Haihua Fu

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.

A retrospective analysis of RET translocation, gene copy number gain and expression in NSCLC patients treated with vandetanib in four randomized Phase III studies

BackgroundTo determine the prevalence of RET rearrangement genes, RET copy number gains and expression in tumor samples from four Phase III non-small-cell lung cancer (NSCLC) trials of vandetanib, a selective inhibitor of VEGFR, RET and EGFR signaling, and to determine any association with outcome to vandetanib treatment.MethodsArchival tumor samples from the ZODIAC (NCT00312377, vandetanib ± docetaxel), ZEAL (NCT00418886, vandetanib ± pemetrexed), ZEPHYR (NCT00404924, vandetanib vs placebo) and ZEST (NCT00364351, vandetanib vs erlotinib) studies were evaluated by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in 944 and 1102 patients.ResultsThe prevalence of RET rearrangements by FISH was 0.7% (95% CI 0.3–1.5%) among patients with a known result. Seven tumor samples were positive for RET rearrangements (vandetanib, n = 3; comparator, n = 4). 2.8% (n = 26) of samples had RET amplification (innumerable RET clusters, or ≥7 copies in > 10% of tumor cells), 8.1% (n = 76) had low RET gene copy number gain (4–6 copies in ≥40% of tumor cells) and 8.3% (n = 92) were RET expression positive (signal intensity ++ or +++ in >10% of tumor cells). Of RET-rearrangement-positive patients, none had an objective response in the vandetanib arm and one patient responded in the comparator arm. Radiologic evidence of tumor shrinkage was observed in two patients treated with vandetanib and one treated with comparator drug. The objective response rate was similar in the vandetanib and comparator arms for patients positive for RET copy number gains or RET protein expression.ConclusionsWe have identified prevalence for three RET biomarkers in a population predominated by non-Asians and smokers. RET rearrangement prevalence was lower than previously reported. We found no evidence of a differential benefit for efficacy by IHC and RET gene copy number gains. The low prevalence of RET rearrangements (0.7%) prevents firm conclusions regarding association of vandetanib treatment with efficacy in the RET rearrangement NSCLC subpopulation.Trial registrationRandomized Phase III clinical trials (NCT00312377, ZODIAC; NCT00418886, ZEAL; NCT00364351, ZEST; NCT00404924, ZEPHYR).

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.

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.

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.

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.

Programmed cell death ligand 1 protein levels predicted survival of non-small cell lung cancer

Objective: To investigate the relationship between programmed death ligand 1 (PD-L1) expression using 5%, 25%, 50% cutoffs in tumor cells (TC) and postsurgical survival in non-small-cell lung cancer (NSCLC) patients. For samples with tumor infiltrating lymphocytes (TIL), correlation between PD-L1 expression in TIL using 1% cutoff and postsurgical survival was also evaluated. Methods: Primary NSCLC tumor surgical samples staging I to IIIA of 126 patients who underwent surgical procedures from September 2009 to August 2012 in Shanghai Chest Hospital, Shanghai Jiao Tong University were retrospectively included. PD-L1 protein expression was detected by immunohistochemistry (IHC) assays. A rabbit anti-human PD-L1 (E1L3N) monoclonal antibody (1:300, CST#13684, Cell Signaling Technology) was used for PD-L1 IHC staining. PD-L1 expression was evaluated both on TC and TIL. Univariate and multivariate analyses for postsurgical survival were done using Kaplan-Meier and Cox regression model, respectively. Results: The median postsurgical survival for all patients was 44.1 months [95% confidence interval (CI): 33.9-70.0 months). The median postsurgical survival for PD-L1 expression percentage 0, 1-50% and ≥50% were 51.9 months (95%CI: 33.9-70.0 months), 33.2 months (95%CI: 20.8-45.6 months) and 14.7 months (95%CI: 1.9-27.6 months), respectively (P = 0.002). Clinical stage and PD-L1 expression in TC (25% cutoff or 50% cutoff values) were found to be independent predictors for longer postsurgical survival in all cohort. Ninety (71.4%) of the 126 samples were identified to concurrent TIL. The median postsurgical survival time was 39.6 months (95% CI: 31.8-47.4 months) in patients with TIL. PD-L1 expression in TC (25% cutoff or 50% cutoff values) was found to be the independent predictor for longer postsurgical survival time in patients with TIL. Conclusion: PD-L1 negative expression in TC at 25% or 50% cutoff values was the independent predictor for longer postsurgical survival time in both NSCLC samples and NSCLC samples with TIL. For patients with PD-L1 high expression at 25% or 50% cutoff values, PD-L1 blocking may be considered.

