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K-ras Point Mutation Detection in Lung Cancer: Comparison of Two Approaches to Somatic Mutation Detection Using ARMS Allele-specific Amplification

¹ AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom.

² Roy Castle International Centre for Lung Cancer Research, 200 London Rd., Liverpool L3 9TA, United Kingdom.

³ Department of Biological Sciences, Manchester Metropolitan University, Manchester M1 5GD, United Kingdom.

⁴ RSOM, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom.

⁵ Molecular Genetics and Oncology Group, Clinical Dental Sciences, University of Liverpool, Liverpool L69 3BX, United Kingdom.
^a Address correspondence to this author at: AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK. Fax 44-1625-514463; e-mail simon.clayton{at}astrazeneca.com

Background: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMS^TM allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples.

Methods: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples.

Results: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04–100%.

Conclusions: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.

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