Performance characteristics of a polymerase chain reaction-based assay for the detection of EGFR mutations in plasma cell-free DNA from patients with non-small cell lung cancer using cell-free DNA collection tubes.

Survival rates in non-small cell lung cancer (NSCLC) are low. Detection of circulating tumor DNA in liquid biopsy (plasma) is increasingly used to identify targeted therapies for clinically actionable mutations, including EGFR mutations in NSCLC. The cobas® EGFR Mutation Test v2 (cobas EGFR test) is FDA-approved for EGFR mutation detection in tissue or liquid biopsy from NSCLC. Standard K2EDTA tubes require plasma separation from blood within 4 to 8 hours; however, Roche Cell-Free DNA (cfDNA) Collection Tubes (Roche cfDNA tube) enable whole blood stability for up to 7 days prior to plasma separation. This analysis assessed performance of Roche cfDNA tubes with the cobas EGFR test for the detection of EGFR mutations in plasma from healthy donors or patients with NSCLC. Overall, test performance was equally robust with either blood collection tube, eg, regarding limit of detection, linearity, and reproducibility, making Roche cfDNA tubes suitable for routine clinical laboratory use in this setting. Importantly, the Roche cfDNA tubes provided more flexibility for specimen handling versus K2EDTA tubes, eg, in terms of tube mixing, plasma separation, and sample stability, and do not require processing of blood within 8 hours thereby increasing the reach of plasma biopsies in NSCLC.

Performance Characteristics of a Real-Time Polymerase Chain Reaction Assay for the Detection of Epidermal Growth Factor Receptor (EGFR) Mutations in Plasma Samples of Non-Small Cell Lung Cancer (NSCLC) Patients.

BACKGROUND: Circulating free DNA in plasma is an alternative source of tumor-derived DNA that can be a surrogate for tissue epidermal growth factor receptor (EGFR) testing. OBJECTIVE: We evaluated the analytical performance of the cobas® EGFR Mutation Test v2 (cobas test), a real-time polymerase chain reaction assay designed to detect defined EGFR gene mutations in plasma from patients with advanced non-small cell lung cancer (NSCLC). METHODS: We used K2-ethylenediaminetetraacetic acid plasma samples from NSCLC patients and healthy donors (HDs), along with cell line DNA. Results from a complete technical performance evaluation are described, including a comparison between NSCLC and HD plasma to support the use of surrogate samples and an independent confirmation of the limit of detection (LoD). RESULTS: The cobas test reported an overall percent agreement of approximately 88% for plasma samples when compared with a next-generation sequencing method. The LoD for all EGFR mutations was ≤ 100 copies/mL for plasma samples. An external study confirmed the LoD for exon 19 deletion, L858R, and T790M at ≤ 100 copies/mL using samples derived from NSCLC patient specimens. The cobas test showed linearity between at least 50 and 10,000 copies/mL for plasma samples. An internal repeatability study reported a correct call accuracy of 99.2% for plasma samples. The performance of the cobas test is equivalent when using sheared or intact cell line DNA diluted into either HD plasma or NSCLC patient plasma. CONCLUSIONS: The cobas test is a sensitive, robust, and accurate assay that delivers reproducible results.

A Highly Verified Assay for KRAS Mutation Detection in Tissue and Plasma of Lung, Colorectal, and Pancreatic Cancer.

CONTEXT.­: KRAS Mutation Test v2 is used for the qualitative detection and identification of 28 mutations in exons 2, 3, and 4 of the human KRAS gene. OBJECTIVE.­: To verify the performance of KRAS Mutation Test v2 and to evaluate its accuracy by correlation with a next-generation sequencing method on Illumina MiSeq. DESIGN.­: In this study, we used formalin-fixed, paraffin-embedded tissue and plasma specimens from non-small cell lung cancer, colorectal cancer, and pancreatic cancer patients. Results of specificity, precision, analytical sensitivity, and accuracy as compared with a MiSeq method are reported. RESULTS.­: The KRAS Mutation Test v2 demonstrated exquisite sensitivity and specificity and broad coverage of KRAS mutations. Precision was 100% (108 of 108) across all samples, operators, and instruments for formalin-fixed, paraffin-embedded tissue and 99.8% (615 of 616) for plasma. Analytical sensitivity was high with detection of 1% mutant sequence in formalin-fixed, paraffin-embedded tissue samples and as low as 25 mutant sequence copies/mL for plasma samples. The test also showed high overall concordance for formalin-fixed, paraffin-embedded tumor tissue as well as for plasma specimens when compared with MiSeq sequencing results. CONCLUSIONS.­: The KRAS Mutation Test v2 is a highly robust, reproducible, and sensitive test for the qualitative detection of 28 mutations in exons 2, 3, and 4 of the KRAS gene in both solid (tissue) and liquid (plasma) biopsies from colorectal cancer, non-small cell lung cancer, and pancreatic cancer, and is a convenient option for KRAS mutation testing.

