Clinical validation of the 50 gene AmpliSeq Cancer Panel V2 for use on a next generation sequencing platform using formalin fixed, paraffin embedded and fine needle aspiration tumour specimens

Summary

The advent of massively parallel sequencing has caused a paradigm shift in the ways cancer is treated, as personalised therapy becomes a reality. More and more laboratories are looking to introduce next generation sequencing (NGS) as a tool for mutational analysis, as this technology has many advantages compared to conventional platforms like Sanger sequencing. In Australia all massively parallel sequencing platforms are still considered in-house in vitro diagnostic tools by the National Association of Testing Authorities (NATA) and a comprehensive analytical validation of all assays, and not just mere verification, is a strict requirement before accreditation can be granted for clinical testing on these platforms. Analytical validation of assays on NGS platforms can prove to be extremely challenging for pathology laboratories. Although there are many affordable and easily accessible NGS instruments available, there are no standardised guidelines as yet for clinical validation of NGS assays. We present an accreditation development procedure that was both comprehensive and applicable in a setting of hospital laboratory for NGS services. This approach may also be applied to other NGS applications in service laboratories.

Introduction

Anatomical pathology laboratories face increasing demands for mutational analysis required for management of multiple tumour streams including lung and colon cancers and malignant melanoma. Since high throughput sequencing is becoming feasible in the clinical setting, a suitable next generation sequencing (NGS) platform was deemed to be the best option for sequencing in our laboratory. As the majority of requests for sequencing involve small biopsies and cytology specimens, the most appropriate platform is one which is not only cheap to run by offering scalability but which also utilises a minimum amount of tumour DNA, gives accurate and reliable results, has a fast turnaround time and is not complicated to run.

The Ion Ampliseq Cancer Panel V2 (ThermoFisher, USA) was the panel of our choice for validation study on the sequencer Ion Torrent Personal Genome Machine (PGM; ThermoFisher). The panel is used to generate a library of amplicons for each sample. The primer pool is used to amplify a minimum of 10 ng of genomic DNA (gDNA) from fresh-frozen or formalin fixed, paraffin embedded (FFPE) tissue sample in a multiplex polymerase chain reaction (PCR) target amplification reaction. The resulting amplicons are treated with FuPa reagents to partially digest the primers and phosphorylate the extremities of the amplicons. The amplicons are then ligated to Ion adapters and purified. The completed libraries are then combined with Ion Sphere Particles (ISPs) for clonal amplification in an emulsion PCR reaction. After the emulsion PCR and enrichment process the template-positive ISPs are loaded into a chip for sequencing in the PGM. The Cancer Panel V2 assay detects mutations on selected hotspot regions of the following 50 genes: ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZF2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, IDH2, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, and VHL. The Cancer Panel V2 detects mutations on 2855 hotspots.

The scope of the validation process included selection of appropriate reference samples with known mutations, DNA extraction and quality control, library preparation and quality control, amplicon sequencing using an Ion Torrent PGM, data analysis using the Torrent suite and Ion Reporter software, variant calling and nomenclature, clinical interpretation and reporting. As part of the validation procedure, taking into consideration NGS guidelines from The National Pathology Accreditation Advisory Council,¹ The Royal College of Pathologists of Australasia,² The American College of Medical Genetics³ and The New York State Department of Health,⁴ we established the analytical sensitivity and specificity, lower limit of detection, accuracy, robustness, inter-instrumental variability and precision (repeatability and reproducibility) of the panel, thereby empirically establishing quality control parameters required to minimise false results from FFPE tumour samples. While these above mentioned guidelines are very comprehensive, little information on how to archive the specifications is provided. Here, we provided a detailed step by step workflow based on a four-stage validation process to address these requirements.