Codon 12 K-ras mutations in plasma DNA are not an indicator of disease in non-small-cell lung cancer (NSCLC) patients

Abstract

7204 Background: Overexpression or mutational activation of the K-ras gene, mostly in codon 12, has been found in 30–80% of NSCLCs. Cell-free circulating DNA genetically identical to that of the primary tumor has been detected in the plasma of cancer patients. K-ras mutations were examined in plasma DNA and paired tumor tissue specimens of patients with NSCLC and also in plasmas from a different cohort of NSCLC patients and healthy control subjects. Methods: An overall number of 48 NSCLC patients and 40 controls participated in the study, after written consent. Peripheral blood was collected in EDTA-coated Vacutainers. The DNA was purified from plasma aliquots of 12 NSCLC and the corresponding tumor tissue samples. The plasma DNA was isolated using both the Qiagen method and the modified Guanidine/Promega Resin (G/R) method according to Wang et al. (Kopreski et al. J Natl Cancer Inst 2000; 92:918–23; Wang et al. Clin Chem 2004; 50:211–3). The DNA was also isolated by the Qiagen method from an additional 76 plasma samples (36 NSCLC and 40 controls, respectively). The isolated DNA was amplified by means of a PCR method that enriches for mutations in codon 12 of the K-ras gene. The assay employs simultaneous restriction enzyme digestion and PCR amplification and is similar to previously reported assays described by Kopreski et al. All amplification assays included a K-Ras mutation-positive control. Results: The presence of K-ras mutations in cancer tissue showed no correlation with the corresponding plasma sample, either extracted by the Qiagen or by the G/R Method: Codon 12 K-ras mutations were present in 2 out of 12 tissue samples, while no mutation or two mutations different from those identified in the corresponding tumor tissue were detected in the plasma using the Quiagen and G/R methods, respectively. K-ras mutations were detected in 15 plasma samples collected from the additional 36 NSCLC patients (41.7%) as well as from 12/40 controls (30.0%). Conclusions: Our results do not support the use of codon 12 K-ras mutations in plasma DNA as a diagnostic marker in NSCLC. (This study was funded by the Assessorato Sanità Regione Piemonte - Progetti Ricerca Sanitaria Finalizzata 2003) No significant financial relationships to disclose.