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Multicenter validation of targeted NGS in CLL
Introduction
Chronic lymphocytic leukemia (CLL), a clinically and biologically heterogeneous B-cell malignancy, was one of the first cancers for which the genomic landscape was uncovered using next-generation sequencing (NGS) tech- nologies, in particular whole-exome/genome sequencing (WES/WGS).1-4 These initial studies led to the discovery of a large number of recurrently mutated genes affecting diverse cellular pathways and processes contributing to the pathobiology of CLL. That said, in a diagnostic or untreated cohort, only a limited number of genes carry mutations at a frequency >5% (i.e., TP53, ATM, SF3B1 and NOTCH1), with the majority of gene mutations detected in only a minor proportion of CLL patients.5,6 To date, more than 2,000 genes have been reported as mutat- ed in CLL, with mutations occurring in classic tumor sup- pressor genes (e.g., TP53 and ATM), signaling pathways (e.g., the Toll-like receptor [MYD88], NF-κB [BIRC3, NFKBIE], and NOTCH [NOTCH1, FBXW7] pathways) as well as genes involved in essential cellular processes such as RNA processing (e.g., SF3B1, RPS15, XPO1).5-10
In addition to mutations or defects in TP53, which have long been associated with a poor prognosis and progres- sive disease, mutations in several of these recently ana- lyzed genes (with the exception of MYD88) have been linked to an aggressive clinical course with a significantly shorter time to treatment and a poor outcome when treat- ed with chemo(immuno)therapy (e.g., ATM, BIRC3, EGR2, NFKBIE, NOTCH1, RPS15, SF3B1, XPO1).8-20 In recent years it has also been reported that patients carry- ing minor clones harboring TP53 mutations (i.e., <10% variant allele frequency [VAF]) may have an outcome equally as poor as patients with TP53 mutations with VAF >10%, the approximate detection limit of Sanger sequenc- ing.21-24 Employing ultra-deep sequencing technologies these minor clones have been detected at frequencies as low as 0.1% and may expand as the disease progresses and/or at treatment relapse. Minor clones carrying other gene mutations may also affect outcome, however there are few studies published to date, hence precluding firm conclusions from being drawn.23 That said, based on cur- rently available data it appears that both the mutational complexity and subclonal diversity strongly influence the evolution of CLL.6,25-29
Owing to the increasing number of clinically relevant gene mutations identified in CLL, the shift from Sanger sequencing to high-throughput technologies is essential and targeted amplicon-based gene panels are a promising option.30,31 These assays have numerous attractive features including the ability to custom design and screen a large number of genes (complete coding sequence or hotspots) and samples simultaneously, ultimately leading to a reduced cost per sample and a higher throughput. Another appealing facet of targeted gene panels is that the sequenc- ing capacity is efficiently utilized, resulting in higher cov- erage of the regions of interest (ROI), and hence allowing for more sensitive detection of low-frequency variants; thus, they are ideally suited for routine diagnostics and monitoring procedures. However, despite the necessity for mutation detection using NGS assays, with numerous technologies available, including both commercial and laboratory developed tests (LDT), important issues that commonly arise concern the specific technique to choose,
and the sensitivity, specificity and reproducibility of indi- vidual methodologies. These concerns are magnified within the diagnostic setting as the test results may impact on clinical decision-making and the therapeutic stratifica- tion of patients.
The European Research Initiative on CLL (ERIC) conduct- ed this multi-center study to better understand the compa- rability of several gene panel assays by assessing various analytical parameters such as coverage, sensitivity and reproducibility; with the overall aim of highlighting the crit- ical parameters users should take into consideration when introducing targeted NGS into the laboratory. In brief, we selected three amplicon-based assays (HaloPlex, TruSeq and Multiplicom), each protocol was tested by two centers, and all sequencing was performed on the Illumina MiSeq system. Our cohort comprised 48 well-characterized CLL cases and all centers sequenced the same samples. We observed high concordance between the various technolo- gies and test centers when considering gene mutations with VAF >5%. Although low-frequency variants were detected by all techniques, greater diversity was observed for muta- tions with a VAF between 1-5%.
Methods
Patient material
Genomic DNA (gDNA), prepared from tumor and germline samples (buccal swabs or CD19-depleted peripheral blood mononuclear cells), was used as the analytical material. Samples were sourced from archival material that had previously been ana- lyzed using established molecular techniques to interrogate either a single gene or numerous targets. Cases were selected based on the available mutational data such that 45 of 48 cases contained a previously identified somatic variant in at least one of the genes included in the panel designs (Online Supplementary Figure S1). Quality control was performed centrally before distribution of the samples to the six participating institutes. With the exception of the coordinating center, details regarding pre-characterized muta- tions were not disclosed to participating laboratories. All cases were diagnosed according to the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) guidelines and displayed a typical CLL immunophenotyped.32 Informed consent was obtained in accordance with the Declaration of Helsinki and ethi- cal approval was granted by local review committees.
Target enrichment and library construction
Three amplicon-based targeted NGS assays were used in this study. Two assays, the HaloPlex Target Enrichment System (Agilent Technologies, Santa Clara, CA) and the Illumina TruSeq Custom Amplicon (TSCA) (Illumina, San Diego, CA), were cus- tomized and targeted 11 genes: full coding sequence (ATM, BIRC3, EGR2, FBXW7, MYD88, NFKBIE, POT1 and TP53) or hotspot regions (NOTCH1 [exon 34], SF3B1 [exons 14-16 and 18] and XPO1 [exons 15-16]). The third assay, the Multiplicom CLL Multiplex MASTR Plus (Agilent Technologies, Santa Clara, CA), is a commercially designed panel targeting the full coding sequence of nine of the genes listed above; NFKBIE and EGR2 are not included in the design. Intron-exon boundaries were covered by all assays to enable the detection of splice-site mutations. A HaloPlexHS capture-based custom-design assay incorporating unique molecular identifiers (UMI) was used to validate and more accurately quantify variants present at low frequencies within the 11 genes detailed above. The specifics of each assay are detailed in the Online Supplementary Appendix.
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