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.

Multiplex testing for Factor II and Factor V mutations in thrombophilia: technical verification and clinical validation of the cobas® Factor II and Factor V test.

Laboratory testing for thrombophilia is complicated but essential for diagnosis. In 2017, the cobas® Factor II and Factor V Test (cobas F2F5 test) was launched for use with the cobas z 480 analyzer. This qualitative polymerase chain reaction test enables multiplex Factor II and Factor V testing with flexible reporting and workflow efficiency. Here, we report the results from studies investigating the performance of the cobas F2F5 test. Technical performance verification, clinical validation, external laboratory performance, and workflow comparison studies were performed. Fresh and frozen whole-blood and genomic DNA (gDNA) samples were tested, and several manual and automated DNA isolation methods were used. Bidirectional Sanger sequencing was used to verify genotypes identified by the cobas F2F5 test. One hundred percent agreement between the cobas F2F5 test and Sanger sequencing was observed for all genotypes. An external laboratory using remnant clinical samples also yielded 100% agreement between cobas F2F5 test results and their routine testing method. The cobas F2F5 test reduced the total sample processing time compared with the LightCycler® 1.2 platform (98.6 vs 420.2 min; 96 samples). Hemoglobin, extraction buffer, and ethanol contamination of the gDNA sample can lead to invalid results. The cobas F2F5 test has a high degree of accuracy for identification of Factor II and Factor V genotypes. This multiplex testing with short sample processing time can reduce handling errors and increase efficiency. Both manual and automated DNA isolation methods can be used with the cobas F2F5 test.

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.