Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis

Alevizos, Ilias; Mahadevappa, M.; Zhang, X; Ohyama, Hiroe; Kohno, Yohko; Posner, Marshall R.; Gallagher, George; Varvares, Mark A.; Cohen, Donald M.; Kim, D; Kent, Ralph; Donoff, R. Bruce; Todd, Randy; Yung, CW; Warrington, Janet A.; Wong, David T.

doi:10.1038/sj.onc.1204685

Cited by 210 publications

(155 citation statements)

References 40 publications

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“…We generated the signature gene set using a widely accepted statistical method, SAM, that selects genes that are differentially expressed between two groups and estimates a false discovery rate (Tusher et al, 2001). Due to the wide variation in published gene signatures even pertaining to the relatively narrow question of transformation in HNSCC, we considered the accurate prediction of an independent validation set essential to evaluating our results (Alevizos et al, 2001;Mendez et al, 2002;Gonzalez et al, 2003;Hwang et al, 2003;Leethanakul et al, 2003;Ntzani and Ioannidis, 2003). Principal-components analysis is also a useful visual tool but is not considered a robust predictor, and in our hands showed a trend differentiating NÀ from N þ .…”

Section: Discussionmentioning

confidence: 99%

“…HNSCC may be a useful system for studying lymphatic metastasis because of the high probability of lymphatic spread and low probability of hematogenous spread. A recent study has identified a gene signature for recurrent disease in HNSCC and several studies have investigated the changes in gene expression from normal tissue to carcinoma, but studies involving lymphatic metastasis have been limited by the small number of genes assessed and/or lack of a rigorous efficacy test of the metastatic signature (Alevizos et al, 2001;Belbin et al, 2002;Mendez et al, 2002;Hwang et al, 2003;Gonzalez et al, 2003;Leethanakul et al, 2003;Nagata et al, 2003;Chung et al, 2004;Ginos et al, 2004;Schmalbach et al, 2004;Warner et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Gene expression signature predicts lymphatic metastasis in squamous cell carcinoma of the oral cavity

et al. 2004

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Metastasis via the lymphatics is a major risk factor in squamous cell carcinoma of the oral cavity (OSCC). We sought to determine whether the presence of metastasis in the regional lymph node could be predicted by a gene expression signature of the primary tumor. A total of 18 OSCCs were characterized for gene expression by hybridizing RNA to Affymetrix U133A gene chips. Genes with differential expression were identified using a permutation technique and verified by quantitative RT-PCR and immunohistochemistry. A predictive rule was built using a support vector machine, and the accuracy of the rule was evaluated using crossvalidation on the original data set and prediction of an independent set of four patients. Metastatic primary tumors could be differentiated from nonmetastatic primary tumors by a signature gene set of 116 genes. This signature gene set correctly predicted the four independent patients as well as associating five lymph node metastases from the original patient set with the metastatic primary tumor group. We concluded that lymph node metastasis could be predicted by gene expression profiles of primary oral cavity squamous cell carcinomas. The presence of a gene expression signature for lymph node metastasis indicates that clinical testing to assess risk for lymph node metastasis should be possible.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Gene expression signature predicts lymphatic metastasis in squamous cell carcinoma of the oral cavity

et al. 2004

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“…4 -8 More recent work 9,10 has shown the feasibility of gene expression profiling studies using pure populations of cells obtained with laser capture microdissection (LCM). 11 We modified established protocols [12][13][14] for analyzing mRNA from microdissected frozen tissues and applied this protocol to ethanol-paraffin tissue. The direct comparison of frozen and ethanol-paraffin tissues provides insight into the advantages and disadvantages of each tissue preservation method.…”

Section: (J Mol Diagn 2004 6:371-377)mentioning

confidence: 99%

Comparison of Snap Freezing versus Ethanol Fixation for Gene Expression Profiling of Tissue Specimens

