Iain Beehuat Tan

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.

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.

Human Innate Lymphoid Cell Subsets Possess Tissue-Type Based Heterogeneity in Phenotype and Frequency

&NA; Animal models have highlighted the importance of innate lymphoid cells (ILCs) in multiple immune responses. However, technical limitations have hampered adequate characterization of ILCs in humans. Here, we used mass cytometry including a broad range of surface markers and transcription factors to accurately identify and profile ILCs across healthy and inflamed tissue types. High dimensional analysis allowed for clear phenotypic delineation of ILC2 and ILC3 subsets. We were not able to detect ILC1 cells in any of the tissues assessed, however, we identified intra‐epithelial (ie)ILC1‐like cells that represent a broader category of NK cells in mucosal and non‐mucosal pathological tissues. In addition, we have revealed the expression of phenotypic molecules that have not been previously described for ILCs. Our analysis shows that human ILCs are highly heterogeneous cell types between individuals and tissues. It also provides a global, comprehensive, and detailed description of ILC heterogeneity in humans across patients and tissues. Graphical Abstract Figure. No caption available. HighlightsComprehensive profiling of human ILCs across tissuesDetailed description of previously defined ILC subsets except helper‐type ILC1ieILC1‐like cells are present in several tissues and functionally similar to NK cellsIdentification of markers expressed on ILCs, including functional IL‐18R &NA; Animal models have highlighted the importance of innate lymphoid cells (ILCs) in multiple immune responses. Simoni et al. (2016) profile human ILCs using mass cytometry across tissues. The results provide a global, comprehensive, and detailed description of ILC populations and their heterogeneity across individuals and tissues.

Reference component analysis of single-cell transcriptomes elucidates cellular heterogeneity in human colorectal tumors

Intratumoral heterogeneity is a major obstacle to cancer treatment and a significant confounding factor in bulk-tumor profiling. We performed an unbiased analysis of transcriptional heterogeneity in colorectal tumors and their microenvironments using single-cell RNA–seq from 11 primary colorectal tumors and matched normal mucosa. To robustly cluster single-cell transcriptomes, we developed reference component analysis (RCA), an algorithm that substantially improves clustering accuracy. Using RCA, we identified two distinct subtypes of cancer-associated fibroblasts (CAFs). Additionally, epithelial–mesenchymal transition (EMT)-related genes were found to be upregulated only in the CAF subpopulation of tumor samples. Notably, colorectal tumors previously assigned to a single subtype on the basis of bulk transcriptomics could be divided into subgroups with divergent survival probability by using single-cell signatures, thus underscoring the prognostic value of our approach. Overall, our results demonstrate that unbiased single-cell RNA–seq profiling of tumor and matched normal samples provides a unique opportunity to characterize aberrant cell states within a tumor.

Signatures of tumour immunity distinguish Asian and non-Asian gastric adenocarcinomas

Objective Differences in gastric cancer (GC) clinical outcomes between patients in Asian and non-Asian countries has been historically attributed to variability in clinical management. However, recent international Phase III trials suggest that even with standardised treatments, GC outcomes differ by geography. Here, we investigated gene expression differences between Asian and non-Asian GCs, and if these molecular differences might influence clinical outcome. Design We compared gene expression profiles of 1016 GCs from six Asian and three non-Asian GC cohorts, using a two-stage meta-analysis design and a novel biostatistical method (RUV-4) to adjust for technical variation between cohorts. We further validated our findings by computerised immunohistochemical analysis on two independent tissue microarray (TMA) cohorts from Asian and non-Asian localities (n=665). Results Gene signatures differentially expressed between Asians and non-Asian GCs were related to immune function and inflammation. Non-Asian GCs were significantly enriched in signatures related to T-cell biology, including CTLA-4 signalling. Similarly, in the TMA cohorts, non-Asian GCs showed significantly higher expression of T-cell markers (CD3, CD45R0, CD8) and lower expression of the immunosuppressive T-regulatory cell marker FOXP3 compared to Asian GCs (p<0.05). Inflammatory cell markers CD66b and CD68 also exhibited significant cohort differences (p<0.05). Exploratory analyses revealed a significant relationship between tumour immunity factors, geographic locality-specific prognosis, and postchemotherapy outcomes. Conclusions Analyses of >1600 GCs suggest that Asian and non-Asian GCs exhibit distinct tumour immunity signatures related to T-cell function. These differences may influence geographical differences in clinical outcome, and the design of future trials particularly in immuno-oncology.

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.

Management of colon cancer: resource-stratified guidelines from the Asian Oncology Summit 2012.

Colon cancer is seen with increasing frequency in the Asia-Pacific region, and it is one of the most important causes of cancer mortality worldwide. This article reviews the available evidence for optimum management of colon cancer-in particular, with respect to screening and early detection of colon cancer, laparoscopic surgical treatment, adjuvant treatment of individuals with high-risk stage II and stage III cancer, palliative treatment of patients with metastatic disease, and management of resectable and potentially resectable metastases-and how these strategies can be applied in Asian countries with different levels of health-care resources and economic development, stratified by basic, limited, enhanced, and maximum resource levels.

Excessive fatty acid oxidation induces muscle atrophy in cancer cachexia

Cachexia is a devastating muscle-wasting syndrome that occurs in patients who have chronic diseases. It is most commonly observed in individuals with advanced cancer, presenting in 80% of these patients, and it is one of the primary causes of morbidity and mortality associated with cancer. Additionally, although many people with cachexia show hypermetabolism, the causative role of metabolism in muscle atrophy has been unclear. To understand the molecular basis of cachexia-associated muscle atrophy, it is necessary to develop accurate models of the condition. By using transcriptomics and cytokine profiling of human muscle stem cell–based models and human cancer-induced cachexia models in mice, we found that cachectic cancer cells secreted many inflammatory factors that rapidly led to high levels of fatty acid metabolism and to the activation of a p38 stress-response signature in skeletal muscles, before manifestation of cachectic muscle atrophy occurred. Metabolomics profiling revealed that factors secreted by cachectic cancer cells rapidly induce excessive fatty acid oxidation in human myotubes, which leads to oxidative stress, p38 activation and impaired muscle growth. Pharmacological blockade of fatty acid oxidation not only rescued human myotubes, but also improved muscle mass and body weight in cancer cachexia models in vivo. Therefore, fatty acid–induced oxidative stress could be targeted to prevent cancer-induced cachexia.