FREQUENT DETECTION OF RAS AND P53 MUTATIONS IN BRUSH CYTOLOGY SAMPLESFROM LUNG-CANCER PATIENTS BY A RESTRICTION FRAGMENT LENGTH POLYMORPHISM-BASED ENRICHED PCR TECHNIQUE

RFLP-mediated PCR has been successfully applied as a reliable tool in the detection of ras mutations in many cancers and provides a basis fo r ''mutant-enriched PCR'' protocols, We have, therefore, modified this technique to the sensitive detection of K-ras codon 12 and also p53 ' 'hot spot'' mutations, which, frequently in lung cancer, affect codons at the positions 157, 175, 245, 248, 249, and 273. With a high sensit ivity of 1 mutant allele in 10(4) normal alleles, our enrichment assay allows the detection of oncogene alleles when only a few tumor cells are present within a normal cell population, Brush cytology material o btained from the tumor site of 20 patients with endoscopically apparen t bronchial carcinoma was compared to macroscopically normal mucosa ta ken from the contralateral bronchus (''control'' cytology), We found K -ras codon 12 mutations in 5 cases (25%) and p53 mutations in 13 cases (65%) in the tumor-derived cell material but, with the exception of t wo cases, not in cell material taken from the control cytology, Sevent y-five % of the samples analyzed showed that at least one of the two o ncogenes was affected, In several cases, two p53 lesions were detected concomitantly, The majority of the mutations could be reconfirmed by an alternative approach exploiting changes in the genomic RFLP pattern induced by these mutations and were also demonstrated in separate dia gnostic biopsies taken, Thus, we conclude that the established enriche d PCR protocol ensures a high sensitivity and preserved specificity fo r the diagnosis of oncogene lesions associated with lung cancer, Becau se conventional techniques normally yield a lower incidence of corresp onding ras and p53 mutations, we think that both the high rate and the heterogeneity of p53 mutations found in some cases are, indeed, relat ed to the increased sensitivity of this new enriched PCR technique.