Clinical validation of a PCR assay for the detection of EGFR mutations in non-small-cell lung cancer: retrospective testing of specimens from the EURTAC trial.

The EURTAC trial demonstrated that the tyrosine kinase inhibitor (TKI) erlotinib was superior to chemotherapy as first-line therapy for advanced non-small cell lung cancers (NSCLC) that harbor EGFR activating mutations in a predominantly Caucasian population. Based on EURTAC and several Asian trials, anti-EGFR TKIs are standard of care for EGFR mutation-positive NSCLC. We sought to validate a rapid multiplex EGFR mutation assay as a companion diagnostic assay to select patients for this therapy. Samples from the EURTAC trial were prospectively screened for EGFR mutations using a combination of laboratory-developed tests (LDTs), and tested retrospectively with the cobas EGFR mutation test (EGFR PCR test). The EGFR PCR test results were compared to the original LDT results and to Sanger sequencing, using a subset of specimens from patients screened for the trial. Residual tissue was available from 487 (47%) of the 1044 patients screened for the trial. The EGFR PCR test showed high concordance with LDT results with a 96.3% overall agreement. The clinical outcome of patients who were EGFR-mutation detected by the EGFR PCR test was very similar to the entire EURTAC cohort. The concordance between the EGFR PCR test and Sanger sequencing was 90.6%. In 78.9% of the discordant samples, the EGFR PCR test result was confirmed by a sensitive deep sequencing assay. This retrospective study demonstrates the clinical utility of the EGFR PCR test in the accurate selection of patients for anti-EGFR TKI therapy. The EGFR PCR test demonstrated improved performance relative to Sanger sequencing.

Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. METHODS: The assay's limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations. RESULTS: A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test performance was not compromised by endogenous substances, therapeutic drugs, necrosis up to 85%, and common micro-organisms. All of the assessed less common mutations except one (exon 19 deletion mutation 2236_2248 > AGAC) were detected at a similar DNA input level as that for the corresponding predominant mutation. CONCLUSION: The cobas EGFR Mutation Test is a sensitive, accurate, rapid, and reproducible assay.

Comparison of molecular testing methods for the detection of EGFR mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer.

AIM: To conduct a methods correlation study of three different assays for the detection of mutations at EGFR gene in human formalin-fixed paraffin-embedded tumour (FFPET) specimens of non-small cell lung carcinomas (NSCLC). METHODS: We conducted a 2-site method comparison study of two european conformity (CE) in vitro diagnostic (IVD)-marked assays, the cobas EGFR Mutation Test and the Therascreen EGFR29 Mutation Kit, and 2× bidirectional Sanger sequencing. We blind-tested 124 NSCLC FFPET specimens with all three methods; the cobas test was performed at both sites. Positive (PPA) and negative percent agreements (NPA) were determined for the cobas test versus each of the other two methods. Specimens yielding discordant test results between methods were further tested using quantitative massively parallel pyrosequencing (MPP). RESULTS: PPA between cobas and Sanger was 98.8%; NPA was 79.3%. Overall there were seven discordant results. MPP confirmed an exon 19 deletion in two cases and L858R mutation in four cases. PPA between cobas and Therascreen was 98.9% and NPA was 100%. There was one discordant result. Reproducibility of the cobas test between the two sites was 99.2%. CONCLUSIONS: The invalid rates for the cobas test and Therascreen were lower than Sanger sequencing. The cobas and Therascreen assays showed a high degree of concordance, and both were more sensitive for the detection of exon 19 deletion and L858R mutations than Sanger. The cobas test was highly reproducible between the two testing sites, used the least amount of DNA input and was the only test with automated results reporting.

Comparison of testing methods for the detection of BRAF V600E mutations in malignant melanoma: pre-approval validation study of the companion diagnostic test for vemurafenib.

