Diagnostic innovations developed by EuroClonality

Two central aims of EuroClonality are innovation and standardization. For almost 30 years, researchers from the laboratories connected to EuroClonality have been collaborating to develop new diagnostic tests (assays) for lymphoid malignancies, and to standardize their use. These innovations are used by laboratories worldwide to obtain more accurate information on diagnosis and prognosis of (potential) leukemia and lymphoma patients, in a shorter timeline. Furthermore, standardization facilitates optimal use of the tests and comparison of results between laboratories. This means that patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma, multiple myeloma and other diseases can be treated faster and more efficiently, resulting in longer life expectancy and better quality of life.

The most important and impactful innovations of EuroClonality are:

Multiplex PCR assays for analysis of IG/TR rearrangements: Gold standard for clonality assessment

These assays were developed in the BIOMED-2 project period, during which the consortium received funding from the European Commission, and later on became the gold standard for clonality assessment worldwide. Sets of primers targeting an IG gene (IGH, IGK, IGL) or TR gene (TRB, TRD, TRG) were smartly designed and optimized. By detecting IG/TR rearrangements, the assays can be used to discriminate between normal (polyclonal) and abnormal/malignant (monoclonal) lymphoproliferations during diagnosis and follow-up of lymphoid malignancies.

The IP related to this invention was licensed to the IVD manufacturer Invivoscribe® in 2002, and the assays have been commercially available since then. Royalty income obtained from this partnership has enabled EuroClonality to continue to contribute to the field with innovations, guidelines, EQA schemes and educational activities.

Main references:

Van Dongen et al, Leukemia 2003;17:2257-317

Langerak et al, Leukemia 2012;26:2159-71

One-tube assay for analysis of TRG rearrangements

Following a EuroClonality publication on new one-tube TRG assays in 2019, the original TRG two-tube assay for clonality assessment has been gradually replaced in diagnostic practice.

In preceding years, EuroClonality designed a one-tube TRG assay with the following specifications: (i) to regroup PCR products within a limited size range, (ii) to generate relatively short PCR products (<200 bp), and (iii) to combine all primers within a single tube. In a multi-center study, EuroClonality compared the performance of this one-tube TRG assay with Invivoscribe’s one-tube TRG2.0 assay and the original EuroClonality/BIOMED-2 two-tube TRG assay.

Compared to the classical EuroClonality/BIOMED-2 two-tube TRG assay, both one-tube assays displayed fairly similar performances and demonstrated easier interpretation as well as a lower risk of false positives from minor peaks in dispersed repertoires. In addition, both assays generate smaller fragments, likely to be better adapted to analysis of formalin-fixed paraffin-embedded tissue samples.

Production and sales of the EuroClonality/BIOMED-2 TRG tube A-B assays has been discontinued by Invivoscribe, and has been replaced by the Invivoscribe TRG2.0 assay.

Reference:

Armand et al, Hemasphere 2019;3:e255

Capture-based NGS integrated lymphoma panel

The EuroClonality-NGS DNA capture (NDC) panel is composed of probes targeting a large number of IG/TR rearrangements, translocations, copy number alterations, and somatic mutations associated with lymphoma. EuroClonality developed this approach with the aim of replacing a variety of genetic tests through a single, integrated assay.

The IP related to this invention was licensed to the IVD manufacturer Univ8 Genomics® (Belfast, UK) in 2020, and following publication of the EuroClonality-NDC assay in Blood Advances in 2021, the capture panel was introduced to the market. Data acquired with the assay can be analyzed with a dedicated module of ARResT/Interrogate, the NGS data analysis software developed by EuroClonality.

References:

Stewart et al, Blood Adv 2021;5:3188-3198

Stewart et al, Methods Mol Biol 2022;2453:133-152

McCafferty et al, Haematologica 2023;108:1313-1321

Amplicon-based NGS IG/TR assays for clonality assessment and MRD marker identification

Over the previous decade, the EuroClonality-NGS Working Group developed amplicon-based NGS assays to analyze IG/TR rearrangements. Based on primers that amplify smaller amplicons, the approach is perfectly suited for clonality assessment in both frozen and formalin-fixed paraffin-embedded tissues with suboptimal DNA quality. Protocols were developed and published for both Ion Torrent and Illumina sequencers. More recent work has focused on recommendations for evaluation of clonality analysis.

The same approach has been validated in a multi-center study for MRD marker identification in acute lymphoblastic leukemia (ALL).

For NGS data analysis and automated report generation, dedicated modules of ARResT/Interrogate were developed.

References:

Brüggemann et al, Leukemia 2019;33:2241-2253

Scheijen et al, Leukemia 2019;33:2227-2240

Van den Brand et al, J Mol Diagn 2021;23:1105-1115

Kotrova et al, Methods Mol Biol 2021;2185:95-111

Pott et al, Methods Mol Biol 2022;2453:101-117

Van Bladel et al, Methods Mol Biol 2022;2453:7-42

Van den Brand et al, J Mol Diagn 2023;25:729-739

Quality control samples

Quality control samples are critical for standardization and quality metrics. Two different quality control samples for IG/TR analysis with the amplicon-based NGS assays of the EuroClonality-NGS Working Group were developed and tested:

A central polytarget quality control (cPT-QC) to monitor primer performance
A central in-tube quality control (cIT-QC) or ‘spike-in’ to be added to an individual sample as a library-specific quality control and calibrator (e.g. for calibration of MRD quantification)

The NGS data analysis software ARResT/Interrogate can automatically take these controls along in the analysis, performing quality control and quantification.

References:

Brüggemann et al, Leukemia 2019;33:2241-2253

Knecht et al, Leukemia 2019;33:2254-2265

IGHV leader primer assay for mutational status assessment and MRD measurement

Mutational status is a prognostic biomarker for chronic lymphocytic leukemia (CLL). For analysis of the mutational status of IG genes in CLL, the EuroClonality-NGS Working Group developed and validated an NGS IGHV leader primer assay in a large multi-center study. The same assay has also been evaluated for use as sensitive MRD assay in CLL, and is currently further validated together with ERIC (European Research Initiative on CLL).

References:

Langlois de Septenville et al, Methods Mol Biol 2022;2453:153-167

Hengeveld et al, Blood 2023;141:519-528

Bioinformatics pipelines for NGS IG/TR data analysis

Antigen Receptors Research Tool (ARResT)/Interrogate is a bioinformatics pipeline for analysis of NGS data on IG/TR rearrangements. It is optimized for data acquired with the EuroClonality-NGS amplicon- and capture-based NGS IG/TR assays for clonality assessment and MRD. It is also compatible with use of our central polytarget quality control (cPT-QC) and central in-tube quality control (cIT-QC) samples.

ARResT/Interrogate was built and extensively tested, optimized and validated by the EuroClonality-NGS Working Group.

References:

Bystry et al, Bioinformatics 2017;33:435-437

Knecht et al, Leukemia 2019;33:2254-2265

Darzentas et al, Methods Mol Biol 2022;2453:571-584

Van den Brand et al, J Mol Diagn 2023;25:729-739