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.

Background

Technologies based on DNA microarrays have the potential to provide detailed information on genomic aberrations in tumor cells. In practice a major obstacle for quantitative detection of aberrations is the heterogeneity of clinical tumor tissue. Since tumor tissue invariably contains genetically normal stromal cells, this may lead to a failure to detect aberrations in the tumor cells.

Principal Finding

Using SNP array data from 44 non-small cell lung cancer samples we have developed a bioinformatic algorithm that accurately models the fractions of normal and tumor cells in clinical tumor samples. The proportion of normal cells in combination with SNP array data can be used to detect and quantify copy number neutral loss-of-heterozygosity (CNNLOH) in the tumor cells both in crude tumor tissue and in samples enriched for tumor cells by laser capture microdissection.

Conclusion

Genome-wide quantitative analysis of CNNLOH using the CNNLOH Quantifier method can help to identify recurrent aberrations contributing to tumor development in clinical tumor samples. In addition, SNP-array based analysis of CNNLOH may become important for detection of aberrations that can be used for diagnostic and prognostic purposes.