The characterized 13 DEGs (2-fold up-regulated), such as v-myb avian myeloblastosis viral oncogene homolog (MYB), recombination activating gene-1 (RAG-1), epithelial cell adhesion molecule (TACSTD1
), Secale cereale 75k gamma secalin gene (75k gamma secalin ), snRNA (U6), microRNA mir-155 (ssc-mir-155), microRNA let-7c (ssc-let-7c), NCK interacting protein with SH3 domain/cadherin, EGF LAG seven-pass G-type receptor 3 (CELSR3,NCKIPSD), cAMP-regulated phosphoprotein, 21kDa (ARPP21), interleukin 20 receptor beta (IL20RB), DNA nucleotidylexotransferase (DNTT), collagen, type V, alpha 2 (COL5A2), and microRNA mir-374a/microRNA mir-374b/microRNA mir-421/microRNA mir-545 (ssc-mir-374a) are shown in Supplementary Table I.
Briefly, reactions were performed with a probe concentration of 200 nmol/L and a 60-s anneal/extend phase at 60 [degrees]C for GUSB,  CK19, CK20, EGFR, MGB1, PTHrP, SCCA, and SFTPB; 62 [degrees]C for CK7, MAGEA-plex, MART1, and MGB2; and 64 [degrees]C for CEA, LUNX, PIP, TACSTD1, TM4SF3, and TYR.
When we used median expression in the tumors as the numerator (median tumor/highest normal), 6 genes (CK7, CK19, TACSTD1, MGB1, MGB2, and PIP) for breast cancer, 5 genes (CEA, CK19, CK20, TACSTD1, and TM4SF3) for colon cancer, 6 genes (CEA, CK19, CK7, TACSTD1, CK20, and TM4SF3) for esophageal cancer, 4 genes (CEA, CK19, SCCA, and PTHrP) for head and neck cancer, 7 genes (CEA, CK7, CK19, LUNX, SCCA, SFTPB, and TACSDT1) for lung cancer, and 3 genes (MAGEA-plex, MART1, and TYR) for melanoma clearly stood out as having mean tumor:highest normal blood ratios ~1500-25,000.
Similarly, the best 2- to 4-marker combination for the other cancer types would be TM4SF3 and TACSTD1 for colon cancer (100% of 28 tumors >1000-fold; minimum, 1.23 x [10.sup.3]), TM4SF3 and CK7 for esophageal cancer (89.5% of 19 tumors; 4.64 x [10.sup.2]), PTHrP, EGFR, and SCCA for head and neck cancer (100% of 26 tumors; 3.28 x [10.sup.3]), CK7, EGFR, SCCA, and SFTPB for lung cancer (100% of 22 tumors; 2.26 x [10.sup.3]), and MAGEA-plex, MART1, and TYR for melanoma (100% of 24 tumors; 3.27 x [10.sup.3]).
Five of these genes--CDX1, CEA, CK19, TACSTD1, and Villin1 (VIL1)--have median tumor/highest benign node ratios >300 and therefore have the potential to detect small foci of tumor while still differentiating negative nodes.
On the basis of expression results in positive nodes and distribution of expression in benign nodes, TACSTD1 expression was, at a minimum, 595-fold higher in positive nodes than the highest expression in benign nodes.