Perlmutter

Best

Gillespie

et al. 2004

The Journal of Molecular Diagnostics

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Frozen tissue specimens are the gold standard for molecular analysis. However, snap freezing presents several challenges regarding collection and storage of tissue, and preservation of histological detail. We evaluate an alternative preservation method, ethanol fixation followed by paraffin embedding, by analyzing expression profiles of microdissected cells on Affymetrix oligonucleotide arrays of three matched benign prostatic hyperplasia (BPH) and tumor samples processed with each preservation method. Frozen samples generated an average present call of 26% of the probe sets, compared to 4.5% in ethanol-paraffin samples. Eighty-eight percent of the probe sets called present in the ethanol-paraffin samples were also present in the frozen specimens. Comparing ethanolparaffin BPH to tumor, 52 probe sets showed a twofold differential expression or higher in at least two cases, 23 of which were also differentially expressed in at least one frozen case. Despite a significant drop in the number of transcripts detectable, the data suggests that the obtainable information in ethanol-fixed samples may be useful for molecular profiling where frozen tissue is not available. However, ethanol fixation and paraffin embedding of tissue specimens is not optimal for high-throughput mRNA expression analysis. Improved methods for transcript profiling of archival samples, and/or tissue processing are still required. Formalin fixation and paraffin embedding is the standard tissue processing method used in histopathology laboratories. This protocol allows for permanent preservation of the tissues, easy storage, and optimal histological quality. Unfortunately, formalin fixation severely compromises analysis of biomolecules, in particular mRNA and proteins. We have recently demonstrated the utility of an alternative fixation method, 70% ethanol followed by paraffin embedding. 1

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“…Although no connection between NMU expression and tumor cachexia (Tisdale, 2005) has yet been documented, such a link would be clinically important, as the design of small molecule inhibitors of NMU receptors is feasible. With regard to tumorigenesis, NMU has been shown to be downregulated in oral tumors (Alevizos et al, 2001), identified as an ovarian cancer-associated antigen (Euer et al, 2005), and acts as a potential autocrine growth factor for human myeloid leukemias (Shetzline et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Neuromedin U is regulated by the metastasis suppressor RhoGDI2 and is a novel promoter of tumor formation, lung metastasis and cancer cachexia

McRoberts

Berr

et al. 2006

Oncogene

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Most deaths from urinary bladder cancer are owing to metastatic disease. A reduction in Rho GDP Dissociation Inhibitor 2 (RhoGDI2) protein has been associated with increased risk of metastasis in patients with locally advanced bladder cancer, whereas in animal models, RhoGDI2 reconstitution in cells without expression results in lung metastasis suppression. Recently, we noted an inverse correlation between tumor RhoGDI2 and Neuromedin U (NMU) expression, suggesting that NMU might be a target of the lung metastasis suppressor effect of RhoGDI2. Here we evaluated whether NMU is regulated by RhoGDI2 and is functionally important in tumor progression. We used small interfering RNA knockdown of endogenous RhoGDI2 in poorly tumorigenic and nonmetastatic human bladder cancer T24 cells and observed increased NMU RNA expression. Although NMU overexpression did not increase the monolayer growth of T24 or related T24T poorly metastatic human bladder cancer cells, it did augment anchorage-independent growth for the latter. Overexpression of NMU in T24 and T24T cells significantly promoted tumor formation of both cell lines in nude mice, but did not alter the growth rate of established tumors. Furthermore, NMU-overexpressing xenografts were associated with lower animal body weight than control tumors, indicating a possible role of NMU in cancer cachexia. NMU overexpression in T24T cells significantly enhanced their lung metastatic ability. Bioluminescent in vivo imaging revealed that lung metastases in T24T grew faster than the same tumors in the subcutaneous microenvironment. In conclusion, NMU is a RhoGDI2-regulated gene that appears important for tumorigenicity, lung metastasis and cancer cachexia, and thus a promising therapeutic target in cancer.

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Oral cancer in vivo gene expression profiling assisted by laser capture microdissection and microarray analysis

Cited by 210 publications

References 40 publications

Gene expression signature predicts lymphatic metastasis in squamous cell carcinoma of the oral cavity

Gene expression signature predicts lymphatic metastasis in squamous cell carcinoma of the oral cavity

Comparison of Snap Freezing versus Ethanol Fixation for Gene Expression Profiling of Tissue Specimens

Neuromedin U is regulated by the metastasis suppressor RhoGDI2 and is a novel promoter of tumor formation, lung metastasis and cancer cachexia

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