Combined DNA flow cytometry and sorting with k‐ras2 mutation spectrum analysis and the prognosis of human sporadic colorectal cancer

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Abstract
Background: Activation of the k‐ras2 pathways and chromosomal instability leading to aneuploidy in human sporadic colorectal cancer (sCRC) is essential to the tumor cell ability to survive, grow, and metastatize. Methods: The study included 135 patients with sCRC who were followed up for a median of 72 months. Multiple fresh‐frozen fragments obtained from superficial and invasive areas of the tumors were mixed and used to detect the degree of DNA aneuploidy (DNA index [DI]) and S‐phase fraction by two scatter signals and 4,6‐diamidino‐2‐phenylindole‐2‐hydrocloride (DAPI) fluorescence flow cytometry (FCM). PCR amplification and k‐ras2 mutation spectrum analysis were performed using enriched epithelial nuclei after sorting DNA aneuploid nuclei and DNA diploid nuclei from which tissue‐infiltrating lymphocytes were absent. Results: DNA aneuploidy was detected in 98 (73%) and k‐ras2 mutations in 54 cases (40%). Univariate analyses of overall survival with both Dukes' A to D or B to C series of cases showed that DNA multiple aneuploidy, k‐ras2 mutations, older age, and distal site, but not increased S‐phase fraction, were predictive of worse outcome. Multivariate Cox models strongly indicated that k‐ras2 mutations, but neither single nor multiple DNA aneuploidy, were an independent prognostic factor in both series of patients. In particular, with B and C Dukes' stage patients (n = 110), the relative risk (RR) of death was above 2.5 with k‐ras2 mutations and above 3 with the G→C/T transversions. Conclusion: Combined FCM and k‐ras2 analysis may be used to predict long‐term increased risk of death in sCRC patients. Cytometry (Clin. Cytometry) 50:216–224, 2002.