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

Elio Geido, Andrea Sciutto, Alessandra Rubagotti, Cristina Oliani, Roberto Monaco, Mauro Risio, Walter Giaretti

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish
Pages (from-to)216-224
Number of pages9
JournalCytometry Part B - Clinical Cytometry
Issue number4
Publication statusPublished - 2002


  • Aneuploidy
  • Chromosome instability
  • Oncogenes
  • Tumor progression

ASJC Scopus subject areas

  • Biophysics
  • Cell Biology
  • Endocrinology
  • Hematology
  • Pathology and Forensic Medicine


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