Tatiana Ivanova

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.

Oncogenic pathway combinations predict clinical prognosis in gastric cancer.

Many solid cancers are known to exhibit a high degree of heterogeneity in their deregulation of different oncogenic pathways. We sought to identify major oncogenic pathways in gastric cancer (GC) with significant relationships to patient survival. Using gene expression signatures, we devised an in silico strategy to map patterns of oncogenic pathway activation in 301 primary gastric cancers, the second highest cause of global cancer mortality. We identified three oncogenic pathways (proliferation/stem cell, NF-kappaB, and Wnt/beta-catenin) deregulated in the majority (>70%) of gastric cancers. We functionally validated these pathway predictions in a panel of gastric cancer cell lines. Patient stratification by oncogenic pathway combinations showed reproducible and significant survival differences in multiple cohorts, suggesting that pathway interactions may play an important role in influencing disease behavior. Individual GCs can be successfully taxonomized by oncogenic pathway activity into biologically and clinically relevant subgroups. Predicting pathway activity by expression signatures thus permits the study of multiple cancer-related pathways interacting simultaneously in primary cancers, at a scale not currently achievable by other platforms.

Methylation Subtypes and Large-Scale Epigenetic Alterations in Gastric Cancer

A large-scale genomic survey of epigenetic alterations in gastric cancer identifies clinically relevant molecular subgroups. The Silent Treatment of Gastric Cancer A new study by Zouridis and colleagues refutes the old adage that “silence is golden”—at least in the realm of gene methylation and epigenetic silencing in cancer. To decipher the effects of “silence” on gastric cancer, the authors analyzed gene methylation patterns in 240 gastric tumors and compared them to those of 94 matched samples of adjacent normal tissue. Gastric cancer is one of the most common types of cancer worldwide—and one of the most deadly, with few effective treatment options available. As a possible source of therapeutic targets, scientists are searching for genetic and epigenetic alteration patterns characteristic of these tumors. Here, the authors extensively characterized methylation patterns in human gastric cancers, which revealed tumor-specific arrangements of hyper- and hypomethylation. Zouridis and colleagues also identified a subset of cancers that fell into the CpG island methylator phenotype (CIMP) subgroup, which is associated with more extensive methylation and lower chances of survival in younger patients. As a possible pharmaceutical intervention, the authors tested the effects of the demethylating drug 5-aza-2′-deoxycytidine in CIMP tumor cell lines and found that their proliferation was significantly decreased when compared with non-CIMP cell lines. A broader analysis of gene regions that undergo modifications in cancers likely will identify new therapeutic targets and corresponding treatments. But for patients with high-risk gastric cancers that fall into the CIMP subgroup, silenced DNA is golden because it serves as a target for currently available drugs. Epigenetic alterations are fundamental hallmarks of cancer genomes. We surveyed the landscape of DNA methylation alterations in gastric cancer by analyzing genome-wide CG dinucleotide (CpG) methylation profiles of 240 gastric cancers (203 tumors and 37 cell lines) and 94 matched normal gastric tissues. Cancer-specific epigenetic alterations were observed in 44% of CpGs, comprising both tumor hyper- and hypomethylation. Twenty-five percent of the methylation alterations were significantly associated with changes in tumor gene expression. Whereas most methylation-expression correlations were negative, several positively correlated methylation-expression interactions were also observed, associated with CpG sites exhibiting atypical transcription start site distances and gene body localization. Methylation clustering of the tumors revealed a CpG island methylator phenotype (CIMP) subgroup associated with widespread hypermethylation, young patient age, and adverse patient outcome in a disease stage–independent manner. CIMP cell lines displayed sensitivity to 5-aza-2′-deoxycytidine, a clinically approved demethylating drug. We also identified long-range regions of epigenetic silencing (LRESs) in CIMP tumors. Combined analysis of the methylation, gene expression, and drug treatment data suggests that certain LRESs may silence specific genes within the region, rather than all genes. Finally, we discovered regions of long-range tumor hypomethylation, associated with increased chromosomal instability. Our results provide insights into the epigenetic impact of environmental and biological agents on gastric epithelial cells, which may contribute to cancer.

