KRAS analysis in colorectal carcinoma: analytical aspects of Pyrosequencing and allele-specific PCR in clinical practice.
BACKGROUND
Epidermal growth factor receptor inhibitor therapy is now approved for treatment of metastatic colorectal carcinomas (CRC) in patients with tumors lacking KRAS mutations. Several procedures to detect KRAS mutations have been developed. However, the analytical sensitivity and specificity of these assays on routine clinical samples are not yet fully characterised.


METHODS
The practical aspects and clinical applicability of a KRAS-assay based on Pyrosequencing were evaluated in a series of 314 consecutive CRC cases submitted for diagnostic KRAS analysis. The performance of Pyrosequencing compared to allele-specific, real-time PCR was then explored by a direct comparison of CE-IVD-marked versions of Pyrosequencing and TheraScreen (DxS) KRAS assays for a consecutive subset (n = 100) of the 314 clinical CRC samples.


RESULTS
Using Pyrosequencing, 39% of the 314 CRC samples were found KRAS-mutated and several of the mutations (8%) were located in codon 61. To explore the analytical sensitivity of the Pyrosequencing assay, mutated patient DNA was serially diluted with wild-type patient DNA. Dilutions corresponding to 1.25-2.5% tumor cells still revealed detectable mutation signals. In clinical practice, our algorithm for KRAS analysis includes a reanalysis of samples with low tumor cell content (< 10%, n = 56) using an independent assay (allele-specific PCR, DxS). All mutations identified by Pyrosequencing were then confirmed and, in addition, one more mutated sample was identified in this subset of 56 samples. Finally, a direct comparison of the two technologies was done by re-analysis of a subset (n = 100) of the clinical samples using CE-IVD-marked versions of Pyrosequencing and TheraScreen KRAS assays in a single blinded fashion. The number of samples for which the KRAS codon 12/13 mutation status could be defined using the Pyrosequencing or the TheraScreen assay was 94 and 91, respectively, and both assays detected the same number of codon 12 and 13 mutations.


CONCLUSIONS
KRAS mutation detection using Pyrosequencing was evaluated on a consecutive set of clinical CRC samples. Pyrosequencing provided sufficient analytical sensitivity and specificity to assess the mutation status in routine formalin-fixed CRC samples, even in tissues with a low tumor cell content.
Molecular Pathology Unit, Department of Genetics and Pathology, Uppsala University, SE-751 85 Uppsala, Sweden.
Magnus
M
Sundström
Karolina
K
Edlund
Monica
M
Lindell
Bengt
B
Glimelius
Helgi
H
Birgisson
Patrick
P
Micke
Johan
J
Botling
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D003062:Codon; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D004252:DNA Mutational Analysis; D005404:Fixatives; D005557:Formaldehyde; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000379:methods; D018631:ras Proteins / Q000235:genetics*
D003062:Codon; D005404:Fixatives; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins