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We attempted to identify potent marker genes using a new statistical analysis and developed a prediction system for individual response to platinum/paclitaxel combination chemotherapy in ovarian cancer patients based on the hypothesis that expression analysis of a set of the key drug sensitivity genes for platinum and paclitaxel could allow us to predict therapeutic response to the combination. From 10 human ovarian cancer cell lines, genes correlative in the expression levels with cytotoxicities of cisplatin (CDDP) and paclitaxel were chosen. We first selected five reliable prediction markers for the two drugs from 22 genes already known as sensitivity determinants and then identified another 8 novel genes through a two-dimensional mixed normal model using oligomicroarray expression data. Using expression data of genes quantified by real-time reverse transcription-PCR, we fixed the best linear model, which converted the quantified expression data into an IC50 of each drug. Multiple regression analysis of the selected genes yielded three prediction formulae for in vitro activity of CDDP and paclitaxel. In the same way, using the same genes selected in vitro, we then attempted to develop prediction formulae for progression-free survival to the platinum/paclitaxel combination. We therefore constructed possible formulae using different sets of 13 selected marker genes (5 known and 8 novel genes): Utility confirmation analyses using another nine test samples seemed to show that the formulae using a set of 8 novel marker genes alone could accurately predict progression-free survival (r = 0.683; P = 0.042). [Mol Cancer Ther 2006;5(3):767–75]

Supplementary Data from Prediction of individual response to platinum/paclitaxel combination using novel marker genes in ovarian cancers

We previously demonstrated that a new statistical analysis of oligomicroarray expression data based on a two-dimensional mixed normal model could provide a way to identify potent markers for drug sensitivity from the expression-sensitivity correlation analysis alone in esophageal squamous cell carcinoma (ESCC), and the selected genes, 3 for 5-FU and 6 for CDDP, were likely better drug-sensitivity markers, some of which may be the novel drug-response determinants, and developed highly predictive formulae in vitro and in vivo using expression data of a set of the all selected marker genes (100th AACR Annual Meeting). But interestingly, among these selected genes, empty spiracles homolog 2 (EMX2) showed discrepancies between chemo-sensitivity in vitro and patient prognosis in vivo, which led us to focus on the gene. EMX2, the homolog to the ‘empty spiracles’ gene in Drosophila, encodes a homeobox-containing transcription factor, which is critical for central nervous system but has not been reported as a prognostic factor in any cancers so far. So, we attempted to clarify the biological function of EMX2 in cancer cells whether it would be a drug-response determinant or a colony formation capacity determinant through the gene transfection analyses and the soft agar colony formation assay in vitro. Gene transfection analyses demonstrated that the ectopic expression of EMX2 did not correlate with the sensitivity to 5-FU in ESCC cell lines, excluding the former capacity. Meanwhile, we found that in 20 ESCC cell lines, while 9 cell lines with EMX2 little expression showed weak or none colony formation capacity, those with high EMX2 expression showed strong capacity in level dependent manner. Furthermore, colony formation capacity was reduced in EMX2 expression level-dependent manner by siRNA-mediated knockdown in cell lines with high EMX2 expression, suggesting a possible capacity as a colony formation capacity determinant. Using multiple regression analyses, we could develop highly predictive formulae of clinical prognosis in the patients treated with 5-FU/CDDP combination therapy using only 2 selected markers including EMX2, instead of using all the selected genes (Disease free survival: R of the model = 0.798, R of test sample: 0.828; Disease specific survival: R of the model = 0.828, R of test sample: 0.951), and confirmed that expression of EMX2 was a poor prognostic factor in the prediction formulae. These results indicate that EMX2 is the indispensable gene of anchorage-independent growth for ESCC, and high EMX2 expression level is associated with the early recurrence in ESCC. We propose that EMX2 is a novel and potent prognostic maker for ESCC, and elucidation of its molecular mechanism may help to develop new molecular targeting drugs that improve the patient prognosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1731.