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

Cytometry. 2002 Aug 15;50(4):216-24. doi: 10.1002/cyto.10109.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Age Factors
  • Aged
  • Aneuploidy
  • Carcinoma / genetics*
  • Carcinoma / metabolism
  • Carcinoma / pathology
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / metabolism
  • Colorectal Neoplasms / pathology
  • DNA / metabolism*
  • Diploidy
  • Female
  • Flow Cytometry / instrumentation
  • Flow Cytometry / methods*
  • Follow-Up Studies
  • Genes, ras / genetics*
  • Humans
  • Male
  • Mutation / genetics
  • Predictive Value of Tests
  • Prognosis
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins p21(ras)
  • S Phase / genetics
  • Sex Factors
  • Spectrum Analysis / instrumentation
  • Spectrum Analysis / methods*
  • Survival Rate
  • ras Proteins


  • KRAS protein, human
  • Proto-Oncogene Proteins
  • DNA
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins