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Poster Papers
primary tumors or a lack of sufficient primary tumor for the experiments.
To address the issue of having insufficient primary tumor available for
research purposes, our lab grew xenografts from fresh primary tumors
in nude mice. The methylation and expression patterns of tumor-derived
xenografts, tumor-derived cell lines, and cell lines derived from normal
oral mucosa were then compared to primary tumors. Design: Genomic
DNA and total RNA were extracted from primary tumors, xenografts,
and cell lines. Genomic DNA samples were analyzed by methylation
microarrays. The RNA samples were analyzed using expression
microarrays. Data from the microarrays were normalized and then
analyzed independently using unsupervised hierarchical clustering. The
data from both microarray platforms were subsequently analyzed using
Significance Analysis of Microarrays (SAM) to generate lists differentially
methylated and differentially expressed genes. Subjects: Genomic DNA
and total RNA were extracted from five HNSCC cell lines (JHU-O11,
JHU-O22, FADU, UM-22A and UM-22B), two cell lines derived from
normal oral mucosa (OKF6 and NOK-SI), four primary HNSCC tumors
and four matched tumor-derived xenografts grown in nude mice.
Results: Unsupervised hierarchical clustering analysis of the normalized
methylation array data demonstrated similar overall methylation in the
primary tumors, xenografts and OKF6 cell line compared to the HNSCC
cell lines and NOK-SI cell line, which was evident at the first branch point
in the dendrogram. Globally, all of the cell lines except the OKF6 cell line
were hypermethylated compared to the primary tumors and xenografts.
Expression analysis demonstrated a clustering of primary tumors and
xenografts into one group and all cell lines into a second group after
the first branch point on the dendrogram. SAM analysis identified 239
genes up-regulated and 941 genes down-regulated in both tumors
and xenografts when compared to the cell lines. Conclusions: Tumorderived
xenografts appear to better represent the methylation and gene
expression signatures seen in primary tumors than HNSCC cell lines.
P006 (COSM Poster #28)
THE ROLE OF MAGEA2 IN THE TUMORIGENIC PATHWAY OF
HNSCC. Chad A Glazer, MD, Ian M Smith, MD, Sheetal Bhan, PhD,
Wenyue Sun, PhD, Steven S Chang, MD, Kavita M Pattani, MD, William
Westra, MD, Zubair Khan, MD, Joseph A Califano, MD; Johns Hopkins
Medical Institutions.
Objective: The disruption of p53 function via missense somatic
mutations is known to be a central factor in the oncogenic pathway
leading to HNSCC; however, many primary HNSCC tumors lack
mutations in the TP53 tumor suppressor gene. It is currently unknown
how the p53 pathway is silenced in this subset of HNSCC. Recent
evidence has shown that the MAGEA family may be involved in the
downstream silencing of p53 gene targets via the MAGEA2-p53 complex
shown to be active in melanoma. In this study, we examine the role of
MAGEA2 in the tumorigenesis of HNSCC. Methods: Primary tissue
microarray data and quantitative RT-PCR (qRT-PCR) in a separate cohort
of primary tissue showed that MAGEA2 is differentially overexpressed in
HNSCC. Anchorage dependent growth studies and cell cycle analysis
using flow cytometry were performed in normal upper aerodigestive cell
lines after transfection with a MAGE2 construct. Small interfering RNAs
(siRNA) were then used to knockdown MAGEA2 in HNSCC cells, and
growth characteristics were examined. qRT-PCR was used to evaluate
expression changes of p53 downstream targets following transfection
of MAGEA2 into normal upper aerodigestive cell lines. Results: Cancer
Outlier Profile Analysis (COPA) was used to analyze 49 HNSCC tumors
and 19 normal upper aerodigestive tissue mRNA expression arrays.
This analysis showed that MAGEA2 was significantly overexpressed
in 15/49 tumors (30%) (p