Maria Pik Wong

The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS

The echinoderm microtubule‐associated protein‐like 4‐anaplastic lymphoma kinase (EML4‐ALK) fusion gene resulting from the chromosome inversion inv(2)(p21;p23) recently was identified in nonsmall cell lung cancer (NSCLC). The authors of this study investigated the frequency, genetic and clinicopathologic profiles of EML4‐ALK in Chinese patients with NSCLC.

Distinct Epidermal Growth Factor Receptor and KRAS Mutation Patterns in Non–Small Cell Lung Cancer Patients with Different Tobacco Exposure and Clinicopathologic Features

Purpose: This study evaluated the mutational profile of epidermal growth factor receptor (EGFR) and KRAS in non–small cell lung cancers in Hong Kong and determined their relation with smoking history and other clinicopathologic features. Experimental Design: Mutational profile of exons 18 to 21 of EGFR and codons 12, 13, and 61 of KRAS were determined in 215 adenocarcinomas, 15 squamous cell (SCC), and 11 EBV-associated lymphoepithelioma-like carcinomas (LELC). Results:EGFR mutations were prevalent in adenocarcinomas (115 of 215), uncommon in LELC (1 of 11), and not found in SCC (P < 0.001). Among adenocarcinomas, mutations were associated with nonsmokers (83 of 111; P < 0.001), female gender (87 of 131; P < 0.001), and well-differentiated (55 of 86) compared with poorly differentiated (11 of 41) tumors (P < 0.001). Decreasing mutation rates with increasing direct tobacco exposure was observed, with 74.8% (83 of 111) in nonsmokers, 61.1% (11 of 18) in passive, 35.7% (10 of 28) in previous, and 19.0% (11 of 58) in current smokers. There were 53% amino acid substitutions, 43% in-frame deletions, and 4% insertions. Complex patterns with 13% double mutations, including five novel substitutions, were observed. For KRAS, mutations occurred in adenocarcinoma only (21 of 215) and were associated with smokers (11 of 58; P = 0.003), men (14 of 84; P = 0.009) and poorly differentiated (7 of 41) compared with well-differentiated (4 of 86) tumors (P = 0.037). EGFR and KRAS mutations occurred in mutually exclusive tumors. Regression analysis showed smoking history was the significant determinant for both mutations, whereas gender was a confounding factor. Conclusion: This study shows EGFR mutations are prevalent in lung adenocarcinoma and suggests that it plays an increasing oncogenic role with decreasing direct tobacco damage.

Non-Small Cell Lung Cancer Cells Expressing CD44 Are Enriched for Stem Cell-Like Properties

Background The cancer stem cell theory hypothesizes that cancers are perpetuated by cancer stem cells (CSC) or tumor initiating cells (TIC) possessing self-renewal and other stem cell-like properties while differentiated non-stem/initiating cells have a finite life span. To investigate whether the hypothesis is applicable to lung cancer, identification of lung CSC and demonstration of these capacities is essential. Methodology/Principal Finding The expression profiles of five stem cell markers (CD34, CD44, CD133, BMI1 and OCT4) were screened by flow cytometry in 10 lung cancer cell lines. CD44 was further investigated by testing for in vitro and in vivo tumorigenecity. Formation of spheroid bodies and in vivo tumor initiation ability were demonstrated in CD44+ cells of 4 cell lines. Serial in vivo tumor transplantability in nude mice was demonstrated using H1299 cell line. The primary xenografts initiated from CD44+ cells consisted of mixed CD44+ and CD44− cells in similar ratio as the parental H1299 cell line, supporting in vivo differentiation. Semi-quantitative Real-Time PCR (RT-PCR) showed that both freshly sorted CD44+ and CD44+ cells derived from CD44+-initiated tumors expressed the pluripotency genes OCT4/POU5F1, NANOG, SOX2. These stemness markers were not expressed by CD44− cells. Furthermore, freshly sorted CD44+ cells were more resistant to cisplatin treatment with lower apoptosis levels than CD44− cells. Immunohistochemical analysis of 141 resected non-small cell lung cancers showed tumor cell expression of CD44 in 50.4% of tumors while no CD34, and CD133 expression was observed in tumor cells. CD44 expression was associated with squamous cell carcinoma but unexpectedly, a longer survival was observed in CD44-expressing adenocarcinomas. Conclusion/Significance Overall, our results demonstrated that stem cell-like properties are enriched in CD44-expressing subpopulations of some lung cancer cell lines. Further investigation is required to clarify the role of CD44 in tumor cell renewal and cancer propagation in the in vivo environment.

