Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues.
A sensitive method was developed and applied to examine the distribution of K-ras gene mutations in histologically differing areas of lung tissues obtained from lung cancer patients. This method, which combines polymerase chain reaction (PCR), mutation allele enrichment (MAE), and denaturing gradient gel electrophoresis (DGGE), allows detection of one K-ras mutant allele present in 10(4) to 10(5) wild-type alleles. It was applied to analyze mutations in codon 12 of the K-ras gene in 43 tissue sites microdissected from paraffin-embedded sections obtained from 8 archival cases of lung cancer, all previously shown to have codon 12 K-ras mutations by direct sequencing. In four cases, mutations were detected only in the tumor, while in the other four cases, the same mutations were also found in tissues adjacent to tumors, using the MAE + DGGE method. No mutations were detected among normal-appearing cells in areas distant from the tumors in any of the cases studied. These findings demonstrate that K-ras mutations can be detected at low frequencies in normal-appearing cells from tissues adjacent to the tumor in some lung cancer cases. In addition, this approach also allowed detection of multiple mutations in colorectal tissues obtained from colorectal cancer patients. Thus, the MAE + DGGE method may be applicable to study of K-ras mutations in premalignant or morphologically suspicious lesions in bronchial mucosa or other types of human cancer.
Keohavong
P
P
Department of Environmental and Occupational Health, University of Pittsburgh, Pennsylvania 15213, USA.
Zhu
D
D
Whiteside
T L
TL
Swalsky
P
P
Bakker
A
A
Elder
E M
EM
Siegfried
J M
JM
Srivastava
S
S
Finkelstein
S D
SD
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D015179:Colorectal Neoplasms / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods* / Q000706:statistics & numerical data; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D011905:Genes, ras; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity
D003062:Codon; D017931:DNA Primers; D004273:DNA, Neoplasm