Detection of BRAF mutations in malignant melanoma and colorectal cancer by SensiScreen® FFPE BRAF qPCR assay.

Mutations in BRAF exon 15 lead to conformational changes in its activation loops, resulting in constitutively active BRAF proteins which are implicated in the development of several human cancer types. Different BRAF inhibitors have been developed and introduced in clinical practice. Identification of BRAF mutations influences the clinical evaluation, treatment, progression and for that reason a sensitive and specific identification of BRAF mutations is on request from the clinic. Here we present the SensiScreen® FFPE BRAF qPCR Assay that uses a novel real-time PCR-based method for BRAF mutation detection based on PentaBases proprietary DNA analogue technology designed to work on standard real-time PCR instruments. The SensiScreen® FFPE BRAF qPCR Assay displays high sensitivity, specificity, fast and easy-to-use. The SensiScreen® FFPE BRAF qPCR Assay was validated on two different FFPE tumour biopsy cohorts, one cohort included malignant melanoma patients previously analyzed by the Cobas® 4800 BRAF V600 Mutation Test, and one cohort from colorectal cancer patients previously analyzed by mutant-enriched PCR and direct sequencing. All BRAF mutant malignant melanoma patients were confirmed with the SensiScreen® FFPE BRAF qPCR Assay and additional four new mutations in the malignant melanoma cohort were identified. All the previously identified BRAF mutations in the colorectal cancer patients were confirmed, and additional three new mutations not identified with direct sequencing were detected. Also, one new BRAF mutation not previously identified with ME-PCR was found. Furthermore, the SensiScreen® FFPE BRAF qPCR Assay identified the specific change in the amino acid. The SensiScreen® FFPE BRAF qPCR Assay will contribute to a more specific, time and cost saving approach to better identify and characterize mutations in patients affected by cancer, and consequently permits a better BRAF characterization that is fundamental for therapy decision.

Binding and orientation of tricyclic antidepressants within the central substrate site of the human serotonin transporter.

Tricyclic antidepressants (TCAs) have been used for decades, but their orientation within and molecular interactions with their primary target is yet unsettled. The recent finding of a TCA binding site in the extracellular vestibule of the bacterial leucine transporter 11 A above the central site has prompted debate about whether this vestibular site in the bacterial transporter is applicable to binding of antidepressants to their relevant physiological target, the human serotonin transporter (hSERT). We present an experimentally validated structural model of imipramine and analogous TCAs in the central substrate binding site of hSERT. Two possible binding modes were observed from induced fit docking calculations. We experimentally validated a single binding mode by combining mutagenesis of hSERT with uptake inhibition studies of different TCA analogs according to the paired mutation ligand analog complementation paradigm. Using this experimental method, we identify a salt bridge between the tertiary aliphatic amine and Asp(98). Furthermore, the 7-position of the imipramine ring is found vicinal to Phe(335), and the pocket lined by Ala(173) and Thr(439) is utilized by 3-substituents. These protein-ligand contact points unambiguously orient the TCA within the central binding site and reveal differences between substrate binding and inhibitor binding, giving important clues to the inhibition mechanism. Consonant with the well established competitive inhibition of uptake by TCAs, the resulting binding site for TCAs in hSERT is fully overlapping with the serotonin binding site in hSERT and dissimilar to the low affinity noncompetitive TCA site reported in the leucine transporter (LeuT).

Clinical performance evaluation of the Idylla NRAS-BRAF mutation test on retrospectively collected formalin-fixed paraffin-embedded colorectal cancer tissue.

AIMS: Understanding the molecular mechanisms of underlying disease has led to a movement away from the one-drug-fits-all paradigm towards treatment tailored to the genetic profile of the patient. The Biocartis Idylla platform is a novel fully automated, real-time PCR-based in vitro diagnostic system. The Idylla NRAS-BRAF mutation test has been developed for the qualitative detection of mutations in NRAS and BRAF oncogenes, facilitating genetic profiling of patients with cancer. The aim of this study was to carry out a formal clinical performance evaluation. METHODS: Two-hundred and forty-two formalin-fixed paraffin-embedded (FFPE) human malignant colorectal cancer (CRC) tissue samples were identified in departmental archives and tested with both the Idylla NRAS-BRAF mutation test and the Agena Bioscience MassARRAY test. RESULTS: The overall concordance between the Idylla NRAS-BRAF mutation test and the MassARRAY comparator reference test result was 241/242 (99.59%, lower bound of one-sided 95% CI=98.1%) for NRAS and 242/242 (lower bound of 95% one-sided 95% CI=98.89%) for BRAF. The Idylla NRAS-BRAF test detected one NRAS mutation that had not been reported by the MassARRAY comparator reference test. Reanalysis of this sample by droplet digital PCR confirmed that the mutation was present, but at an allelic frequency below the stated sensitivity level of the MassARRAY system. CONCLUSION: These results confirm that the Idylla NRAS-BRAF mutation test has high concordance with a widely used NRAS-BRAF test, and is therefore suitable for use as an in vitro diagnostic device for this application.