A KRAS Mutation Profile in Colorectal Carcinomas: Mutation Detection Technique May Affect Patient Selection for Anti-EGFR Therapy
Huseyin Baloglu, Ismail Yilmaz, Zafer Kucukodaci. GATA-HEH, Istanbul, Turkey
Background: Mutant allele detection sensitivity and the tumor percentage of test material seem to have a crucial impact on KRAS mutation ratios in colorectal carcinomas. All FDA approved KRAS detection kits are very sensitive, and some even detect a single mutant allele in 106 wild-type background. On the other hand, commonly used and widely available conventional dideoxysequencing methods have limited sensitivity. Although there is no precise data about the presence of lower KRAS mutation ratios and the efficacy of anti-EGFR therapy, it is widely accepted to select only the wild type KRAS patients for rational therapy. In the present study, by using the same extracted DNA to exclude the possible effect of tumor content ratio, we performed both a sensitive pyrosequencing and a conventional dideoxysequencing technique to detect KRAS mutations to realize whether the detection methods effect the patient selection for anti-EGFR therapy.
Design: In a series of 227 colorectal carcinomas 121 mutant tumor were previously detected by a sensitive pyrosequencing method (Qiagen PyroMark Q24 system and KRAS Pyro Kit, CE, IVD). Mutations were located in codon 12 (n=56, 46,2%), codon 13 (n=26, 21,4%), codon 61 (n=26, 21,4%) and more then one codon in 13 cases (10,7%). Mutant allele ratios were between 3%-52 % and were <20% in 66 cases (54.5%). The same DNA samples of the 121 mutant cases were reanalyzed by conventional dideoxysequencing technique for KRAS exon 2 mutations (ABI 3730 DNA Analyzer).
Results: Reanalyzing the same DNA samples by dideoxysequencing technique revealed 49 cases with KRAS mutations out of previously detected 121 (40,5%). In the remaining 72 cases, mutant allele ratios were between 3%-23% by pyrosequencing. While KRAS mutation ratio by pyrosequencing method in our series of 227 colorectal carcinomas was 53,3%, dideoxysequencing technique detected only 49 of these 121 mutant tumor cases.
Conclusions: KRAS mutation analysis technique directly affects patient selection for anti-EGFR therapy. According to our experience, widely available conventional dideoxysequencing techniques can be applied as a first-line screening method. Detection of any activating mutation may exclude the patient from anti-EGFR therapy. If no mutation is detected, a second-line more sensitive method, such as pyrosequencing, should be applied for more reliable, targeted therapy decision.
Tuesday, March 20, 2012 1:00 PM
Poster Session IV # 68, Tuesday Afternoon