Inhibition of gastric cancer invasion and metastasis by PLA2G2A, a novel β-catenin/TCF target gene

Elevated expression of the PLA2G2A phospholipase in gastric cancer (GC) is associated with improved patient survival. To elucidate function and regulation of PLA2G2A in GC, we analyzed a panel of GC cell lines. PLA2G2A was specifically expressed in lines with constitutive Wnt activity, implicating beta-catenin-dependent Wnt signaling as a major upstream regulator of PLA2G2A expression. The invasive ability of PLA2G2A-expressing AGS cells was enhanced by PLA2G2A silencing, whereas cellular migration in non-PLA2G2A-expressing N87 cells was inhibited by enforced PLA2G2A expression, indicating that PLA2G2A is both necessary and sufficient to function as an inhibitor of GC invasion in vitro. We provide evidence that antiinvasive effect of PLA2G2A occurs, at least in part, through its ability to inhibit the S100A4 metastasis mediator gene. Consistent with its invasion inhibitor role, PLA2G2A expression was elevated in primary gastric, colon, and prostrate early-stage tumors, but was decreased in metastatic and late-stage tumors. There was a strong association between PLA2G2A promoter methylation status and PLA2G2A expression, suggesting that the loss of PLA2G2A expression in late-stage cancers may be due to epigenetic silencing. Supporting this, among the non-PLA2G2A-expressing lines, pharmacologic inhibition of epigenetic silencing reactivated PLA2G2A in Wnt-active lines, but in non-Wnt-active lines, a combination of Wnt hyperactivation and inhibition of epigenetic silencing were both required for PLA2G2A reactivation. Our results highlight the complexity of PLA2G2A regulation and provide functional evidence for PLA2G2A as an important regulator of invasion and metastasis in GC.

Comprehensive genomic meta-analysis identifies intra-tumoural stroma as a predictor of survival in patients with gastric cancer

Objective Gastric adenocarcinoma (gastric cancer, GC) is a major cause of global cancer mortality. Identifying molecular programmes contributing to GC patient survival may improve our understanding of GC pathogenesis, highlight new prognostic factors and reveal novel therapeutic targets. The authors aimed to produce a comprehensive inventory of gene expression programmes expressed in primary GCs, and to identify those expression programmes significantly associated with patient survival. Design Using a network-modelling approach, the authors performed a large-scale meta-analysis of GC transcriptome data integrating 940 gastric transcriptomes from multiple independent patient cohorts. The authors analysed a training set of 428 GCs and 163 non-malignant gastric samples, and a validation set of 288 GCs and 61 non-malignant gastric samples. Results The authors identified 178 gene expression programmes (‘modules’) expressed in primary GCs, which were associated with distinct biological processes, chromosomal location patterns, cis-regulatory motifs and clinicopathological parameters. Expression of a transforming growth factor β (TGF-β) signalling associated ‘super-module’ of stroma-related genes consistently predicted patient survival in multiple GC validation cohorts. The proportion of intra-tumoural stroma, quantified by morphometry in tissue sections from gastrectomy specimens, was also significantly associated with stromal super-module expression and GC patient survival. Conclusion Stromal gene expression predicts GC patient survival in multiple independent cohorts, and may be closely related to the intra-tumoural stroma proportion, a specific morphological GC phenotype. These findings suggest that therapeutic approaches targeting the GC stroma may merit evaluation.