Expression of Nicotinic Acetylcholine Receptor Subunit Genes in Non–Small-Cell Lung Cancer Reveals Differences between Smokers and Nonsmokers

Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChR) on bronchial epithelial cells, can regulate cellular proliferation and apoptosis via activating the Akt pathway. Delineation of nAChR subtypes in non-small-cell lung cancers (NSCLC) may provide information for prevention or therapeutic targeting. Expression of nAChR subunit genes in 66 resected primary NSCLCs, 7 histologically non-involved lung tissues, 13 NSCLC cell lines, and 6 human bronchial epithelial cell lines (HBEC) was analyzed with quantitative PCR and microarray analysis. Five nonmalignant HBECs were exposed to nicotine in vitro to study the variation of nAChR subunit gene expression with nicotine exposure and removal. NSCLCs from nonsmokers showed higher expression of nAChR alpha6 (P < 0.001) and beta3 (P = 0.007) subunit genes than those from smokers, adjusted for gender. In addition, nAChR alpha4 (P < 0.001) and beta4 (P = 0.029) subunit gene expression showed significant difference between NSCLCs and normal lung. Using Affymetrix GeneChip U133 Sets, 65 differentially expressed genes associated with NSCLC nonsmoking nAChR alpha6beta3 phenotype were identified, which gave high sensitivity and specificity of prediction. nAChR alpha1, alpha5, and alpha7 showed significant reversible changes in expression levels in HBECs upon nicotine exposure. We conclude that between NSCLCs from smokers and nonsmokers, different nAChR subunit gene expression patterns were found, and a 65-gene expression signature was associated with nonsmoking nAChR alpha6beta3 expression. Finally, nicotine exposure in HBECs resulted in reversible differences in nAChR subunit gene expression. These results further implicate nicotine in bronchial carcinogenesis and suggest targeting nAChRs for prevention and therapy in lung cancer.

Expression of vascular endothelial growth factor and its receptors in the anaplastic progression of astrocytoma, oligodendroglioma, and ependymoma

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic factor, which is known to be upregulated in most cases of glioblastoma multiforme (GBM). The expression of VEGF and its receptors in ependymomas, oligodendrogliomas, and particularly the expression during anaplastic progression of these three types of gliomas has not been studied extensively. Fifty-six gliomas, consisting of 10 ependymomas, 12 oligodendrogliomas, 3 anaplastic oligodendrogliomas, 6 astrocytomas grade II, 5 anaplastic astrocytomas, and 20 glioblastoma multiformes, were investigated for VEGF and receptor expression using in situ hybridization (ISH) and reverse transcription polymerase chain reaction (RT-PCR). Results showed that VEGF was moderately to strongly expressed in 8 of 10 ependymomas and in all anaplastic oligodendrogliomas and glioblastoma multiforme cases. These tumors displayed similar degrees of extensive necrosis and vascular proliferation, with VEGF expression consistently seen in tumor cells around necrotic areas. The VEGF expression, although present at a lower level, also was shown in 4 of 12 oligodendrogliomas, in 3 of 6 astrocytomas grade II, and in 2 of 5 anaplastic astrocytomas, with a regional rather than diffuse pattern of positive result. The findings from the in situ hybridization study correlated with the expression index, as determined by reverse transcription polymerase chain reaction. Expression of VEGF was correlated significantly with vascular proliferation (p < 10(-5)) and necrosis (p < 10(-5)), as well as with microvessel density (p = 0.002, rs = 0.41). The VEGF receptors, kinase domain region (KDR) and Fms-like-tyrosine kinase (Flt-1), also were upregulated in the tumor vasculature of glioblastoma multiforme, anaplastic oligodendrogliomas, and ependymomas with necrosis, whereas the astrocytomas grade II, anaplastic astrocytomas, and oligodendroglioma tumors tended to express a weak to nondetectable signal. Anaplastic progression in all three types of gliomas is heralded by the occurrence of small zones of VEGF-expressing cells and early vascular proliferation, followed by an accelerated phase of angiogenesis closely associated with VEGF induction around areas of necrosis and with the expression of VEGF receptors in the tumor vasculature.

Monocyte chemoattractant protein-1 expression and macrophage infiltration in gliomas

Abstract While the number of reports on macrophage infiltration of gliomas is increasing, the extent and mechanisms of macrophage recruitment remain unclear. To investigate whether monocyte chemoattractant protein-1 (MCP-1) plays a role in this process, in situ hybridisation (ISH) was performed for 22 glioblastomas (GBM), 1 anaplastic astrocytoma (AA) and 4 grade II fibrillary astrocytomas (AII) and reverse transcription-polymerase chain reaction was performed in 13 GBM, 1 AA and 3 AII. High levels of MCP-1 mRNA were detectable in most GBM, while a lower level was detected in AII. Many tumour-associated macrophages (TAM) could be demonstrated by immunohistochemistry (IHC) in most GBM, while the AII contained a lower number of TAM. The positive correlation between the MCP-1 level and abundance of TAM suggested that MCP-1 has a role in TAM recruitment. By combining ISH and IHC, high levels of MCP-1 mRNA were shown both in tumour cells and TAM. Along tumour borders, reactive astrocytes and microglia also expressed MCP-1. In areas with T lymphocyte infiltration, larger numbers of MCP-1-positive cells with an enhanced level of expression could be identified. We propose that the mechanism of macrophage recruitment is, at least partly, effected by constitutive expression and T cell-mediated up-regulation of MCP-1 in tumour cells and TAM. The production of MCP-1 by TAM establishes a positive amplification circuit for macrophage recruitment in gliomas.