Multisite analytic performance studies of a real-time polymerase chain reaction assay for the detection of BRAF V600E mutations in formalin-fixed, paraffin-embedded tissue specimens of malignant melanoma.

CONTEXT: A polymerase chain reaction-based companion diagnostic (cobas 4800 BRAF V600 Mutation Test) was recently approved by the US Food and Drug Administration to select patients with BRAF-mutant metastatic melanoma for treatment with the BRAF inhibitor vemurafenib. OBJECTIVES: (1) To compare the analytic performance of the cobas test to Sanger sequencing by using screening specimens from phase II and phase III trials of vemurafenib, and (2) to assess the reproducibility of the cobas test at different testing sites. DESIGN: Specimens from 477 patients were used to determine positive and negative percent agreements between the cobas test and Sanger sequencing for detecting V600E (1799T>A) mutations. Specimens were evaluated with a massively parallel pyrosequencing method (454) to resolve discordances between polymerase chain reaction and Sanger results. Reproducibility of the cobas test was assessed at 3 sites by using 3 reagent lots and an 8-member panel of melanoma samples. RESULTS: A valid cobas result was obtained for all eligible patients. Sanger sequencing had a failure rate of 9.2% (44 of 477). For the remaining 433 specimens, positive percent agreement was 96.4% (215 of 223) and negative percent agreement, 80% (168 of 210). Among 42 cobas mutation-positive/Sanger V600E-negative specimens, 17 were V600E positive and 24 were V600K positive by 454. The cobas test detected 70% of V600K mutations. In the reproducibility study, a correct interpretation was made for 100% of wild-type specimens and specimens with greater than 5% mutant alleles; V600E mutations were detected in 90% of specimens with less than 5% mutant alleles. CONCLUSIONS: The cobas test (1) had a lower assay failure rate than that of Sanger, (2) was more sensitive in detecting V600E mutations, (3) detected most V600K mutations, and (4) was highly reproducible.

Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma.

Melanomas frequently harbor BRAFV600 mutations. Vemurafenib (RG7204/PLX4032), a small-molecule inhibitor of mutant BRAF, has shown striking clinical efficacy in BRAFV600 mutant melanoma, creating the need for a well-validated companion diagnostic to select patients for treatment. We describe analytic performance characteristics of the cobas 4800 BRAF V600 Mutation Test, the test used to select patients for the pivotal vemurafenib trials. This real-time polymerase chain reaction assay was designed to detect the V600E (1799T>A) mutation DNA from formalin-fixed paraffin-embedded tissue samples. Sensitivity was assessed using blends of cell lines or tumor DNA, and tumor specimens with low levels of mutant alleles, as determined by 454 sequencing (a quantitative next-generation pyrosequencing method). A >96% hit rate was obtained across all specimen types with 5% mutant alleles at a DNA input of 125 ng, an amount readily obtained from one 5-µm section. The cobas test showed a higher sensitivity and specificity than direct bidirectional sequencing in a panel of 219 melanoma specimens. Cross reactivity with V600K and V600D was observed. Repeated testing of 5 specimens by 2 operators, using different instruments and reagent lots, yielded correct calls in 158/160 tests (98.8%). A set of 26 highly pigmented samples were identified that gave invalid test results. A simple 1:2 dilution resulted in a valid test result of 76% in such cases. The cobas test is a reproducible assay that detects some non-V600E mutations and is more accurate than direct sequencing in detecting BRAFV600E.

Analytical and clinical performance evaluation of the cobas TaqScreen MPX Test for use on the cobas s 201 system.

Clinical specificity, analytical and clinical sensitivities, reproducibility, and subtype/genotype coverage of the cobas TaqScreen MPX Test, a multiplex nucleic acid test with expanded coverage of HIV variants, were determined. A total of 72,281 blood donations were evaluated. The 95% limit of detection (LOD) for the MPX Test inclusive viruses was determined by testing six dilutions of WHO or Roche standards. Over 3000 high-risk and confirmed seropositive specimens were tested with the MPX and COBAS AmpliScreen Tests. Ten subtypes of HIV-1 Group M, HIV-1 Group O, HIV-2 A and B, HBV genotypes A-H, and HCV genotypes 1-6 were tested with the MPX Test. Reproducibility panels were evaluated at three testing sites across three lots. Clinical specificity in pools was 99.99%. There was one HBV yield case. The LODs for HIV-1 Group M, HCV, and HBV were 49, 11, and 3.8 IU/mL, respectively, and 89 and 59.3 copies/mL for HIV-1 Group O and HIV-2, respectively. Concordance between the MPX and the AmpliScreen Tests was 94.9%. Clinical sensitivity based on AmpliScreen comparison was 97.8-99.5%. All genotype/subtype replicates were detected at three times the LOD. Reproducibility was 98.3-100%. In conclusion, the MPX Test is robust and covers HIV-1 Group O and HIV-2.