Abstract 5140: PD-L1 expression and its relationship with other driver genes in non-small cell lung cancer (NSCLC)

Aims: In order to understand the potential patient population who could benefit from anti PD-1/PD-L1 mono or combinational therapies, this study aimed to profile a panel of immune-mediated therapy for cancer (IMT-C) related biomarkers (CD8, PD-1, PD-L1 and CTLA-4) and molecular targeted therapy biomarkers (EGFRmut, KRASmut, ALK, ROS1 and MET) in NSCLC patients. Methods: Tumor samples from 356 Chinese NSCLC patients including 191 adenocarcinomas (AD) and 142 squamous carcinomas (SCC) were analyzed in this study. There were 221 early stage (I to IIIa) and 113 late stage (IIIb and IV) patients. PD-L1, PD-1, CTLA-4 and MET expression were detected using immunohistochemical (IHC) staining on formalin fixed paraffin embedded (FFPE) tissues. PD-L1 and CD8 expression were also simultaneously observed using a dual-color immunofluorescence (IF) assay. EGFR and KRAS mutations were detected by sequencing analysis. ALK and ROS1 gene rearrangements were defined by break-apart FISH assays, and MET gene amplification was also detected using a FISH assay. Results: Among 356 NSCLC patient samples, 145 (40.7%) had PD-L1 positive staining on ≥5% tumor cells (TC). The PD-L1 positive rate on TC was significantly higher in SCC than in AD (47.9% vs 34.0%, p = 0.011). In AD patients, PD-L1 expression was significantly elevated in males (p = 0.0059), smokers (p = 0.0003), higher tumor grade (p Conclusion: This study showed almost half of NSCLC patients have PD-L1 positive expression on TC, which to some extent overlaps with EGFR/KRAS mut, ALK/ROS1 rearrangement and MET alteration. As a potential patient selection biomarker, the PD-L1 positive prevalence in NSCLC TC and/or tumor infiltrating IC provides a strong evidence for PD-1/PD-L1 immune therapy in single agent or in combination with the molecular targeted agents. Furthermore, CD8+ lymphocyte’ infiltration in the tumor center and its PD-L1 expression status might also be considered as part of patient selection biomarker strategy. Citation Format: Liyan Jiang, Xinying Su, Tianwei Zhang, Xiaolu Yin, Meizhuo Zhang, Haihua Fu, Hulin Han, Yun Sun, Lili Dong, Jialin Qian, Yanhua Xu, Xuan Fu, Paul Gavine, Yanbin Zhou, Tian Kun, Jiaqi Huang, Haiyi Jiang, Yihong Yao, Baohui Han, Yi Gu. PD-L1 expression and its relationship with other driver genes in non-small cell lung cancer (NSCLC). [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 5140.

Abstract 4954: Serine/arginine splicing factor 1 (SRSF1) mediates DNA repair and chemo-sensitivity and drives growth in small cell lung cancer

Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Despite a high response rate to chemotherapy, more than 95% of patients eventually die from SCLC. We have identified that Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression in tumor is strongly associated with poor survival based on whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Here, SRSF1 is evaluated as a tumor driver in SCLC. Treatment of SCLC cell lines in vitro with a low dose of cisplatin or topotecan (two of the most common standard of care in SCLC) only induced a modest decrease of cell growth. However, knockdown of SRSF1 with siRNA along with a sub-lethal dose of cisplatin or topotecan enhanced the overall growth inhibition effect compared to the chemotherapy alone. SRSF1 siRNA alone induced modest but significant caspase-3 activation, similar to cisplatin or topotecan treatment alone. The combination of SRSF1 siRNA with chemotherapy treatments produced a significantly higher caspase induction. DNA-damage induction as a potential mechanism of SRSF1 knockdown was assessed. Phosphorylation of H2AX and Chk2, established markers of DNA-strand breaks and DNA-repair response, was consistently induced upon SRSF1 abrogation in cells, and further increased the phosphorylation of these proteins when combined with cisplatin or topotecan treatment. The knockdown of SRSF1 in SCLC cells also resulted in significant growth inhibition when cells were grown as 3D spheroids. Cells transfected with non-targeting siRNA produced large and well-organized spheroids; in contrast, cells transfected with SRSF1 siRNA did not form well-organized structures but mainly existed as single cells with poor viability. Results were confirmed by colony formation assays and could be rescued with a siRNA-resistant SRSF1 expression construct. Finally, we investigated the impact of SRSF1 loss on kinase signaling pathways in SCLC cells through phospho-kinase array profiling. Strong phospho-AKT and ERK signals were observed in control siRNA-transfected cells, and were abrogated by SRSF1 siRNA. Western blot confirmed this in several SCLC cell lines. These targeting studies demonstrate that SRSF1 plays a key role in DNA repair, chemo-sensitivity and cell proliferation. Together, these data reveal SRSF1 as a therapeutic target in SCLC and provide a rationale for personalized therapy in SCLC. Citation Format: Sarah J. Conley, Xin Yao, Jiaqi Huang, Brandon Higgs, Zhibin Hu, Zhan Xiao, Haihong Zhong, Zheng Liu, Philip Brohawn, Xiaoxiao Ge, Meggan Czapiga, Vaheh Oganesyan, Haihua Fu, David Tice, Ronald Herbst, Xinying Su, Yi Gu, Jianren Gu, Baohui Han, Laura Richman, Bahija Jallal, Liyan Jiang, Hongbing Shen, Yihong Yao. Serine/arginine splicing factor 1 (SRSF1) mediates DNA repair and chemo-sensitivity and drives growth in small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4954. doi:10.1158/1538-7445.AM2015-4954

Abstract 928: Volitinib (HMPL504), a novel, selective and potent cMET inhibitor, is efficacious in primary tumor models of cMET-driven gastric cancer.

Gastric cancer (GC) incidence rates are amongst the highest in Asian countries including; China, Japan and Korea(1). 5-year survival rates in early stage disease have improved through aggressive combinations of surgery and chemo/radiotherapy. In late stage disease however, despite Her2 molecular segmentation and trastuzumab therapy, prognosis remains dismal and novel therapeutic options are urgently required(2). The MET oncogene encodes the receptor tyrosine kinase for hepatocyte growth factor and controls genetic programs leading to cell growth, invasion and survival. Dysregulation of MET signaling is a common feature of a diverse range of human tumor types and thus represents a highly competitive and attractive therapeutic target. Volitinib (HMPL-504) is an orally bioavailable, highly selective and potent small molecule ATP-competitive inhibitor, which inhibits cMET autophosphorylation and downstream signaling (3). Volitinib is currently in Phase I clinical development. To evaluate the utility of volitinib in treating patients with GC, we established the incidence of cMET amplification and overexpression and attempted to correlate this with response to volitinib in primary GC tumor models. Elevated cMET gene copy number (amplification 5%, high polysomy 13%), and protein overexpression (12%, cases > IHC 2+, 16% > IHC1+) were detected in a cohort of 217 Chinese GC samples using fluorescent in situ hybridization (FISH) and immunohistochemical (IHC) approaches. In vitro proliferation assays were performed on a panel of 26 GC cell lines and profound sensitivity to volitinib was demonstrated in lines harboring elevated cMET gene copy number (GI 50 range 6 to 49nM). In the cMET amplified GC xenograft model, Hs746t, once-daily oral dosing of volitinib (2.5mg/kg) elicited potent anti-tumour activity at well-tolerated doses (97% tumour growth inhibition after 16 days dosing, P Citation Format: Paul R. Gavine, Shiming Fan, Haihua Fu, Lu Han, Yuan Jie Liu, Jing Lv, Weiguo Qing, Yongxin Ren, Weiguo Su, Xinying Su, Huiying Wang, Liang Xie, Shirlian Xu, Wen Xu, XiaoLu Yin, Yongjuan Yu, Tianwei Zhang, Q.May Wang. Volitinib (HMPL504), a novel, selective and potent cMET inhibitor, is efficacious in primary tumor models of cMET-driven 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 928. doi:10.1158/1538-7445.AM2013-928