BACKGROUND: The cobas 4800 BRAF V600 Mutation Test is a CE-marked and FDA-approved in vitro diagnostic assay used to select patients with metastatic melanoma for treatment with the selective BRAF inhibitor vemurafenib. We describe the pre-approval validation of this test in two external laboratories. METHODS: Melanoma specimens were tested for BRAF V600 mutations at two laboratories with the: cobas BRAF Mutation Test; ABI BRAF test; and bidirectional direct sequencing. Positive (PPA) and negative (NPA) percent agreements were determined between the cobas test and the other assays. Specimens with discordant results were tested with massively parallel pyrosequencing (454). DNA blends with 5% mutant alleles were tested to assess detection rates. RESULTS: Invalid results were observed in 8/116 specimens (6·9%) with Sanger, 10/116 (8·6%) with ABI BRAF, and 0/232 (0%) with the cobas BRAF test. PPA was 97·7% for V600E mutation for the cobas BRAF test and Sanger, and NPA was 95·3%. For the cobas BRAF test and ABI BRAF, PPA was 71·9% and NPA 83·7%. For 16 cobas BRAF test-negative/ABI BRAF-positive specimens, 454 sequencing detected no codon 600 mutations in 12 and variant codon 600 mutations in four. For eight cobas BRAF test-positive/ABI BRAF-negative specimens, four were V600E and four V600K by 454 sequencing. Detection rates for 5% mutation blends were 100% for the cobas BRAF test, 33% for Sanger, and 21% for the ABI BRAF. Reproducibility of the cobas BRAF test was 111/116 (96%) between the two sites. CONCLUSIONS: It is feasible to evaluate potential companion diagnostic tests in external laboratories simultaneously to the pivotal clinical trial validation. The health authority approved assay had substantially better performance characteristics than the two other methods. The overall success of the cobas BRAF test is a proof of concept for future biomarker development.

Labeling tumor cells with fluorescent nanocrystal-aptamer bioconjugates.

Aptamers that bind to prostate specific membrane antigen (PSMA) were conjugated to luminescent CdSe and CdTe nanocrystals for cell-labeling studies. The aptamer-nanocrystal conjugates showed specific targeting of both fixed and live cells that overexpressed PSMA. More importantly, aptamers were able to label cells dispersed in a collagen gel matrix simulating tissue. The specific binding abilities and synthetic accessibility of aptamers combined with the photostability and small size of semiconductor nanocrystals offers a powerful and general tool for cellular imaging.

General shape control of colloidal CdS, CdSe, CdTe quantum rods and quantum rod heterostructures.

We report a general synthetic method for the formation of shape-controlled CdS, CdSe and CdTe nanocrystals and mixed-semiconductor heterostructures. The crystal growth kinetics can be manipulated by changing the injection rate of the chalcogen precursor, allowing the particle shape-spherical or rodlike-to be tuned without changing the underlying chemistry. A single injection of precursor leads to isotropic spherical growth, whereas multiple injections promote epitaxial growth along the length of the c-axis. This method was extended to produce linear type I and type II semiconductor nanocrystal heterostructures.

Challenges in quantum dot-neuron active interfacing.

Semiconductor nanocrystal quantum dots (qdots) are now being explored in applications requiring active cellular interfaces, such as biosensing and therapeutics in which information is passed from the qdot to the biological system, or vice versa, to perform a function. These applications may require surface coating chemistry that is different from what is commonly employed for passive interface applications like labeling (i.e., thick polymer coatings such as poly(ethylene glycol) (PEG)), in which the only concern is nonspecific sticking to cells and biocompatibility. The thick insulating coatings that are generally needed for labeling are generally not suitable for active qdot-cell interface applications. There is currently little data regarding the interactions between viable cells and qdots under physiological conditions. Our initial investigations using mercaptoacetic acid-coated CdS and CdTe qdots as a simple model to interface with neuron cell surface receptors under physiological conditions uncovered two significant technological hurdles: nonspecific binding and endocytosis. Nonspecific binding can be extensive and in general there appears to be greater nonspecific binding for larger particle sizes, but this also depends sensitively on the particle surface characteristics and the type of neuron, possibly indicating a detailed relationship between particle-cell affinity and cell membrane chemistry. More importantly, qdot endocytosis occurs rapidly at physiological temperature for the different nerve cell types studied, within the first five minutes of exposure to both CdS and CdTe qdots, regardless of whether the molecular coatings specifically recognize cell surface receptors or not. As a consequence, new strategies for tagging cell surface recognition groups for long-term active interfacing with cells under physiological conditions are needed, which requires more sophisticated ligands than MAA but also the absence of thick insulating coatings.