Free DNA and carcinoembryonic antigen serum levels: an important combination for diagnosis of colorectal cancer.

PURPOSE: The identification of new molecular markers for the early detection of colorectal cancer has become an important objective. We compared the sensitivity and specificity of free circulating DNA with that of the more conventional carcinoembryonic antigen (CEA) and evaluated the two markers in combination. EXPERIMENTAL DESIGN: The study was carried out on 75 healthy donors and 75 colorectal cancer patients. Free DNA was determined in serum with quantitative PCR analysis. The diagnostic accuracy of each assay was calculated using receiver operating characteristic (ROC) curves. The diagnostic relevance of the two-marker combination was analyzed by the logistic regression model. RESULTS: Median free DNA concentration was approximately 5-fold higher in patients than in healthy donors (P < 0.001). The area under the ROC curve was 0.86, and when 12.5 ng/mL was used as cutoff, 81.3% sensitivity and 73.3% specificity were observed for the overall series. As CEA and free DNA provided independent diagnostic information, they were also considered in combination. ROC curve analysis of the combined CEA and free DNA algorithms showed a higher diagnostic capacity (area under the ROC curve, 0.92) than that of markers considered singly, with 84% sensitivity and 88% specificity. CONCLUSIONS: Free circulating DNA, especially when used in combination with CEA, represents a potentially useful tool for the diagnosis of early-stage colorectal cancer.

Mutation analysis of p53, K-ras, and BRAF genes in colorectal cancer progression.

Gene mutations in APC, K-ras, and p53 are thought to be essential events for colorectal cancer development. Recent data seem to indicate that K-ras and p53 mutations rarely co-exist in the same tumor, indicating that these alterations do not represent a synergistic evolutionary pathway. Moreover, an inverse relation between K-ras gene activation and BRAF mutations has been demonstrated, suggesting alternative pathways for colorectal cancer transformation. To reconstruct the chronological modulation of these gene mutations during cell transformation and colorectal cancer progression, mutations of p53, K-ras, and BRAF genes were analyzed by Single Strand Conformation Polymorphism (SSCP) or sequencing analysis in 100 colorectal cancer samples, evenly distributed among different Dukes' stages. We found mutations in p53, K-ras, and BRAF genes in 35%, 30%, and 4% of tumors, respectively, and observed a minimal or no co-presence of these gene alterations. Moreover, the frequency of molecular p53 mutations increased as tumor stage increased, suggesting an important role for this gene in the progression of colorectal cancer. Conversely, K-ras or BRAF genes were not related to tumor stage or location. These data seem to indicate the absence of a co-presence of the genes, highlighting the possibility of multiple pathways for colorectal tumor progression. Moreover, mutations in p53, K-ras, and BRAF are not present in about one-third of colorectal cancers and therefore other gene mutations need to be investigated to better understand molecular mechanisms at the basis of cell transformation and the progression of colorectal cancer.

Detection of colorectal cancer by a quantitative fluorescence determination of DNA amplification in stool.

DNA amplification of exfoliated cells in stool represents an inexpensive and rapid test, but has only 50% to 60% sensitivity. A new quantitative method, called fluorescence long DNA, was developed and validated in our laboratory on stool obtained from 86 patients with primary colorectal cancer and from 62 healthy individuals. It consists of the amplification of stool DNA with fluorescence primers and the quantification of the amplification using a standard curve. Results are arbitrarily expressed in nanograms. The potential of the new method compared to the conventional approach was analyzed in a subgroup of 94 individuals (56 patients and 38 healthy volunteers). In the present series, DNA amplification analysis showed a specificity of 97% and a sensitivity of only 50%. Conversely, fluorescence DNA evaluation, using the best cutoff of 25 ng, showed a sensitivity of about 76% and a specificity of 93%. Similar sensitivity was observed regardless of Dukes stage, tumor location, and size, thus also permitting the detection of early-stage tumors. The present study seems to indicate that quantitative fluorescence DNA determination in stool successfully identifies colorectal cancer patients with a sensitivity comparable, if not superior, to that of multiple gene analysis but at a lower cost and in a shorter time.