Heng Nung Koong

Brivanib Alaninate, a Dual Inhibitor of Vascular Endothelial Growth Factor Receptor and Fibroblast Growth Factor Receptor Tyrosine Kinases, Induces Growth Inhibition in Mouse Models of Human Hepatocellular Carcinoma

Purpose: Hepatocellular carcinoma (HCC) is the fifth most common primary neoplasm; surgery is the only curative option but 5-year survival rates are only 25% to 50%. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) are known to be involved in growth and neovascularization of HCC. Therefore, agents that target these pathways may be effective in the treatment of HCC. The aim of this study was to determine the antineoplastic activity of brivanib alaninate, a dual inhibitor of VEGF receptor (VEGFR) and FGF receptor (FGFR) signaling pathways. Experimental Design: Six different s.c. patient-derived HCC xenografts were implanted into mice. Tumor growth was evaluated in mice treated with brivanib compared with control. The effects of brivanib on apoptosis and cell proliferation were evaluated by immunohistochemistry. The SK-HEP1 and HepG2 cells were used to investigate the effects of brivanib on the VEGFR-2 and FGFR-1 signaling pathways in vitro. Western blotting was used to determine changes in proteins in these xenografts and cell lines. Results: Brivanib significantly suppressed tumor growth in five of six xenograft lines. Furthermore, brivanib–induced growth inhibition was associated with a decrease in phosphorylated VEGFR-2 at Tyr1054/1059, increased apoptosis, reduced microvessel density, inhibition of cell proliferation, and down-regulation of cell cycle regulators. The levels of FGFR-1 and FGFR-2 expression in these xenograft lines were positively correlated with its sensitivity to brivanib-induced growth inhibition. In VEGF-stimulated and basic FGF stimulated SK-HEP1 cells, brivanib significantly inhibited VEGFR-2, FGFR-1, extracellular signal-regulated kinase 1/2, and Akt phosphorylation. Conclusion: This study provides a strong rationale for clinical investigation of brivanib in patients with HCC.

Sorafenib and rapamycin induce growth suppression in mouse models of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. Vascular endothelial growth factor, platelet derived growth factor and the Raf/mitogen‐activated protein kinase/extracellular signal regulated kinase (Raf/MEK/ERK) signalling pathway regulates the growth, neovascularization, invasiveness and metastatic potential of HCC. In this study, we investigated the in vivo antitumour activity and mechanisms of action of sorafenib tosylate on four patient‐derived HCC xenografts. Sorafenib dosed at 50 mg/kg and 100 mg/kg inhibited tumour growth by 85% and 96%, respectively. Sorafenib‐induced growth suppression and apoptosis were associated with inhibition of angiogenesis, down‐regulation of phospho‐platelet‐derived growth factor receptor β Tyr1021, phospho‐eIF4E Ser209, phospho‐c‐Raf Ser259, c‐Raf, Mcl‐1, Bcl‐2, Bcl‐x and positive cell cycle regulators, up‐regulation of apoptosis signalling kinase‐1, p27 and p21. Expression of IGF‐1Rβ and phosphorylation of c‐Raf Ser338, MEK1/2 Ser217/221 and ERK1/2 Thr202/Tyr204 were increased by sorafenib treatment. Phosphorylation of mammalian target‐of‐rapamycin (mTOR) targets (p70S6K, S6R and 4EBP1) was reduced by sorafenib in sorafenib‐sensitive lines but activated in sorafenib‐less‐sensitive 10–0505 xenograft. Sorafenib‐induced phosphorylation of c‐met, p70S6K and 4EBP1 was significantly reduced when 10–0505 cells were co‐treated with anti‐human anti‐HGF antibody, suggesting that treatment with sorafenib leads to increased HGF secretion and activation of c‐met and mTOR targets. Treatment of 10–0505 tumours with sorafenib plus rapamycin resulted in growth inhibition, inhibition of vascular endothelial growth factor receptor‐2 phosphorylation, increased apoptosis and completely blocked sorafenib‐induced phosphorylation of mTOR targets and cyclin B1 expression. These data also provide a strong rationale for clinical investigation of sorafenib in combination with mTOR inhibitors in patients with HCC.

AZD6244 enhances the anti-tumor activity of sorafenib in ectopic and orthotopic models of human hepatocellular carcinoma (HCC).

