¹Charité – Universitätsmedizin Berlin, Campus Virchow-Klinikum, Medizinische Klinik und Poliklinik m.S. Hämatologie und Onkologie, Augustenburger Platz 1, 13353 Berlin, Germany

Correspondence: J Hänfler, E-mail: joachim.haenfler@charite.de

Received 26 July 2004; Revised 27 October 2004; Accepted 8 November 2004; Published online 11 January 2005.

Abstract

In the present study, we combined the PCR-clamping approach with melting curve analysis using mutant specific hybridisation probes and wild-type specific peptide nucleic acids (PNAs) to determine the genotypes of the most frequent point mutation in codon 12 of the proto-oncogene Ki-ras in tissue and plasma samples of patients with pancreatic cancer. The sensitivity of our assay was 1–5 10^-5. The melting curve analysis of tissue samples of four patients revealed two valine mutations, one none-valine mutation and one wild-type sequence. Ki-ras alterations were found in 28% of DNAs (18 out of 64) of nonrelated plasma samples of 10 patients with ductal adenocarcinoma of the pancreas. The valine mutation was the predominantly detected gene alteration (83%). Out of ten patients investigated, four patients (40%) became positive during clinical observation with respect to Ki-ras mutation. All four patients exhibited progressive disease and high levels of tumour marker CA 19-9. In conclusion, the one-step procedure discribed may be a useful clinical tool for analysing Ki-ras point mutations in tissue and plasmas samples. In addition, this method can be adapted for simultanous detection of multiple mutations and quantitation.