Integrated epigenomics identifies BMP4 as a modulator of cisplatin sensitivity in gastric cancer

Objective Cisplatin is a widely used gastric cancer (GC) chemotherapy; however, genetic factors regulating GC responses to cisplatin remain obscure. Identifying genes regulating cisplatin resistance could aid clinicians in tailoring treatments, by distinguishing cisplatin sensitive patients from those who might benefit from alternative platinum therapies, and highlight novel targeted strategies for overcoming cisplatin resistance. Here integrated epigenomics is applied to identify genes associated with GC cisplatin resistance. Design 20 GC cell lines were subjected to gene expression profiling, DNA methylation profiling and drug response assays. The molecular data were integrated to identify genes highly expressed and unmethylated specifically in cisplatin-resistant lines. Candidate genes were functionally tested by several in vitro and in vivo assays. Clinical impact of candidate genes was also assessed in a cohort of 197 GC patients. Results Epigenomic analysis identified bone morphogenetic protein 4 (BMP4) as an epigenetically regulated gene highly expressed in cisplatin-resistant lines. Functional assays confirmed that BMP4 is necessary and sufficient for the expression of several prooncogenic traits, likely mediated through stimulation of the epithelial-mesenchymal transition. In primary tumours, BMP4 promoter methylation levels were inversely correlated with BMP4 expression, and patients with high BMP4-expressing tumours exhibited significantly worse prognosis. Therapeutically, targeted genetic inhibition of BMP4 caused significant sensitisation of GC cells to cisplatin. Notably, BMP4-expressing GCs also did not exhibit cross resistance to oxaliplatin. Conclusions BMP4 epigenetic and expression status may represent promising biomarkers for GC cisplatin resistance. Targeting BMP4 may sensitise GC cells to cisplatin. Oxaliplatin, a clinically acceptable cisplatin alternative, may represent a potential therapeutic option for BMP4-positive GCs.

Abstract PR1: Genomic discovery of CD44-SLC1A2 gene fusions in gastric cancer

Among the various genomic abnormalities associated with cancers, fusion genes and transcripts are particularly notable due to their cancer-specific nature and translational potential as diagnostic and therapeutic targets. In pediatric acute lymphoblastic leukemia (ALL), assays detecting the AML-ETO and PML-RAR fusion genes are routinely used to diagnose particular clinical subtypes, and treatment of chronic myelogenous leukemia (CML), a disease once associated with extremely poor prognosis, has been revolutionized by imatinib, an inhibitor of the BCR-ABL fusion gene. While fusion genes are well- known in hematological malignancies, our knowledge of recurrent fusion events in solid epithelial tumors is far less mature. To date, only two fusion genes have been identified in solid tumors, including TMPRSS2-ERG in prostate cancer, and EML4-ALK in non-small cell lung cancer. No recurrent fusion genes have been so far identified in common gastrointestinal cancers such as stomach, colon and esophageal adenocarcinoma. In this study, to identify genes affected by genome rearrangements in gastric cancer (GC), which is the second highest cause of global cancer mortality, we analyzed chromosomal imbalances in a cohort of 123 primary GCs and cell lines, and discovered several tumors exhibiting recurrent genomic breakpoints in the SLC1A2/EAAT2 glutamate transporter. 5’ RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) analysis of a GC cell line with SLC1A2 breakpoints (SNU16) revealed expression of a CD44-SLC1A2 fusion transcript, computationally predicted to result in a truncated but functional SLC1A2 protein. In an independent tumor panel, CD44-SLC1A2 gene fusions were detected in 4% to 5% of GCs (2/43), but not in adjacent matched normal gastric tissues. Using custom-designed fusion-specific siRNAs, we show that silencing of CD44-SLC1A2 in fusion-positive SNU16 cells significantly reduced cellular proliferation, invasion, and colony formation, but not in cell lines lacking CD44-SLC1A2 expression. CD44-SLC1A2 silencing also significantly reduced intracellular glutamate levels and sensitized SNU16 cells to cisplatin. Fusions of SLC1A2 to upstream CD44 regulatory elements likely causes SLC1A2 transcriptional dysregulation, as tumors expressing abnormally high SLC1A2 levels also tended to be CD44-SLC1A2 positive. CD44-SLC1A2 gene fusions may be one mechanism used by cancers to establish a pro-oncogenic metabolic milieu favoring tumor growth and survival. We believe that our study will be of great interest to the gastric cancer community, as it represents the first recurrent fusion transcript identified in this disease (and for that matter any common gastrointestinal cancer). Citation Information: Clin Cancer Res 2010;16(14 Suppl):PR1.