A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue

F. H. Sarkar, M. Valdivieso, J. Borders, K. L. Yao, M. M T Raval, S. K. Madan, P. Sreepathi, R. Shimoyama, Z. Steiger, Daniel W Visscher, J. D. Crissman

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized non-radioisotopic detection system using a single 6-μm tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non- small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.

Original languageEnglish (US)
Pages (from-to)266-273
Number of pages8
JournalDiagnostic Molecular Pathology
Volume4
Issue number4
StatePublished - 1995
Externally publishedYes

Fingerprint

ras Genes
p53 Genes
Non-Small Cell Lung Carcinoma
Paraffin
Formaldehyde
Mutation
Single-Stranded Conformational Polymorphism
Codon
Polymerase Chain Reaction
Exons
Adenocarcinoma
Genes
Oligonucleotide Probes
Sequence Deletion
Gene Silencing
DNA-Directed DNA Polymerase
Amino Acid Substitution
Southern Blotting
Tumor Suppressor Genes
DNA Sequence Analysis

Keywords

  • K-ras gene
  • Mutational analysis
  • Mutations
  • Non-small-cell lung cancer
  • p53 tumor suppression gene
  • Polymerase chain reaction

ASJC Scopus subject areas

  • Pathology and Forensic Medicine

Cite this

Sarkar, F. H., Valdivieso, M., Borders, J., Yao, K. L., Raval, M. M. T., Madan, S. K., ... Crissman, J. D. (1995). A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue. Diagnostic Molecular Pathology, 4(4), 266-273.

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue. / Sarkar, F. H.; Valdivieso, M.; Borders, J.; Yao, K. L.; Raval, M. M T; Madan, S. K.; Sreepathi, P.; Shimoyama, R.; Steiger, Z.; Visscher, Daniel W; Crissman, J. D.

In: Diagnostic Molecular Pathology, Vol. 4, No. 4, 1995, p. 266-273.

Research output: Contribution to journalArticle

Sarkar, FH, Valdivieso, M, Borders, J, Yao, KL, Raval, MMT, Madan, SK, Sreepathi, P, Shimoyama, R, Steiger, Z, Visscher, DW & Crissman, JD 1995, 'A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue', Diagnostic Molecular Pathology, vol. 4, no. 4, pp. 266-273.
Sarkar, F. H. ; Valdivieso, M. ; Borders, J. ; Yao, K. L. ; Raval, M. M T ; Madan, S. K. ; Sreepathi, P. ; Shimoyama, R. ; Steiger, Z. ; Visscher, Daniel W ; Crissman, J. D. / A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue. In: Diagnostic Molecular Pathology. 1995 ; Vol. 4, No. 4. pp. 266-273.
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abstract = "The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized non-radioisotopic detection system using a single 6-μm tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18{\%}) of non- small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33{\%} of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5{\%}) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.",
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AU - Yao, K. L.

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