FREQUENT DETECTION OF RAS AND P53 MUTATIONS IN BRUSH CYTOLOGY SAMPLESFROM LUNG-CANCER PATIENTS BY A RESTRICTION FRAGMENT LENGTH POLYMORPHISM-BASED ENRICHED PCR TECHNIQUE
M. Behn et al., FREQUENT DETECTION OF RAS AND P53 MUTATIONS IN BRUSH CYTOLOGY SAMPLESFROM LUNG-CANCER PATIENTS BY A RESTRICTION FRAGMENT LENGTH POLYMORPHISM-BASED ENRICHED PCR TECHNIQUE, Clinical cancer research, 4(2), 1998, pp. 361-371
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.