1. Introduction
The EuroClonality-NDC is a capture-based targeted next generation sequencing (NGS) assay with customised bioinformatics pipeline for research use only, designed and developed by the EuroClonality-NGS working group (an independent subdivision of EuroClonality).
The EuroClonality-NDC assay has undergone a large multi-centre validation, performed across seven European laboratories, in a wide range of samples from more than 15 categories of lymphoid malignancies and including high molecular weight (HMW) and FFPE gDNA1.
These results highlight the robustness of the assay as performed in academic or clinical laboratories with varying degree of experience with DNA capture hybridization protocols.
In addition, EuroClonality-NDC can now be used in conjunction with our highly streamlined data analysis app. Powered by one of the most secure platforms for genomic data analysis (Illumina’s BioInsights and BaseSpace Sequence HubTM) the analysis process is set up to support our customers with security and data protection compliance.
Stewart JP, Gazdova J, Darzentas N, Wren D, Proszek P, Fazio G, Songia S, Alcoceba M, Sarasquete ME, Villarese P, van der Klift MY, Heezen KC, McCafferty N, Pal K, Catherwood M, Kim CS, Srivastava S, Kroeze LI, Hodges E, Stamatopoulos K, Klapper W, Genuardi E, Ferrero S, van den Brand M, Cazzaniga G, Davi F, Sutton LA, Garcia-Sanz R, Groenen PJTA, Macintyre EA, Brüggemann M, Pott C, Langerak AW, Gonzalez D; EuroClonality-NGS Working Group. Validation of the EuroClonality-NGS DNA capture panel as an integrated genomic tool for lymphoproliferative disorders. Blood Adv. 2021 Aug 24;5(16):3188-3198. doi: 10.1182/bloodadvances.2020004056. PMID: 34424321.
2. Scope
| Gene Name | Region Covered |
|---|---|
| ABL1 | Exons 4-9 |
| ARID1A | All coding |
| ASXL1 | Hotspot in last exon |
| ATM | All coding |
| B2M | All coding |
| BCL2 | All coding |
| BIRC3 | Exons 8 and 9 |
| BRAF | Exons 11 and 15 |
| BTG1 | All coding |
| BTK | All coding |
| CARD11 | Exons 2-16 |
| CCND1 | Exon 1 |
| CCND3 | Exon 5 |
| CD79A | Exons 4 & 5 |
| CD79B | Exons 4-6 |
| CDKN2A | All coding |
| CDKN2B | All coding |
| CDKN2C | All coding |
| CKS1B | All coding |
| CREBBP | Exons 2-30 |
| CXCR4 | Hotspot in exon 2 |
| DIS3 | Exons 1-3 and 9-18 |
| DNMT3A | Exons 8-23 |
| EGR2 | Hotspot in exon 2 |
| EP300 | All coding |
| ERG | All coding |
| EZH2 | Exons 15-17 |
| FAT1 | All coding |
| FBXW7 | Exons 1-12 |
| FOXO1 | Exon 1 |
| H1-2 | All coding |
| H1-3 | All coding |
| H1-4 | All coding |
| H1-5 | All coding |
| ID3 | All coding |
| IDH1 | Exon 4 |
| IDH2 | Exons 4 and 7 |
| IKZF1 | Exons 2-7 |
| IL7R | Exon 6 |
| IRF4 | Exons 2-3 |
| JAK1 | Exons 12-15 |
| JAK2 | Exons 12-15 |
| JAK3 | Exons 11-24 |
| KIT | Exons 9, 11, 13, 17 and 18 |
| KLF2 | All coding |
| KMT2D | All coding |
| KRAS | Exons 2, 3 & 4 |
| MAP2K1 | Exons 1-8 |
| MAP3K14 | All coding |
| MAPK1 | All coding |
| MEF2B | Exons 2-4 |
| MYC | All coding |
| MYD88 | Exon 3-5 |
| NFKBIE | Exon 1 |
| NOTCH1 | Exons 26, 27 & 34 |
| NOTCH2 | Exon 34 |
| NRAS | Exons 2-4 |
| NT5C2 | Exons 9-15 |
| PAX5 | All coding |
| PHF6 | All coding |
| PIK3CA | Exons 2, 5 10 & 21 |
| PLCG1 | Exons 1, 11, 15-17, 19 and 29 |
| PLCG2 | Exons 19, 20 & 24 |
| POT1 | All coding |
| PTEN | All coding |
| PTPRD | All coding |
| RHOA | All coding |
| RUNX1 | All coding |
| SAMHD1 | All coding |
| SF3B1 | Exons 12-17 |
| SOCS1 | All coding |
| STAT3 | Exons 10-16 and 20-21 |
| STAT5B | Exons 13-19 |
| STAT6 | Exons 5, 9, 11, 12 and 14 |
| TCF3 | All coding |
| TENT5C | All coding |
| TET2 | All coding |
| TNFAIP3 | All coding |
| TP53 | All coding |
| TRAF2 | All coding |
| TRAF3 | All coding |
| WT1 | All coding |
| XPO1 | Exons 15-16 |
| Chromosome | Gene Name | Chromosome | Gene Name |
|---|---|---|---|
| chr1 | STIL | chr11 | CCND1 |
| chr2 | ALK | chr11 | BIRC3 |
| chr3 | BCL6 | chr11 | KMT2A |
| chr6 | DUSP22 | chr18 | BCL2 |
| chr8 | MYC | chr19 | TCF3 |
| chr9 | CD274 | chrX | CRLF2 |
| chr9 | PDCD1LG2 |
| 9pTEL chr9 | 9pCEN chr9 | 11qCEN chr11 | 11qTEL chr11 | RB1 chr13 |
| 13qMDRa chr13 | 13qTEL chr13 | 17pTEL chr17 | 17pCEN chr17 |
a Probes span a minimally deleted region (MDR) comprising the DLEU1 and DLEU2 genes
3. Output
The table below shows the approximate expected metrics of the EuroClonality-NDC assay based on 22 samples per hybridisation reaction (plus controls) and run on a NextSeq500/550 (Mid-Output 150 cycles cartridge) at 2 x 75bp. These can vary depending on DNA quality and quantity, technical factors in each laboratory, differences in equipment, etc.