GWASdb: a database for human genetic variants identified by genome-wide association studies

Recent advances in genome-wide association studies (GWAS) have enabled us to identify thousands of genetic variants (GVs) that are associated with human diseases. As next-generation sequencing technologies become less expensive, more GVs will be discovered in the near future. Existing databases, such as NHGRI GWAS Catalog, collect GVs with only genome-wide level significance. However, many true disease susceptibility loci have relatively moderate P values and are not included in these databases. We have developed GWASdb that contains 20 times more data than the GWAS Catalog and includes less significant GVs (P < 1.0 × 10−3) manually curated from the literature. In addition, GWASdb provides comprehensive functional annotations for each GV, including genomic mapping information, regulatory effects (transcription factor binding sites, microRNA target sites and splicing sites), amino acid substitutions, evolution, gene expression and disease associations. Furthermore, GWASdb classifies these GVs according to diseases using Disease-Ontology Lite and Human Phenotype Ontology. It can conduct pathway enrichment and PPI network association analysis for these diseases. GWASdb provides an intuitive, multifunctional database for biologists and clinicians to explore GVs and their functional inferences. It is freely available at http://jjwanglab.org/gwasdb and will be updated frequently.

Vascular endothelial growth factor is up-regulated in the early pre-malignant stage of colorectal tumour progression.

Angiogenesis is an essential requirement for the development, progression and metastasis of malignant tumours. Studies on transgenic mouse models have shown that angiogenesis begins in the pre‐malignant phase of oncogenesis, when dysplastic lesions acquire an increased microvasculature. To investigate the relationship between the expression of vascular endothelial growth factor (VEGF) and colorectal tumour progression, we have studied VEGF expression level and splice variant pattern by semi‐quantitative RT‐PCR and the cellular source of VEGF expression by in situ hybrization (ISH) in a range of lesions that modelled the tumour‐development pathway from normal colon to invasive colorectal adenocarcinomas. Colonic adenomas showed a statistically significant up‐regulation of VEGF expression over normal tissues, with a further increase during the development of adenocarcinomas. Tumour cells formed the major source of VEGF expression, with a minor contribution from mononuclear cells in the tumour stroma and enhanced expression in tumour cells around necrotic regions. The comparable expression level in both the in situ and invasive components in the same tumours indicated that a high VEGF expression capacity had been acquired prior to establishment of the invasive phenotype. Our findings support activation of VEGF as the molecular basis for the discrete induction of angiogenesis in the pre‐malignant phase of colorectal tumour development. Int. J. Cancer81:845–850, 1999.

Tissue-specific expression pattern of vascular endothelial growth factor isoforms in the malignant transformation of lung and colon.

Angiogenesis, a prerequisite for tumor growth and progression, results from a shift in the equilibrium between angiogenic factors and angiogenic inhibitors. Vascular endothelial growth factor (VEGF) has been identified as one of the most important factors mediating angiogenesis in physiological and pathological conditions. Through alternative splicing, four isoforms of VEGF are formed, consisting of 206, 189, 165, and 121 amino acids, respectively. VEGF206 and VEGF189 differ from VEGF165 and VEGF121 in their bioavailability, with the longer forms being matrix-bound and the shorter forms freely diffusible. To investigate the relative importance of the VEGF isoforms in neoplastic transformation, we studied the pattern of splice variant expression by reverse transcription polymerase chain reaction (RT-PCR) in 18 lung and 11 colonic carcinomas and their corresponding normal tissues, respectively. The findings showed a significant upregulation of VEGF in both carcinomas, with VEGF165 and VEGF121 being the predominant forms; VEGF189 was significantly expressed in normal lung but not colon; and VEGF206 was not detected in any specimen. The findings indicate that during malignant progression, an angiogenic switch favoring the shorter diffusible isoforms is likely to confer on the tumor a growth advantage. From the differential expression of VEGF isoforms in normal lung and colonic tissues, different functional roles of the splice variants is suggested. In particular, VEGF189 may be important for the maintenance of the vascular integrity of the lung.

A novel KIF5B-ALK variant in nonsmall cell lung cancer

The anaplastic lymphoma kinase (ALK) gene is involved frequently in chromosomal translocations, resulting in fusion genes with different partners found in various lymphoproliferative conditions. It was recently reported in nonsmall cell lung cancer (NSCLC) that the fusion protein encoded by echinoderm microtubule‐associated protein‐like 4‐ALK (EML4‐ALK) fusion gene conferred oncogenic properties. The objective of the current study was to identify other possible ALK fusion genes in NSCLC.