Intratumor heterogeneity and tissue distribution of KRAS mutation in non-small cell lung cancer: implications for detection of mutated KRAS oncogene in exhaled breath condensate

Abstract

Purpose: Mutated KRAS oncogene in exhaled breath condensate (EBC) can be a genetic marker of non-small cell lung cancer (NSCLC). However, a possibility of inhomogeneous distribution in cancer tissue and intratumor heterogeneity of KRAS mutation may decrease its significance. We investigated a status of KRAS point mutation and its sequence at codon 12 in 51 NSCLC patients after tumor resection. The comparison of KRAS mutation status between EBC-DNA and cancer tissue was performed in 19 cases.

Methods: Five cancer tissue samples from disparate tumor regions and one from normal lung were harvested at surgery. EBC was collected for DNA analysis the previous day. KRAS point mutations at codon 12 were detected using mutant-enriched PCR technique and pyrosequenced.

Results: Forty-six cancers revealed concordance of KRAS mutation status: 27 contained mutated KRAS and 19 had only wild KRAS. Five NSCLCs revealed inhomogeneous distribution of KRAS mutation. Two different mutations were found in 14 NSCLCs and the most frequent one was G12D and G12V (n = 8). No mutated KRAS was found in normal lung. The concordance ratios of KRAS sequence in codon 12 between EBC-DNA and cancer were 18/19 for NSCLC patients and 11/12 for KRAS mutation positive NSCLC.

Conclusions: Intratumor heterogeneity and inhomogeneous distribution of KRAS point mutation in codon 12 in cancer tissue can occur in NSCLCs. There was a high accordance between KRAS mutation status in EBC-DNA and cancer tissue in NSCLC patients what suggests usefulness of monitoring KRAS mutation in EBC-DNA as a biomarker of NSCLC.

Keywords: Exhaled breath condensate; Genetic biomarkers; Heterogeneity; KRAS oncogene mutation; Non-small cell lung cancer.

Fig. 1

a Cross section of resected right lung with squamous cell carcinoma from a 74-year-old male patient. White squares A, B, C, D and E indicate loci of cancer tissue harvesting (samples of about 60–100 mg of wet mass). Normal lung parenchyma was collected from the place marked with square F. b Hematoxylin–eosin stained slide (fivefold magnification) prepared from a cancer tissue sample collected from locus A. The black perimeter shows the zone containing more than 90% of cancer cells which was excised for DNA isolation and KRAS mutation analysis. Slides prepared from cancer samples collected from remaining four loci revealed very similar histological images