Han Chong Toh

A comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co-occurrence among distinct therapeutic targets

Objective Gastric cancer is a major gastrointestinal malignancy for which targeted therapies are emerging as treatment options. This study sought to identify the most prevalent molecular targets in gastric cancer and to elucidate systematic patterns of exclusivity and co-occurrence among these targets, through comprehensive genomic analysis of a large panel of gastric cancers. Design Using high-resolution single nucleotide polymorphism arrays, copy number alterations were profiled in a panel of 233 gastric cancers (193 primary tumours, 40 cell lines) and 98 primary matched gastric non-malignant samples. For selected alterations, their impact on gene expression and clinical outcome were evaluated. Results 22 recurrent focal alterations (13 amplifications and nine deletions) were identified. These included both known targets (FGFR2, ERBB2) and also novel genes in gastric cancer (KLF5, GATA6). Receptor tyrosine kinase (RTK)/RAS alterations were found to be frequent in gastric cancer. This study also demonstrates, for the first time, that these alterations occur in a mutually exclusive fashion, with KRAS gene amplifications highlighting a clinically relevant but previously underappreciated gastric cancer subgroup. FGFR2-amplified gastric cancers were also shown to be sensitive to dovitinib, an orally bioavailable FGFR/VEGFR targeting agent, potentially representing a subtype-specific therapy for FGFR2-amplified gastric cancers. Conclusion The study demonstrates the existence of five distinct gastric cancer patient subgroups, defined by the signature genomic alterations FGFR2 (9% of tumours), KRAS (9%), EGFR (8%), ERBB2 (7%) and MET (4%). Collectively, these subgroups suggest that at least 37% of gastric cancer patients may be potentially treatable by RTK/RAS directed therapies.

Intrinsic Subtypes of Gastric Cancer, Based on Gene Expression Pattern, Predict Survival and Respond Differently to Chemotherapy

BACKGROUND & AIMS Gastric cancer (GC) is a heterogeneous disease comprising multiple subtypes that have distinct biological properties and effects in patients. We sought to identify new, intrinsic subtypes of GC by gene expression analysis of a large panel of GC cell lines. We tested if these subtypes might be associated with differences in patient survival times and responses to various standard-of-care cytotoxic drugs. METHODS We analyzed gene expression profiles for 37 GC cell lines to identify intrinsic GC subtypes. These subtypes were validated in primary tumors from 521 patients in 4 independent cohorts, where the subtypes were determined by either expression profiling or subtype-specific immunohistochemical markers (LGALS4, CDH17). In vitro sensitivity to 3 chemotherapy drugs (5-fluorouracil, cisplatin, oxaliplatin) was also assessed. RESULTS Unsupervised cell line analysis identified 2 major intrinsic genomic subtypes (G-INT and G-DIF) that had distinct patterns of gene expression. The intrinsic subtypes, but not subtypes based on Laurens histopathologic classification, were prognostic of survival, based on univariate and multivariate analysis in multiple patient cohorts. The G-INT cell lines were significantly more sensitive to 5-fluorouracil and oxaliplatin, but more resistant to cisplatin, than the G-DIF cell lines. In patients, intrinsic subtypes were associated with survival time following adjuvant, 5-fluorouracil-based therapy. CONCLUSIONS Intrinsic subtypes of GC, based on distinct patterns of expression, are associated with patient survival and response to chemotherapy. Classification of GC based on intrinsic subtypes might be used to determine prognosis and customize therapy.

Epstein-Barr virus-associated smooth muscle tumors are distinctive mesenchymal tumors reflecting multiple infection events: a clinicopathologic and molecular analysis of 29 tumors from 19 patients.

Epstein-Barr virus-associated smooth muscle tumors (EBV-SMT) are rare lesions that occur in immunocompromised patients. Because they have not been fully characterized pathologically or at the molecular level, we have studied 29 tumors from 19 patients, the largest series to date. Cases coded as EBV-SMT were identified in 19 patients from consultation files and from the renal transplant database at Singapore General Hospital. EBV-SMT occurred in adults (mean age 39 years; range, 21-57 years) and predominantly affected males (12 male, 7 female). Causes of immunocompromise were renal transplantation (10), AIDS (8), and steroid therapy (1). Tumors were located in soft tissue (5), lung (5), liver (4), and miscellaneous sites (15). In 13 patients (68%), the tumors were multiple. Infection with EBV was confirmed in all cases by in situ hybridization for EBV early RNAs (EBER). EBV-SMT were typically well-differentiated smooth muscle tumors with little atypia and usually a low level of mitotic activity. Unlike classic leiomyosarcomas, they lacked significant pleomorphism but frequently displayed primitive round cell areas and prominent intratumoral T lymphocytes. No consistent relationship between histologic features and clinical outcome was noted. All expressed actin (29 of 29) and less frequently desmin (14 of 26). Multiple tumors in a given patient were clonally distinct as assessed by the long terminal repeat region of the virus, supporting the view that multifocal tumors arise from multiple infection events rather than from metastasis. Strain typing by analysis of the EBNA-3C gene confirmed the presence of EBV type 2. Two of four tumors assessed were positive for a 30-bp deletion in the LMP1 gene. EBV copy number per cell ranged greatly between patients and between tumors from the same patient. Follow-up information was available in 18 of 19 patients (mean, 25 months; range, 1-105 months). Fifteen patients were alive: 11 with disease and 4 without. Three patients died, 1 due to disease. We conclude that EBV-SMT are histologically distinct from classic soft tissue smooth muscle tumors, are not readily evaluated by means of conventional histologic criteria, and in the case of multifocal tumors are the result of multiple infection events rather than metastasis. EBV-2 can transform smooth muscle cells, independent of the presence of the LMP1 deletion associated with greater virulence.

Inflammatory tumour microenvironment is associated with superior survival in hepatocellular carcinoma patients

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is an aggressive malignancy with few treatment options. As the status of the tumour immune microenvironment can affect progression of established tumours, we evaluated potential immune mechanisms associated with survival in HCC. METHODS Immune gene expression profiles were analyzed in tumour and non-tumour liver tissues from resected HCC patients using quantitative PCR and immunohistochemistry. Tumour-infiltrating leukocytes (TILs) were isolated to verify the expression of immune genes and to identify proliferating TILs. These parameters were analyzed statistically in relation with patient survival and tumour phenotype (apoptosis and proliferation). RESULTS The immune microenvironment within tumours was found to be heterogeneous, although globally more inert compared to the adjacent non-tumour liver tissue. Univariate analysis in 61 patients identified a group of innate immune genes whose expression within tumours is positively associated with patient survival. TNF, IL6 and CCL2 are the most significant genes, with TNF being an independent predictor of survival in multivariate analysis. The gene set includes macrophage and NK-associated molecules such as TLR4, TLR3, CCR2, NCR3. Most of these molecules are expressed by TILs. Importantly, proliferating immune cells, predominantly NK and T cells, are present in tumours of patients with longer survival, and exclusively in areas devoid of proliferating tumour cells. NK and CD8(+) T cell densities are correlated positively with tumour apoptosis, and negatively with tumour proliferation. CONCLUSIONS Hence, an inflammatory immune microenvironment within HCC tumours could be an important means to control tumour progression via TIL activation and proliferation.

Chemokine-driven lymphocyte infiltration: an early intratumoural event determining long-term survival in resectable hepatocellular carcinoma

Objective Hepatocellular carcinoma (HCC) is a heterogeneous disease with poor prognosis and limited methods for predicting patient survival. The nature of the immune cells that infiltrate tumours is known to impact clinical outcome. However, the molecular events that regulate this infiltration require further understanding. Here the ability of immune genes expressed in the tumour microenvironment to predict disease progression was investigated. Methods Using quantitative PCR, the expression of 14 immune genes in resected tumour tissues from 57 Singaporean patients was analysed. The nearest-template prediction method was used to derive and test a prognostic signature from this training cohort. The signature was then validated in an independent cohort of 98 patients from Hong Kong and Zurich. Intratumoural components expressing these critical immune genes were identified by in situ labelling. Regulation of these genes was analysed in vitro using the HCC cell line SNU-182. Results The identified 14 immune-gene signature predicts patient survival in both the training cohort (p=0.0004 and HR=5.2) and the validation cohort (p=0.0051 and HR=2.5) irrespective of patient ethnicity and disease aetiology. Importantly, it predicts the survival of patients with early disease (stages I and II), for whom classical clinical parameters provide limited information. The lack of predictive power in late disease stages III and IV emphasises that a protective immune microenvironment has to be established early in order to impact disease progression significantly. This signature includes the chemokine genes CXCL10, CCL5 and CCL2, whose expression correlates with markers of T helper 1 (Th1), CD8+ T and natural killer (NK) cells. Inflammatory cytokines (tumour necrosis factor α, interferon γ) and Toll-like receptor 3 ligands stimulate intratumoural production of these chemokines which drive tumour infiltration by T and NK cells, leading to enhanced cancer cell death. Conclusion A 14 immune-gene signature, which identifies molecular cues driving tumour infiltration by lymphocytes, accurately predicts survival of patients with HCC especially in early disease.

RAD001 (everolimus) inhibits tumour growth in xenograft models of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and highly resistant to available chemotherapies. Mammalian target of rapamycin (mTOR) functions to regulate protein translation, angiogenesis and cell cycle progression in many cancers including HCC. In the present study, subcutaneous patient‐derived HCC xenografts were used to study the effects of an mTOR inhibitor, RAD001 (everolimus), on tumour growth, apoptosis and angiogenesis. We report that oral administration of RAD001 to mice bearing patient‐derived HCC xenografts resulted in a dose‐dependent inhibition of tumour growth. RAD001‐induced growth suppression was associated with inactivation of downstream targets of mTOR, reduction in VEGF expression and microvessel density, inhibition of cell proliferation, up‐regulation of p27Kip1 and down‐regulation of p21Cip1/Waf1, Cdk‐6, Cdk‐2, Cdk‐4, cdc‐25C, cyclin B1 and c‐Myc. Our data indicate that the mTOR pathway plays an important role in angiogenesis, cell cycle progression and proliferation of liver cancer cells. Our study provides a strong rationale for clinical investigation of mTOR inhibitor RAD001 in patients with HCC.

