Colorectal cancer (CRC) is the fourth most common non-cutaneous malignancy in the United States and the second most frequent cause of cancer-related death. One of the most important determinants of CRC survival is lymph node metastasis. To determine whether molecular markers might be prognostic for lymph node metastases, we measured by quantitative real-time RT -PCR the expression levels of 15 cancer-associated genes in formalin-fixed paraffin-embedded primary tissues derived from stage I -IV CRC patients with (n ¼ 20) and without (n ¼ 18) nodal metastases. Using the mean of the 15 genes as an internal reference control, we observed that low expression of b 2 microglobulin (B2M) was a strong prognostic indicator of lymph node metastases (area under the curve (AUC) ¼ 0.85; 95% confidence interval (CI) ¼ 0.69 -0.94). We also observed that the expression ratio of B2M/Spint2 had the highest prognostic accuracy (AUC ¼ 0.87; 95% CI ¼ 0.71 -0.96) of all potential two-gene combinations. Expression values of Spint2 correlated with the mean of the entire gene set at an R 2 value of 0.97, providing evidence that Spint2 serves not as an independent prognostic gene, but rather as a reliable reference control gene. These studies are the first to demonstrate a prognostic role of B2M at the mRNA level and suggest that low B2M expression levels might be useful for identifying patients with lymph node metastasis and/or poor survival.

Abstract. Colorectal cancer (CRC) is the second most frequent cause of cancer-related death in the United States. To determine whether certain molecular markers might be prognostic for survival, we measured by quantitative realtime RT-PCR the expression levels of 15 previously studied genes that are known to be up-regulated or down-regulated in the progression of epithelial cancers. The tumor samples were extracted from formalin-fixed paraffin-embedded primary tissues derived from patients with Stage II CRC who developed disease recurrence within two years (n=10), or were disease-free for at least 4 years (n=12). We were able to determine, by AUC curve analysis, that the ratio of microtubule associated protein 7 (Map7)/B2M was predictive of outcome in our sample set. Further, using Kaplan-Meier survival analysis, we observed significantly different curves as a function of marker positivity for the Map7/B2M (p=0.0001; HR=11) expression ratio. This suggests that the expression ratio of Map7/B2M may serve as a valuable prognostic marker in patients with Stage II colon cancer, and potentially guide therapeutic decision making.