| Parameter | Expected HMW-DNA | Expected FFPE-DNA |
|---|---|---|
| Library yield (ng) | 3,000 ng (range: 500-6,000) | 3,000 ng (range: 500-6,000) |
| Library average fragment size (bp) | 330 bp (range: 275 – 450 bp) | 290 bp (range: 245 – 330 bp) |
| Average on/near target bases (%) | 80% (range: 70 - 85%) | 80% (range: 70 - 85%) |
| Duplicate reads (%) | 40% (range: 30 – 50%) | 40% (range: 30 – 50%) |
| Mean unique target coverage (reads) | 1,000x (range: 500 – 1,500x) | 1,000x (range: 500 – 1,500x) |
This improved version of the EuroClonality-NDC assay and workflow underwent an analytical verification bridging study with gDNA extracted from 22 FFPE and 44 HMW samples which were included in the original validation study¹.
The following table represents the observed analytical performance:
| Analytical Sensitivity | Analytical Specificity | Limit of Detection | |
|---|---|---|---|
| IG/TR Rearrangements | >97% | >97% | 5% VAF |
| Translocation | >95% | >99% | 5% VAF |
| SNV | >99% | >99% | 4% VAF |
| CNV* | >95% | >97% | 40%** |
SNV: single nucleotide variant; CNV: copy number variation. *CNV have been validated in HMW-DNA for trisomy 12 and deletions of 11q, 13q and 17p. No performance data available for FFPE or other genomic regions. **Deletions can be detected if present in ≥ 40% of the cells. The threshold baseline fold-change for deletions detected by EuroClonality-NDC is 0.8 and 1.3 for losses and gains, and 0.7 and 2.0 for deletions and amplifications, respectively.
4. Performance
The validation and performance characterisation of the EuroClonality-NDC was carried out on 280 samples comprising a range of different B and T cell malignancies using a different version of library preparation and hybridisation methodologies.
The EuroClonality-NDC assay and analysis workflow have been updated since to improve performance and target coverage. Overall performance of the EuroClonality-NDC assay was assessed by identifying underperforming regions using DNA extracted from a panel of 91 normal tissue samples. Regions were classed as underperforming if coverage was >2 standard deviations (SD) below the mean in ≥50% samples. Only 10 underperforming coding regions have been identified that could affect mutation detection in the genes and exons shown in the table below.
| Gene Name | Chromosome | Exon |
|---|---|---|
| ARID1A | chr1 | 6 |
| CARD11 | chr7 | 12 |
| JAK2 | chr9 | 15 |
| TRAF2 | chr9 | 1 |
| PAX5 | chr9 | 10 |
| STAT5B | chr17 | 18 |
| TCF3 | chr19 | 7 |
| BTK | chrX | 18 |
| PHF6 | chrX | 6 |
| PHF6 | chrX | 3 |
5. Publications
The authors demonstrate that applying the EuroClonality-NDC assay to DNA samples from peripheral blood and bone marrow reliably detects diagnostically and prognostically important genetic factors in MCL patients, facilitating genetic characterization in clinical routine.
Describes the molecular relationship between BCP-ALL and BCP-LBL using whole exome sequencing / transcriptomics. EuroClonality-NDC was used for IG/TR rearrangement analysis.
Commentary/Editorial on the liquid biopsy manuscript below.
6. Resources
This quick reference document is intended to guide the users through the instructions of use for the EuroClonality-NDC assay and associated laboratory procedures.
This document is intended to guide the users through the analysis of the results generated by the EuroClonality-NDC assay and associated analysis tools.
Examples of EuroClonality-NDC output files from selected cell lines.
Queen’s University Belfast
BELFAST, UK
Universitätsklinikum Schleswig-Holstein (UKSH)
KIEL, GERMANY
CEITEC – Central European Technology Institute
BRNO, CZECH REPUBLIC
The Royal Marsden NHS Trust
LONDON, UK
Centro Maria Letizia Verga
MONZA, ITALY
Hospital Universitario de Salamanca
SALAMANCA, SPAIN
Hopital Necker-Enfants Malades
PARIS, FRANCE
Erasmus MC, University Medical Center Rotterdam
ROTTERDAM, THE NETHERLANDS
Centre for Research and Technology Hellas
THESSALONIKI, GREECE
University of Torino
TORINO, ITALY
Radboud University Medical Centre Nijmegen
NIJMEGEN, THE NETHERLANDS
Hopital Pitié-Salpétrière
PARIS, FRANCE
7. Enquire
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For Research Use Only. Not for use in diagnostic procedures.