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is a particularly vascularized solid tumor where the Raf/MEK/ERK pathway is activated; suggesting that inhibition of this pathway may have therapeutic potential. METHODS We treated patient-derived HCC xenografts with (i) sorafenib, (ii) AZD6244 (ARRY-142886), and (iii) sorafenib plus AZD6244. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant to both angiogenesis and MEK signaling. Apoptosis, microvessel density, and cell proliferation were analyzed by immunohistochemistry. RESULTS We report here that sorafenib treatment resulted in suppression of tumor growth, reduction in cell proliferation, induction of apoptosis and inhibition of mTOR targets. Sorafenib-induced elevation of the insulin-like growth factor receptor 1 (IGF-1R), phospho-c-Raf Ser338, phospho-MEK Ser217/221 and phospho-ERK Thr202/Tyr204 was attenuated by co-treating cells with anti-human IGF-1R antibody or over-expression of activated mutant p70S6K. Pharmacological inhibition of the MEK/ERK pathway by AZD6244 enhanced the anti-tumor effect of sorafenib in both orthotopic and ectopic models of HCC. Such inhibition led to a further increase in pro-apoptotic Bim, apoptosis and a profound inhibition of cell proliferation. CONCLUSION Our findings underscore the potential of a combined therapeutic approach with sorafenib and MEK inhibitors in the treatment of HCC.

Genomic Profiles Specific to Patient Ethnicity in Lung Adenocarcinoma

Purpose: East-Asian (EA) patients with non–small-cell lung cancer (NSCLC) are associated with a high proportion of nonsmoking women, epidermal growth factor receptor (EGFR)-activating somatic mutations, and clinical responses to tyrosine kinase inhibitors. We sought to identify novel molecular differences between NSCLCs from EA and Western European (WE) patients. Experimental Design: A total of 226 lung adenocarcinoma samples from EA (n = 90) and WE (n = 136) patients were analyzed for copy number aberrations (CNA) by using a common high-resolution SNP (single nucleotide polymorphism) microarray platform. Univariate and multivariate analyses were carried out to identify CNAs specifically related to smoking history, EGFR mutation status, and ethnicity. Results: The overall genomic profiles of adenocarcinomas from EA and WE patients were highly similar. Univariate analyses revealed several CNAs significantly associated with ethnicity, EGFR mutation, and smoking, but not to gender, and KRAS or p53 mutations. A multivariate model identified four ethnic-specific recurrent CNAs—significantly higher rates of copy number gain were observed on 16p13.13 and 16p13.11 in EA tumors, whereas higher rates of genomic loss on 19p13.3 and 19p13.11 were observed in tumors from WE patients. We identified several potential driver genes in these regions, showing a positive correlation between cis-localized copy number changes and transcriptomic changes. Conclusion: 16p copy number gains (EA) and 19p losses (WE) are ethnic-specific chromosomal aberrations in lung adenocarcinoma. Patient ethnicity should be considered when evaluating future NSCLC therapies targeting genes located on these areas. Clin Cancer Res; 17(11); 3542–50. ©2011 AACR.

Using Whole Genome Amplification (WGA) of Low-Volume Biopsies to Assess the Prognostic Role of EGFR, KRAS, p53, and CMET Mutations in Advanced-Stage Non-small Cell Lung Cancer (NSCLC)

Background: Progression of non-small cell lung cancer (NSCLC) from early- to late-stage may signify the accumulation of gene mutations. An advanced-stage tumor’s mutation profile may also have prognostic value, guiding treatment decisions. Mutation detection of multiple genes is limited by the low amount of deoxyribonucleic acid extracted from low-volume diagnostic lung biopsies. We explored whole genome amplification (WGA) to enable multiple molecular analyses. Methods: Eighty-eight advanced-stage NSCLC patients were enrolled. Their low-volume lung biopsies underwent WGA before direct sequencing for epidermal growth factor receptor (EGFR), KRAS (rat sarcoma virus), p53, and CMET (mesenchymal-epithelial transition factor) mutations. Overall survival impact was examined. Surgically-resected tumors from 133 early-stage NSCLC patients were sequenced for EGFR, KRAS and p53 mutations. We compared the mutation frequencies of both groups. Results: It is feasible for low-volume lung biopsies to undergo WGA for mutational analysis. KRAS and CMET mutations have a deleterious effect on overall survival, hazard ratios 5.05 (p = 0.009) and 23.65 (p = 0.005), respectively. EGFR and p53 mutations, however, do not have a survival impact. There also does not seem to be significant differences in the frequency of mutations in EGFR, KRAS, and p53 between early- and advanced-stage disease: 20% versus 24% (p = 0.48), 29% versus 27% (p = 0.75), 10% versus 6% (p = 0.27), respectively. Conclusions: In advanced-stage NSCLC, KRAS, and CMET mutations suggest poor prognosis, whereas EGFR and p53 mutations do not seem to have survival impact. Mutations in EGFR, KRAS and p53 are unlikely to be responsible for the progression of NSCLC from early- to late-stage disease. WGA may be used to expand starting deoxyribonucleic acid from low-volume lung biopsies for further analysis of advanced-stage NSCLC.