MicroRNA-224 is up-regulated in hepatocellular carcinoma through epigenetic mechanisms

MicroRNA‐224 (miR‐224) is one of the most commonly up‐regulated microRNAs in hepatocellular carcinoma (HCC), which affects crucial cellular processes such as apoptosis and cell proliferation. In this study, we aim to elucidate the molecular mechanism that leads to the overexpression of miR‐224 in HCC. We examined the transcript expression of miR‐224 and neighboring miR‐452 and genes on chromosome Xq28 in tumor and paired adjacent nontumorous tissues from 100 patients with HCC and found that miR‐224 is coordinately up‐regulated with its neighboring microRNA (miRNA) and genes. This coordinated up‐regulation of miRNAs and genes at the Xq28 locus can be mimicked in nontransformed immortalized human liver cells by the introduction of histone deacetylase (HDAC) inhibitors, which resulted in a corresponding increase in histone H3 acetylation in this region. This miR‐224‐residing locus in Xq28 is reciprocally regulated by HDAC1, HDAC3, and histone acetylase protein, E1A binding protein p300 (EP300). Notably, in HCC tumors that significantly overexpress microRNA‐224, EP300 is also overexpressed and displays increased binding to the Xq28 locus. In transformed HCC cells, high miR‐224 expression can be attenuated through the inhibition of EP300, using either siRNA or the specific drug C646. In summary, overexpression of EP300 may account, in part, for the up‐regulation of miR‐224 expression in patients with HCC.—Wang, Y., Toh, H. C., Chow, P., Chung, A. Y. F., Meyers, D. J., Cole, P. A., Ooi, L. L. P. J., Lee, C. G. L. MicroRNA‐224 is up‐regulated in hepatocellular carcinoma through epigenetic mechanisms. FASEB J. 26, 3032–3041 (2012). www.fasebj.org

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.

Aspirin as adjuvant therapy for colorectal cancer—reinterpreting paradigms

A high-quality body of evidence supports the use of aspirin in reducing sporadic and hereditary adenomatous polyps, and numerous observational studies point to a reduction in colorectal cancer (CRC) risk. However, using aspirin as an adjuvant therapy in established CRC was until recently inconceivable. Now, evidence from both observational and clinical trials of aspirin for other indications suggests that aspirin initiation after (or before) the diagnosis of CRC improves CRC-specific mortality. These exciting findings need to be confirmed in prospective randomized trials that are underway. The recent failure of adjuvant irinotecan, bevacizumab, and cetuximab clinical trials compels us to reconsider our assumptions and paradigms for treating CRC. In this Review, we summarize clinical and preclinical evidence supporting aspirin use in established CRC and outline a framework for better understanding aspirin activity in the pathogenesis of CRC. We describe the data supporting adjuvant aspirin in resected CRC, including the issues of dose, duration and toxicity, and discuss potential biomarkers that may help better select patients for aspirin therapy.

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

BACKGROUND/AIMS Hepatocellular carcinoma is a leading cause of global cancer mortality, with standard chemotherapy being minimally effective in prolonging survival. We investigated if combined targeting of vascular endothelial growth factor protein and expression might affect hepatocellular carcinoma growth and angiogenesis. METHODS We treated patient-derived hepatocellular carcinoma xenografts with (i) bevacizumab; (ii) rapamycin; and (iii) bevacizumab plus rapamycin. Western blotting was employed to determine changes in the proteins. Apoptosis, vascular endothelial growth factor expression, microvessel density, and cell proliferation were analyzed by immunohistochemistry. RESULTS Hepatocellular carcinoma growth was inhibited by bevacizumab plus rapamycin treatment to a significantly greater degree than bevacizumab or rapamycin monotherapy. Reductions in tumor growth by bevacizumab plus rapamycin were associated with inhibition of downstream targets of the mammalian target-of-rapamycin pathway, reductions in vascular endothelial growth factor expression, and tumor microvessel density. Potentially additive effects of bevacizumab plus rapamycin included reductions in vascular endothelial growth factor expression, cyclin D1, and cyclin B1. In an intra-peritoneal model of hepatocellular carcinoma, bevacizumab plus rapamycin potently inhibited both intra-liver and intra-abdominal tumor growth, reduced ascites levels, and significantly prolonged mouse survival. CONCLUSIONS Bevacizumab and rapamycin, which are both clinically approved drugs, may represent a novel molecularly-targeted combination treatment for hepatocellular carcinoma.