ hybridization (FISH) and immunohistochemical (IHC) tissue microarray (TMA) analysis of Chinese GC (n = 170), and anti-tumor efficacy testing and PD analysis of gastric PDX models using volitinib. RESULTS: The incidence of cMET gene amplification and protein overexpression within Chinese patient GC tumors was 6% and 13%, respectively. Volitinib displayed a highly selective profile across a gastric cell line panel, potently inhibiting cell growth only in those lines with dysregulated cMET (EC50 values 0.6 nM/L-12.5 nM/L). Volitinib treatment led to pharmacodynamic modulation of cMET signaling and potent tumor stasis in 3/3 cMET-dysregulated GC PDX models, but had negligible activity in a GC control model. CONCLUSIONS: This study provides an assessment of tumor cMET gene copy number changes and protein overexpression incidence in a cohort of Chinese GC patients. To our knowledge, this is the first study to demonstrate anti-tumor efficacy in a panel of cMET-dysregulated gastric cancer PDX models, using a novel selective cMET-inhibitor (volitinib). Thus, the translational science presented here provides strong rationale for the investigation of volitinib as a therapeutic option for patients with GC tumors harboring amplified cMET.

An evaluation and recommendation of the optimal methodologies to detect RET gene rearrangements in papillary thyroid carcinoma.

To recommend a reliable and clinically realistic RET/PTC rearrangement detection assay for papillary thyroid carcinoma (PTC), we compared multiplex quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). RET/PTC rearrangement was detected using either RET break-apart FISH followed by multicolor FISH to confirm CCDC6/RET or NCOA4/RET fusions, or by multiplex qPCR to detect 14 RET/PTC subtypes with simultaneous RET mRNA expression. RET protein expression was detected by IHC. The specificity and sensitivity of multiplex qPCR and IHC were calculated using break-apart FISH as a reference. Among 73 PTC patients with sufficient tissue available for FISH and multiplex qPCR, 10 cases were defined as RET/PTC positive by both assays, including eight CCDC6/RET and two NCOA4/RET fusions with relatively high RET mRNA. In addition, multiplex qPCR identified another two CCDC6/RET fusion positive cases, but with low RET mRNA expression. IHC staining identified 11 RET positive cases among 39 patients with available samples. In comparison to FISH, multiplex qPCR displayed 100% sensitivity and 97% specificity to detect RET/PTC fusions, while IHC was neither sensitive nor specific. Our data reveal that both multiplex qPCR and FISH assays are equally applicable for detection of RET/PTC rearrangements. Break-apart FISH methodology is highly recommended for the wider screening of RET rearrangements (regardless of partner genes), while multiplex qPCR is preferred to identify all known fusion types using one assay, provided mRNA expression is also measured. IHC analysis could potentially provide an additional method of fusion detection dependent on further optimization of assay conditions and scoring cutoffs.

Establishment and characterization of esophageal squamous cell carcinoma patient-derived xenograft mouse models for preclinical drug discovery.

The purpose of this study was to establish and characterize patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mice for utilization in antitumor drug discovery. A total of 96 esophageal squamous cell carcinoma (ESCC) tissues from Chinese patients were transplanted subcutaneously into immunodeficient mice. Histology, EGFR, K-ras, B-raf, and PIK3CA mutations, and HER2 gene amplifications were analyzed in both patient tumors and mouse xenograft tissues using immunohistochemistry, mutant-enriched liquid chip sequencing and fluorescence in situ hybridization assays, respectively. Furthermore, in vivo efficacy studies using five PDECX mice harboring a variety of genetic aberrations were performed using the chemotherapy agents 5-fluorouracil (5-FU) and cisplatin. Thirty-seven PDECX mouse models were successfully established in immunodeficient mice. Pathological analysis revealed similar histological architecture and degrees of differentiation between patient ESCC and xenografted tumors. No mutations were identified in EGFR, K-ras, and B-raf genes in either xenograft models or patient ESCC tissues. In contrast, PIK3CA gene mutations were detected in 12.5% (12/96) ESCC patients and 18.9% (7/37) PDECX models. Interestingly, patient ESCC tissues exhibiting HER2 overexpression or gene amplification were unable to survive in immunodeficient mice. Further analysis showed that PDECX models carrying HER2 2+ expression had no response to 5-FU/cisplatin, compared with HER2-negative models. In conclusion, a panel of PDECX mouse models, which include PIK3CA mutant and HER2-positive models, was established and characterized thus mimicking the current clinical genetic setting of esophageal carcinoma. The sensitivity of HER2-negative ESCC models to chemotherapy supports stratification approaches in the treatment of esophageal carcinoma patients and warrants further investigation of the impact of PI3KCA on treatment response.

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.

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.

BACKGROUND: Patient-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. METHODS: NSCLC 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. RESULTS: Ten 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. CONCLUSIONS: Ten patient-derived NSCLC xenograft models were established containing a variety of genetic aberrations including EGFR activating mutation, KRAS mutation, and FGFR1 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.

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.

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 (GI(50) = 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.

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

BACKGROUND: Trastuzumab 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. METHODS: PDECX 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. RESULTS: None 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. CONCLUSIONS: This 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.