AZD6244 (ARRY-142886) enhances the therapeutic efficacy of sorafenib in mouse models of gastric cancer

Gastric cancer is a deadly disease for which current therapeutic options are extremely limited. Vascular endothelial growth factor receptors and platelet-derived growth factor receptors regulate gastric cancer cell proliferation, invasion, and tumor angiogenesis. In the present study, we report that sorafenib therapy effectively inhibited tumor growth and angiogenesis in tumor xenografts. These were associated with reduction in the phosphorylation of vascular endothelial growth factor receptor-2 Tyr951, c-Kit Tyr568/570, platelet-derived growth factor receptor-β Tyr1021, and Akt Ser473 and Thr308, down-regulation of positive cell cycle regulators, increased apoptosis, and up-regulation of p27. Sorafenib treatment also caused up-regulation of p-c-Raf Ser338 and p-extracellular signal-regulated kinase (ERK) Thr202/Tyr204 in gastric cancer xenografts. The combination of sorafenib and MAP/ERK kinase inhibitor AZD6244 enhances the effectiveness of each compound alone. Potential effect of sorafenib/AZD6244 included increase in proapoptotic Bim. Our data show that MAP/ERK kinase inhibition enhances the antitumor activity of sorafenib in vivo, supporting a rationale for multitargeted suppression of the angiogenesis and ERK signaling network in gastric cancer therapy. [Mol Cancer Ther 2009;8(9):2537–45]

Sunitinib (SUTENT, SU11248) Suppresses Tumor Growth and Induces Apoptosis in Xenograft Models of Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common and third deadliest primary neoplasm. Since HCC is a particularly vascular solid tumor, we determined the antitumor and antiangiogenic activities of sunitinib malate, a potent inhibitor of two receptors involved in angiogenesis - vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR). In the present study, we reported that treatment of HepG2 and SK-Hep-1 cells with sunitinib led to growth inhibition and apoptosis in a dose-dependent fashion. Sunitinib inhibited phosphorylation of VEGFR-2 at Tyr951 and PDGFR-beta at Tyr1021 both in vitro and in vivo. Sunitinib also suppressed tumor growth of five patient-derived xenografts. Sunitinib-induced tumor growth inhibition was associated with increased apoptosis, reduced microvessel density and inhibition of cell proliferation. This study provides a strong rationale for further clinical investigation of sunitinib in patients with hepatocellular carcinoma.

Early Detection of Breast Cancer through Population-Based Mammographic Screening in Asian Women: A Comparison Study between Screen-Detected and Symptomatic Breast Cancers

Abstract:  The first nation‐wide mammographic screening program in Asia, BreastScreen Singapore (BSS), was launched in Singapore in January 2002. This study compared the presentation and results of screen‐detected breast cancers with symptomatic breast cancers in two affiliated high‐volume institutions, one of which was an assessment centre for BSS. The medical records of patients diagnosed with primary breast cancer at the Department of General Surgery, Singapore General Hospital and the Department of Surgical Oncology, National Cancer Centre, Singapore, during the period January 2002 to December 2003 were reviewed. Clinical and pathological comparisons were made between screen‐detected lesions and symptomatic lesions. Of a total of 767 cases, 640 (83.4%) were invasive carcinomas and 127 (16.6%) were ductal carcinoma in‐situ (DCIS) lesions. Only 13.4% of them were screen‐detected. Compared to symptomatic cancers, screen‐detected lesions were of smaller size (median size 18 versus 23 mm), a lower stage (stages 0–2, 95 versus 83.2%) and histologic grade (grade 1–2, 71 versus 60%), with a higher incidence of DCIS (31.0 versus 14.3%) and had higher rates of breast conservation (45.6 versus 28.2%) (all p‐values <0.05). By multivariate analysis, tumor palpability, tumor size >20 mm, nodal involvement, cerbB2 overexpression, and advanced disease stage were independent poor prognostic factors for disease‐free survival, whereas nodal involvement, advanced disease, and recurrence predicted poor cancer‐specific survival. However, there was no statistically significant difference in disease‐free survival or cancer‐specific survival between the two groups at a median follow‐up of 38 months. Screening mammography has allowed the detection of smaller and hence oncologically more favorable lesions in Asian women. Although no significant survival benefit was demonstrated in our study, a longer period of follow‐up is essential before the benefit of mortality reduction, as a result of mammography screening becomes evident in our population.

