Validation of companion diagnostic for detection of mutations in codons 12 and 13 of the KRAS gene in patients with metastatic colorectal cancer: analysis of the NCIC CTG CO.17 trial.

CONTEXT: The therascreen KRAS RGQ polymerase chain reaction kit is being developed as a companion diagnostic to aid clinicians, through detection of KRAS mutations, in the identification of patients with metastatic colorectal cancer (mCRC) who are more likely to benefit from cetuximab. OBJECTIVES: To assess whether KRAS mutation status, determined by using the therascreen KRAS kit, is a predictive marker of cetuximab efficacy. DESIGN: Tissue samples were obtained from patients with mCRC treated on the National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) CO.17 phase 3 study of cetuximab plus best supportive care (BSC) versus BSC alone. Tumor DNA samples were assessed for the presence of KRAS mutations by using the therascreen KRAS kit. Efficacy and safety were assessed to determine whether mutation status was predictive of outcomes. Results.-Evaluable samples were available from 453 patients (79.2%) enrolled in the NCIC CTG CO.17 trial. The KRAS wild-type subset represented 54.1% (245 of 453) of the evaluated population. Median overall survival of patients with KRAS wild-type tumors was 8.6 months among those who received cetuximab plus BSC and 5.0 months among patients who received BSC alone (hazard ratio [HR], 0.63; P = .002). Among patients with KRAS mutant mCRC, no meaningful difference in overall survival was observed between arms (HR, 0.91; P = .55). These results are consistent with a previous report that analyzed patient tumor samples by using bidirectional sequencing. CONCLUSIONS: These data support the utility of the therascreen KRAS kit as a means of selecting patients who may benefit from cetuximab therapy.

A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting.

Evidence that activating mutations of the KRAS oncogene abolish the response to anti-epidermal growth factor receptor therapy has revolutionized the treatment of advanced colorectal cancer. This has resulted in the urgent demand for KRAS mutation testing in the clinical setting to aid choice of therapy. The aim of this study was to evaluate six different KRAS mutation detection methodologies on two series of primary colorectal cancer samples. Two series of 80 frozen and 74 formalin-fixed paraffin-embedded tissue samples were sourced and DNA was extracted at a central site before distribution to seven different testing sites. KRAS mutations in codons 12 and 13 were assessed by using single strand conformation polymorphism analysis, pyrosequencing, high resolution melting analysis, dideoxy sequencing, or the commercially available TIB Molbiol (Berlin, Germany) or DxS Diagnostic Innovations (Manchester, UK) kits. In frozen tissue samples, concordance in KRAS status (defined as consensus in at least five assays) was observed in 66/80 (83%) cases. In paraffin tissue, concordance was 46/74 (63%) if all assays were considered or 71/74 (96%) using the five best performing assays. These results demonstrate that a variety of detection methodologies are suitable and provide comparable results for KRAS mutation analysis of clinical samples.

Design and use of scorpions fluorescent signaling molecules.

A number of probe systems exist for the real-time detection of PCR products. Scorpions are a unique method wherein primer and probe are combined in a single oligonucleotide. During the PCR, the probe element becomes linked directly to its complementary target site with beneficial consequences. In particular, the unimolecular mechanism of probe/target hybridization ensures rapid, reliable, and robust probing of a chosen amplicon. We discuss the design and use of Scorpions and compare their use with similar systems.

Multiplexed assays for detection of mutations in PIK3CA.

BACKGROUND: Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. METHODS: We combined Amplification Refractory Mutation System (ARMS; AstraZeneca) allele-specific PCR and Scorpions (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). RESULTS: These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. CONCLUSIONS: Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.

The quality of DNA extracted from liquid or dried blood is not adversely affected by storage at 4 degrees C for up to 24 h.

BACKGROUND: A consistent and stable source of DNA is an essential requirement for many Biobanks. An important pre-analytical variable is the delay between sample collection and sample processing. METHODS: Fresh blood samples (n = 80) were collected and either processed immediately or after storage for 24 h. The samples were either stored as liquid blood at 4 degrees C or as dried blood spots at ambient temperature on three types of paper-based storage media. The quality of the DNA extracted from the samples was measured. RESULTS: No difference was observed between fresh and stored blood samples. CONCLUSIONS: The quality of DNA extracted from liquid or dried blood is not adversely affected by storage at 4 degrees C for up to 24 h.

Genetic susceptibility to childhood common acute lymphoblastic leukaemia is associated with polymorphic peptide-binding pocket profiles in HLA-DPB1*0201.

In a previous study, we obtained preliminary evidence in a small series of patients (n = 63) suggesting that susceptibility to childhood common acute lymphoblastic leukaemia (c-ALL) was associated with an allele at the HLA-DPB1 locus, DPB1*0201. We have now tested this hypothesis by comparing the frequency of children with leukaemia (n = 982) who typed for specific DPB1 alleles and two groups of non-leukaemic children, one consisting of children with solid tumours, excluding lymphomas (n = 409), the other consisting of normal infants (n = 864). We found that significantly more children with c-ALL and T-ALL, but not pro-B ALL or acute non-ALL typed for DPB1*0201 as compared with children with solid tumours [odds ratio (OR), 95% confidence interval (CI) for c-ALL: 1.76, 1.20-2.56; T-ALL: 1.93, 1.01-3.80] and normal infants (OR, 95% CI for c-ALL: 1.83, 1.34-2.48; T-ALL: 2.00, 1.10-3.82). In childhood c-ALL, significantly more children than those with solid tumours or normal infants typed for DPB1 alleles coding specific polymorphic amino acids lining the antigen-binding site of the DPbeta1*0201 allotypic protein, suggesting that susceptibility to childhood c-ALL may be influenced by DPbeta ABS amino acid polymorphisms shared by DPbeta1*0201 and other DPbeta1 allotypes. These results point to a mechanism of c-ALL susceptibility that involves the presentation of specific antigenic peptides, possibly derived from infectious agents, by DPbeta1*0201-related allotypic proteins, leading to the activation of helper T cells mediating proliferative stress on preleukaemic cells.