2003 Publication

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	Fig. 1. A schematic overview of the information flow in genomic/proteomic identification and validation of candidate clinical markers for distinguishing ovarian and colon cancers.
	Fig. 2. Clustered Image Map of seven colon and six ovarian carcinoma cell lines for the top 600 candidate clones. Entries are color-coded expression ratio (E) values [log10(CH2FL/CH1FL)]. Red indicates a high positive value; blue indicates a high negative value. There are two distinct expression patterns, corresponding to the two cell classes.
	Fig. 3. A log10 mRNA expression ratio for seven colon and six ovarian carcinoma cell lines. B, top, the IMAGE clone sequence alignment for villin mRNA. Exons are indicated by solid boxes. The two mRNAs are represented below. Positions of translation initiation codons (AUG), stop codons (UGA), and polyadenylation signals (AAUAA or AAUAAA) are indicated. The alignment of the IMAGE clone (791 bp) is indicated by a solid bar. The difference between the two mRNAs in the 3_-noncoding region is thought to be generated by alternative choice of polyadenylation signal rather than by alternative splicing of exons (27). Bottom, the IMAGE clone sequence alignment of moesin mRNA. Positions of initiation, stop, and polyadenylation codons are indicated as described for villin, except that the stop codon is UAA.
	Fig. 4. Staining with human anti-villin (a_d) and moesin (e_h) antibodies on TARP tissue microarrays. Overview of colon (a and e) and ovarian (b and f) array specimens. With antivillin antibody, there is strong staining of colon epithelium (c) but no significant staining of anything in most of the ovarian sections (d). As shown at higher magnification, stromal cells in the colon sections are positively stained with moesin (g), but the tumor epithelium is largely unstained. In contrast, higher magnification of an ovarian specimen (h) shows strong epithelial staining. Because of the stromal staining by moesin low power in (e) do not appear impressively negative.

Abstract: Colon and ovarian cancers can be difficult to distinguish in the abdomen, and the distinction is important because it determines which drugs will be used for therapy. To identify molecular markers for that differential diagnosis, we developed a multistep protocol starting with the 60 human cancer cell lines used by the National Cancer Institute to screen for new anticancer agents. The steps included: (a) identification of candidate markers using cDNA microarrays; (b) verification of clone identities by resequencing; (c) corroboration of transcript levels using Affymetrix oligonucleotide chips; (d) quantitation of protein expression by “reversephase” protein microarray; and (e) prospective validation of candidate markers on clinical tumor sections in tissue microarrays. The two best candidates identified were villin for colon cancer cells and moesin for ovarian cancer cells. Because moesin stained stromal elements in both types of cancer, it would probably not have been identified as a marker if we had started with mRNA or protein profiling of bulk tumors. Villin appears at least as useful as the currently used colon cancer marker cytokeratin 20, and moesin also appears to have utility. The multistep process introduced here has the potential to produce additional markers for cancer diagnosis, prognosis, and therapy.