Clinical Validation of a Customized Multiple Signature Microarray for Breast Cancer

Purpose: Current histopathologic systems for classifying breast tumors require evaluation of multiple variables and are often associated with significant interobserver variability. Recent studies suggest that gene expression profiles may represent a promising alternative for clinical cancer classification. Here, we investigated the use of a customized microarray as a potential tool for clinical practice. Experimental Design: We fabricated custom 188-gene microarrays containing expression signatures for three breast cancer molecular subtypes [luminal/estrogen receptor (ER) positive, human epidermal growth factor receptor 2 (HER2), and “basaloid”], the Nottingham prognostic index (NPI-ES), and low histologic grade (TuM1). The reliability of these multiple-signature arrays (MSA) was tested in a prospective cohort of 165 patients with primary breast cancer. Results: The MSA-ER signature exhibited a high concordance of 90% with ER immunohistochemistry reported on diagnosis (P < 0.001). This remained unchanged at 89% (P < 0.001) when the immunohistochemistry was repeated using current laboratory standards. Expression of the HER2 signature showed a good correlation of 76% with HER2 fluorescence in situ hybridization (FISH; ratio ≥2.2; P < 0.001), which further improved to 89% when the ratio cutoff was raised to ≥5. A proportion of low-level FISH-amplified samples (ratio, 2.2-5) behaved comparably to FISH-negative samples by HER2 signature expression, HER2 quantitative reverse transcription-PCR, and HER2 immunohistochemistry. Luminal/ER+ tumors with high NPI-ES expression were associated with high NPI scores (P = 0.001), and luminal/ER+ TuM1-expressing tumors were significantly correlated with low histologic grade (P = 0.002) and improved survival outcome in an interim analysis (hazard ratio, 0.2; P = 0.019). Conclusion: The consistency of the MSA platform in an independent patient population suggests that custom microarrays could potentially function as an adjunct to standard immunohistochemistry and FISH in clinical practice.

Impact of Smoking and Brain Metastasis on Outcomes of Advanced EGFR Mutation Lung Adenocarcinoma Patients Treated with First Line Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors.

Objectives This purpose of this study was to examine clinical-pathologic factors – particularly smoking and brain metastases – in EGFR mutation positive (M+) lung adenocarcinoma (ADC) to determine their impact on survival in patients treated with first line EGFR TKI. Methods A retrospective review of EGFR mutation reflex testing experience for all ADC diagnosed at a tertiary Asian cancer centre from January 2009 to April 2013. Amongst this cohort, patients with advanced EGFR M+ ADC treated with first line EGFR TKI were identified to determine factors that influence progression free and overall survival. Results 444/742 (59.8%) ADC reflex tested for EGFR mutations were EGFR M+. Amongst never-smokers (n=468), EGFR M+ were found in 74.5% of females and 76.3% of males, and amongst ever smokers (n=283), in 53.3% of females and 35.6% of males. Exon 20 mutations were found more commonly amongst heavy smokers (> 50 pack years and > 20 pack years, Pearson’s chi square p=0.044, and p=0.038 respectively). 211 patients treated with palliative first line TKI had a median PFS and OS of 9.2 and 19.6 months respectively. 26% of patients had brain metastasis at diagnosis. This was significantly detrimental to overall survival (HR 1.85, CI 1.09-3.16, p=0.024) on multivariate analysis. There was no evidence that smoking status had a significant impact on survival. Conclusions The high prevalence of EGFR M+ in our patient population warrants reflex testing regardless of gender and smoking status. Smoking status and dosage did not impact progression free or overall survival in patients treated with first line EGFR TKI. The presence of brain metastasis at diagnosis negatively impacts overall survival.