Validation of the Oncomine™ focus panel for next-generation sequencing of clinical tumour samples.

The clinical utility of next-generation sequencing (NGS) for a diverse range of targets is expanding, increasing the need for multiplexed analysis of both DNA and RNA. However, translation into daily use requires a rigorous and comprehensive validation strategy. The aim of this clinical validation was to assess the performance of the Ion Torrent Personal Genome Machine (IonPGM™) and validate the Oncomine™ Focus DNA and RNA Fusion panels for clinical application in solid tumour testing of formalin-fixed, paraffin-embedded (FFPE) tissue. Using a mixture of routine FFPE and reference material across a variety of tissue and specimen types, we sequenced 86 and 31 samples on the Oncomine™ Focus DNA and RNA Fusion assays, respectively. This validation considered a number of parameters including the clinical robustness of the bioinformatics pipeline for variant detection and interpretation. The Oncomine™ Focus DNA assay had a sample and variant-based sensitivity of 99.1 and 97.1%, respectively, and an assay specificity of 100%. The Oncomine™ Focus Fusion panel had a good sensitivity and specificity based upon the samples assessed, however requires further validation to confirm findings due to limited sample numbers. We observed a good sequencing performance based upon amplicon, gene (hotspot variants within gene) and sample specific analysis with 92% of clinical samples obtaining an average amplicon coverage above 500X. Detection of some indels was challenging for the routine IonReporter™ workflow; however, the addition of NextGENe® software improved indel identification demonstrating the importance of both bench and bioinformatic validation. With an increasing number of clinically actionable targets requiring a variety of methodologies, NGS provides a cost-effective and time-saving methodology to assess multiple targets across different modalities. We suggest the use of multiple analysis software to ensure identification of clinically applicable variants.

Could molecular pathology testing in lung cancer be more cost-effective?

AIMS: EGFR and ALK analysis is routinely undertaken prior to targeted treatment of non-squamous non-small cell lung carcinoma (NSCLC). Increasingly, limited resources require molecular pathology services to be cost-effective without detriment to patient care. METHODS: Data from an audit of molecular pathology testing in the South East Scotland Cancer Network (SCAN) network have been used to explore different testing strategies with the aim of reducing costs; including investigation of thyroid transcription factor 1 (TTF1) expression as a negative predictor for EGFR mutations. RESULTS: TTF1 immunohistochemistry had a high negative predictive value for EGFR mutations (99%). Reflex testing all non-squamous NSCLC, as expected, had the highest costs, whereas limiting testing to those who might be considered for treatment would lead to a cost reduction of only 7.5%; however, a serial testing model could save 32.7%. CONCLUSIONS: Testing only patients being considered for EGFR and ALK inhibitors represented small savings; more significant savings would be achievable if testing algorithms used known associations between clinical biomarkers.

Molecular pathology in lung cancer: a guide to the techniques used in clinical practice.

Five year survival rates for lung cancer patients are poor; however the development of new therapeutic options, which benefit subsets of the population, offer hope of improvement. These novel therapies frequently rely upon the analysis of biomarkers in pathology samples; in lung cancer patients, testing is now routinely carried out to identify small mutations and chromosomal rearrangements in order to predict response to treatment. The recent increase in biomarker analyses in pathology samples has lead to the development of a new specialty, molecular pathology. The use of molecular pathology assays in clinical samples is largely under the control of the histopathologist; who is likely to be asked, as a minimum, to select tissue sections for molecular analysis and mark areas of H&E stained slides for macro or microdissection. Many histopathologists will also be involved in the sourcing and implementation of new assays. This review aims to provide a guide to some of the most commonly used molecular pathology methods - their advantages and their limitations.

Making the most of pathological specimens: molecular diagnosis in formalin-fixed, paraffin embedded tissue.

The development of commercial reagents designed specifically for use with formalin-fixed paraffin-embedded (FFPE) tissue has unlocked the diagnostic potential of this prolific resource. The availability of archival FFPE tissue and tissue from current patients make it an ideal resource for molecular testing. Despite its stability and ability to preserve morphological information, FFPE provides a number of technical challenges to the study of biomolecules. In particular, the cross-linking and processing present problems in the extraction and isolation of DNA, RNA and protein and affect their use in downstream analysis. Here we will discuss some of the problems of FFPE tissue, how they can be overcome and how FFPE material can be used within clinical molecular diagnostics.

The reliable detection of Barley yellow and mild mosaic viruses using real-time PCR (TaqMan).

Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV) are economically-important, soil-borne pathogens of winter barley. Until recently, laboratory diagnosis of these pathogens has relied upon ELISA using polyclonal antiserum. However, due to the unstable nature of these viruses, combined with the inability to sap transmit BaYMV, high quality antiserum has been difficult to obtain and as a result the performance of ELISA is often unsatisfactory. As an alternative approach two TaqMan assays (one BaYMV-specific and the other BaMMV-specific) have been developed. These assays have been validated for three seasons, by testing samples in parallel with ELISA. This testing indicates that TaqMan is a more reliable detection method than ELISA, especially with late-season and mixed infection samples. Data is also provided on a comparison of using simplex assays versus a multiplex assay for detecting BaYMV and BaMMV. The results indicate that while multiplexing does lead to a small reduction in sensitivity, it can be used reliably to streamline routine diagnosis. Further improvements in the development of a routine diagnostic procedure are also described including details of a rapid automated extraction procedure (Kingfisher) and the use of a novel cereal-specific control assay.

The detection of tuber necrotic isolates of Potato virus Y, and the accurate discrimination of PVY(O), PVY(N) and PVY(C) strains using RT-PCR.

Potato tuber necrotic ringspot disease (PTNRD) is a damaging disease of potatoes, causing unsightly necrotic rings on the surface of tubers. The causal agent is thought to be tuber necrotic isolates of Potato virus Y, known as PVY(NTN). The disease spoils tubers for processing and table use, and the lack of a diagnostic method makes control especially difficult. The development of an RT-PCR assay for the reliable detection of PVY(NTN) and discrimination of all the main strains of PVY (PVY(O), PVY(N) and PVY(C)) is described. An assay was developed, exploiting a recombination site in the coat protein of PVY(NTN), allowing more reliable diagnosis of these isolates. Although the conserved nucleotide differences observed between the strains was very small, competitive PCR and mutagenically separated PCR were both employed in the development of a robust assay. The assay was found to be more reliable than the most commonly used RT-PCR method, and should prove to be an important tool in the confirmation of symptoms and for the detection of PVY(NTN) in symptomless tissue, in disease surveys and seed health schemes.