Version 2.0 qualitative and quantitative AMPLICOR reverse transcription-PCR tests for HCV were designed to improve on the performance of first version of the hepatitis C virus (HCV) tests. The new tests were calibrated in international units, the new commonly accepted standard unit of measurement for HCV RNA. The sensitivity of the qualitative tests was enhanced by modifying the specimen processing procedure to achieve a limit of detection 50 IU/ml. The limit of detection for the quantitative tests was 600 IU/ml. Modifications to the amplification reaction mixture and thermal cycling conditions enabled all genotypes to be amplified with similar efficiency. The quantitative tests exhibited a linear range extending from 500 to 500,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 18 to 39%, within the linear range. These data indicate that the version 2.0 AMPLICOR HCV tests will improve diagnosis of HCV infection and will yield more-accurate titers for prognosis and for monitoring therapeutic efficacy, particularly at low viral loads. Furthermore, it will be possible to compare the performance characteristics and viral load measurements of AMPLICOR tests to those of other tests that adopt the international unit as the standard of measurement.

Mutations in the Kras gene that lead to its constitutive activation are quite common in many tumor types including NSCLC. We describe the analytic performance of a TaqMelt PCR assay (cobas® KRAS Mutation Test) that detects 19 mutations in codons 12, 13 and 61 which generates test results within 8 hours. Using a panel of DNA blends from 18 FFPET samples of NSCLC, tested at 4 genomic DNA input levels, the correct mutation call was observed at 5% mutant alleles from the prescribed 50 ng of gDNA/PCR down to 12.5 ng of gDNA/PCR. A separate panel of 194 FFPET samples of NSCLC was tested with the PCR test and compared to Sanger sequencing. There were 15 discordant samples, 9 PCR (+), Sanger (-) and 6 PCR (-), Sanger (+) samples, that were further analyzed using a quantitative 454 sequencing method. All Sanger discordant samples except 1 PCR (+), Sanger (-) sample yielded 454 results that agreed with the PCR result. The PPA, NPA, and overall agreement with sequencing (Sanger resolved by 454) are 100.0%, 98.9% and 99.5%, respectively. Sanger/454 Sequencing MUT WT Total MUT 99 1 100 cobas® KRAS Mutation Test WT 0 94 94 Total 99 95 194* *Includes 1 result for double mutations by Sanger and excludes 1 invalid result by PCR The same panel of 194 specimens tested with another lot of the PCR test gave similar performance. When a panel of 8 FFPET samples of NSCLC (including 3 near the limit of analytical sensitivity) was repeatedly tested by 2 operators, using multiple instruments and reagent lots, a correct result was observed for 100% of tests. PCR test results were not affected by repeated freezing and thawing of the DNA sample or test reagents, or by addition of hemoglobin or triglycerides. These studies show that this PCR test is a robust and reproducible assay that 1) detects 19 mutations in codons 12, 13 and 61, 2) requires a small amount of DNA (which can generally be obtained from 1x5 μm section), and 3) is more sensitive and specific than Sanger sequencing. Citation Format: Sung C. Lee, Jianli Cao, Kiran Adhikary, Lilia Corona, Nitta Lee, Jingchuan Li, Theresa May, Yiqiao Wu, Victoria Brophy. The analytic performance of a TaqMelt PCR assay for the detection of KRAS mutations in formalin-fixed paraffin-embedded tissue (FFPET) samples of Non-Small Cell Lung Cancer (NSCLC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2399. doi:10.1158/1538-7445.AM2013-2399