Assessment of common somatic mutations of EGFR, KRAS, BRAF, NRAS in pulmonary non-small cell carcinoma using iPLEX® HS, a new highly sensitive assay for the MassARRAY® System.

Increased early detection and personalized therapy for lung cancer have coincided with greater use of minimally invasive sampling techniques such as endobronchial ultrasound-guided biopsy (EBUS), endoscopic ultrasound-guided biopsy (EUS), and navigational biopsy, as well as thin needle core biopsies. As many lung cancer patients have late stage disease and other comorbidities that make open surgical procedures hazardous, the least invasive biopsy technique with the highest potential specimen yield is now the preferred first diagnostic study. However, use of these less invasive procedures generates significant analytical challenges for the laboratory, such as a requirement for robust detection of low level somatic mutations, particularly when the starting sample is very small or demonstrates few intact tumor cells. In this study, we assessed 179 clinical cases of non-small cell lung carcinoma (NSCLC) that had been previously tested for EGFR, KRAS, NRAS, and BRAF mutations using a novel multiplexed analytic approach that reduces wild-type signal and allows for detection of low mutation load approaching 1%, iPLEX® HS panel for the MassARRAY® System (Agena Bioscience, San Diego, CA). This highly sensitive system identified approximately 10% more KRAS, NRAS, EGFR and BRAF mutations than were detected by the original test platform, which had a sensitivity range of 5-10% variant allele frequency (VAF).

Ultrasensitive Detection of Multiplexed Somatic Mutations Using MALDI-TOF Mass Spectrometry.

Multiplex detection of low-frequency mutations is becoming a necessary diagnostic tool for clinical laboratories interested in noninvasive prognosis and prediction. Challenges include the detection of minor alleles among abundant wild-type alleles, the heterogeneous nature of tumors, and the limited amount of available tissue. A method that can reliably detect minor variants <1% in a multiplexed reaction using a platform amenable to a variety of throughputs would meet these requirements. We developed a novel approach, UltraSEEK, for high-throughput, multiplexed, ultrasensitive mutation detection and used it for detection of mutant sequence mixtures as low as 0.1% minor allele frequency. The process consisted of multiplex PCR, followed by mutation-specific, single-base extension using chain terminators labeled with a moiety for solid phase capture. The captured and enriched products were then identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. For verification, we successfully analyzed ultralow fractions of mutations in a set of characterized cell lines, and included a direct comparison to droplet digital PCR. Finally, we verified the specificity in a set of 122 paired tumor and circulating cell-free DNA samples from melanoma patients. Our results show that the UltraSEEK chemistry is a particularly powerful approach for the detection of somatic variants, with the potential to be an invaluable resource to investigators in saving time and material without compromising analytical sensitivity and accuracy.

Combined genomarkers approach to Salmonella characterization reveals that nucleotide sequence differences in the phase 1 flagellin gene fliC are markers for variation within serotypes.

The characterization and tracking of pathogenic micro-organisms in the clinical laboratory and public health environment demand schemes that are easy to standardize and use, are automated and high-throughput, and provide portable data. A combined genomarkers approach for Salmonella enterica based on comparative sequence analysis by mass spectrometry has been developed. The scheme targets genes encoding synthesis and assembly of antigens, metabolic pathway enzymes, virulence factors and fluoroquinolone resistance, covering the essential sequences that distinguish between and identify variation within serotypes. This study demonstrated how this single method could replace the combination of methods currently required to determine serotypes, subtypes, antibiotic resistance profiles and the genomic relatedness of Salmonella isolates. The results revealed genomic variation within seven serotypes previously unreported. This variation can be detected by using nucleotide sequence differences in the Salmonella flagellin gene fliC as markers that are not detected by traditional serotyping methods.

Replacing reverse line blot hybridization spoligotyping of the Mycobacterium tuberculosis complex.

Spoligotyping is a tool for the molecular characterization/typing of Mycobacterium tuberculosis complex (MTBC) strains based on target sequences (spacers) in the direct repeat (DR) region (14). The standard spoligotyping assay involves the hybridization of amplified sample DNA to nylon membrane-immobilized oligonucleotides whose sequences are representative of 43 spacer regions. Variations in the number of spacers as a result of deletions of adjacent blocks of repetitive units allow the differentiation of clinical isolates. In the present study, we developed a new multiplexed primer extension-based spoligotyping assay using automated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that improves the classical reverse line blot hybridization assay with respect to reproducibility, throughput, process flow, ease of use, and data analysis. Validation of the MALDI-TOF MS-based spoligotyping assay with two sample sets with a total of 326 samples resulted in 96.6% concordance (315/326) when the full spoligotype patterns were compared with the results of standard spoligotyping and 99.9% concordance when the results were compared with those of individual primer extension assays. Ten strains (including two Mycobacterium canettii strains) showed discordant results with one or two spacer differences from the membrane-based spoligotyping result. Most discordant samples were identified to be the result of ambiguities in the interpretation of weak hybridization signals in the reverse line blot assay and sequence variations in the spacer regions. We established a new automated primer extension assay and successfully validated it for use for the routine typing of MTBC strains in the research and public health laboratory environments. The present multiplex levels of up to 30 are extendable and allow the additional incorporation of controls and antibiotic resistance markers.