Next-Generation Sequencing–Based Antigen-Receptor Gene Clonality Assays
2021; Elsevier BV; Volume: 23; Issue: 9 Linguagem: Inglês
10.1016/j.jmoldx.2021.07.002
ISSN1943-7811
Autores Tópico(s)Lymphoma Diagnosis and Treatment
ResumoMassively parallel sequencing or next-generation sequencing (NGS) is rapidly becoming the method of choice in molecular diagnosis. One of its appeals is the ability to utilize the same platform for a variety of assays, such as targeted mutation detection, RNA-based gene fusion detection and expression analyses, and immunoglobulin gene (IGH/IGK) and T-cell receptor (TRB/TRG) clonal rearrangement studies. IGH/IGK clonal rearrangement studies by NGS-based assay are the subject of this commentary. A recent commentary in the July issue of The Journal of Molecular Diagnostics discussed T-cell receptor clonality testing using NGS.1Greiner T.C. Bagg A. Langerak A.W. Transitioning T-cell clonality testing to high-throughput sequencing.J Mol Diagn. 2021; 23: 781-783Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar In all types of immunoglobulin (IG) clonality studies, primer sets are designed to bind to specific regions of the IGH or IGK genes to enable PCR amplification of the rearranged genes. Historically, electrophoresis-based assays [mostly heteroduplex/gel or capillary electrophoresis (CE) based] separate the amplified products on the basis of length. The main advantages of this clonality approach are that through the efforts of the BIOMED-2 (now EuroClonality) group, primer sets have been optimized, while interpretative criteria and reporting terminologies have also been standardized.2van Dongen J.J.M. Langerak A.W. Brüggemann M. Evans P.A.S. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H.J.M. Droese J. González D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 concerted action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2387) Google Scholar,3Langerak A.W. Groenen P.J.T.A. Brüggemann M. Beldjord K. Bellan C. Bonello L. Boone E. Carter G.I. Catherwood M. Davi F. Delfau-Larue M.H. Diss T. Evans P.A.S. Gameiro P. Garcia Sanz R. Gonzalez D. Grand D. Håkansson Å. Hummel M. Liu H. Lombardia L. MacIntyre E.A. Milner B.J. Montes-Moreno S. Schuuring E. Spaargaren M. Hodges E. Van Dongen J.J.M. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.Leukemia. 2012; 26: 2159-2171Crossref PubMed Scopus (283) Google Scholar Such standardization facilitated the wide adoption of the BIOMED-2/EuroClonality assays in molecular laboratories all over the world in the past two decades. Now, with the availability of NGS methods, the critical questions arise: Do the advantages of NGS-based clonality testing compel molecular laboratories to adopt this technique in preference to electrophoresis-based assays? Are NGS-based clonality assays standardized enough for wide adoption in clinical settings? Instead of separating PCR amplification products by length, NGS-based clonality assays identify the products on the basis of the actual sequences of each DNA fragment. Because of the data output format from sequencing instruments, software pipelines can be developed to automate at least a portion of the analyses. For example, to take into consideration factors such as PCR errors and sequencing artifacts, highly similar sequences are usually grouped together to facilitate the recognition and quantification of clonotypes. At the same time, clonally related DNA fragments with high sequence homology can be identified on the basis of their VDJ annotations and sequence comparisons, even if they differ in length, an analysis approach that would not be possible with the CE-based assays. The results are also highly quantitative, with each clonotype usually represented by its relative proportion of reads within the entire sample. Such presentation of data theoretically allows more objective clonality assessments than the CE assays. On the other hand, NGS-based assays are currently costlier than CE-based assays, with longer turnaround time, and require bioinformatics expertise for data analyses. Having been aware of the potential advantages of the NGS-based assay, the EuroClonality group has embarked on the task of developing a standardized assay over the past few years, from proof-of-concept studies to assay validation, to development of bioinformatics tools that simplify analyses.4Brüggemann M. Kotrová M. Knecht H. Bartram J. Boudjogrha M. Bystry V. et al.Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study.Leukemia. 2019; 33: 2241-2253Crossref PubMed Scopus (79) Google Scholar, 5Knecht H. Reigl T. Kotrová M. Appelt F. Stewart P. Bystry V. Krejci A. Grioni A. Pal K. Stranska K. Plevova K. Rijntjes J. Songia S. Svatoň M. Froňková E. Bartram J. Scheijen B. Herrmann D. García-Sanz R. Hancock J. Moppett J. van Dongen J.J.M. Cazzaniga G. Davi F. Groenen P.J.T.A. Hummel M. Macintyre E.A. Stamatopoulos K. Trka J. Langerak A.W. Gonzalez D. Pott C. Brüggemann M. Darzentas N. Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS.Leukemia. 2019; 33: 2254-2265Crossref PubMed Scopus (40) Google Scholar, 6Scheijen B. Meijers R.W.J. Rijntjes J. van der Klift M.Y. Möbs M. Steinhilber J. Reigl T. van den Brand M. Kotrová M. Ritter J.M. Catherwood M.A. Stamatopoulos K. Brüggemann M. Davi F. Darzentas N. Pott C. Fend F. Hummel M. Langerak A.W. Groenen P.J.T.A. Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS.Leukemia. 2019; 33: 2227-2240Crossref PubMed Scopus (35) Google Scholar, 7Bystry V. Reigl T. Krejci A. Demko M. Hanakova B. Grioni A. Knecht H. Schlitt M. Dreger P. Sellner L. Herrmann D. Pingeon M. Boudjoghra M. Rijntjes J. Pott C. Langerak A.W. Groenen P.J.T.A. Davi F. Brüggemann M. Darzentas N. ARResT/interrogate: an interactive immunoprofiler for IG/TR NGS data.Bioinformatics. 2017; 33: 435-437PubMed Google Scholar In the two EuroClonality-NGS articles on this subject in the current issue of The Journal of Molecular Diagnostics, van den Brand et al8van den Brand M. Rijntjes J. Möbs M. Steinhilber J. van der Klift M.Y. Heezen K.C. Kroeze L.I. Reigl T. Porc J. Darzentas N. Luijks J.A.C.W. Scheijen B. Davi F. ElDaly H. Liu H. Anagnostopoulos I. Hummel M. Fend F. Langerak A.W. Groenen P.J.T.A. on behalf of the E-NWG Next generation sequencing-based clonality assessment of immunoglobulin gene rearrangements: a multicenter validation study by EuroClonality-NGS.J Mol Diagn. 2021; 23: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar present a clinical validation of their NGS-based IG clonality assay using IGH and IGK primer sets, with comparison to the BIOMED-2 primer sets and GeneScan, a widely used CE method. Leenders et al9Leenders A.M. Kroeze L.I. Rijntjes J. Luijks J. Hebeda K.M. Darzentas N. Langerak A.W. van den Brand M. Groenen P.J.T.A. Multiple immunoglobulin kappa rearrangements within a single clone unraveled by NGS-based clonality assessment.J Mol Diagn. 2021; 23: 1097-1104Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar illustrate an interesting clinical application of the IGK primer sets of their NGS-based assay in clarifying the mechanism behind complex IGK rearrangement patterns detected but not easily resolved by BIOMED-2/GeneScan. In the study by van den Brand et al,8van den Brand M. Rijntjes J. Möbs M. Steinhilber J. van der Klift M.Y. Heezen K.C. Kroeze L.I. Reigl T. Porc J. Darzentas N. Luijks J.A.C.W. Scheijen B. Davi F. ElDaly H. Liu H. Anagnostopoulos I. Hummel M. Fend F. Langerak A.W. Groenen P.J.T.A. on behalf of the E-NWG Next generation sequencing-based clonality assessment of immunoglobulin gene rearrangements: a multicenter validation study by EuroClonality-NGS.J Mol Diagn. 2021; 23: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar the EuroClonality-NGS group presented a validation study in four different laboratories, of >200 samples of B-cell neoplasm and reactive lymphoid proliferation samples (most of which were formalin-fixed, paraffin-embedded tissue), using four different primer sets: IGHV-IGHJ, IGHD-IGHJ, IGKV-IGKJ, IGKV-KDE/intronRSS-KDE. The forward primer sets in IGHV target the framework (FR) 3 region only, and do not include those that target FR1 and FR2, as in the BIOMED-2/GeneScan design. The primary reason for the design difference appears to be the desire to generate smaller amplicons with the IGHV-IGHJ primer sets, which would increase the chance of successful testing using samples with suboptimal DNA, such as DNA extracted from formalin-fixed, paraffin-embedded tissue. Although primer sets targeting FR1 and FR2 of IGHV were not used, the use of IGHD and IGK primers (which are also available in the BIOMED-2 primer sets) complemented the clonality detection, and the overall concordance between BIOMED-2/GeneScan and the NGS assay was approximately 98%, with interlaboratory concordance of 99%. In terms of analysis tools, through collaborations with other institutions, the group previously facilitated the development of a free, publicly available, web-based application called ARResT/Interrogate,7Bystry V. Reigl T. Krejci A. Demko M. Hanakova B. Grioni A. Knecht H. Schlitt M. Dreger P. Sellner L. Herrmann D. Pingeon M. Boudjoghra M. Rijntjes J. Pott C. Langerak A.W. Groenen P.J.T.A. Davi F. Brüggemann M. Darzentas N. ARResT/interrogate: an interactive immunoprofiler for IG/TR NGS data.Bioinformatics. 2017; 33: 435-437PubMed Google Scholar which provided VDJ gene segment annotations for each clonotype, and information on the complementarity determining region 3, on the basis of the International ImMunoGeneTics nomenclature. The software allows the users to sort, filter, and visualize the data in each case, as well as compare results with other cases. Similar to the interpretative guidelines for BIOMED-2/GeneScan,2van Dongen J.J.M. Langerak A.W. Brüggemann M. Evans P.A.S. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. García-Sanz R. van Krieken J.H.J.M. Droese J. González D. Bastard C. White H.E. Spaargaren M. González M. Parreira A. Smith J.L. Morgan G.J. Kneba M. Macintyre E.A. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 concerted action BMH4-CT98-3936.Leukemia. 2003; 17: 2257-2317Crossref PubMed Scopus (2387) Google Scholar,3Langerak A.W. Groenen P.J.T.A. Brüggemann M. Beldjord K. Bellan C. Bonello L. Boone E. Carter G.I. Catherwood M. Davi F. Delfau-Larue M.H. Diss T. Evans P.A.S. Gameiro P. Garcia Sanz R. Gonzalez D. Grand D. Håkansson Å. Hummel M. Liu H. Lombardia L. MacIntyre E.A. Milner B.J. Montes-Moreno S. Schuuring E. Spaargaren M. Hodges E. Van Dongen J.J.M. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.Leukemia. 2012; 26: 2159-2171Crossref PubMed Scopus (283) Google Scholar the EuroClonality-NGS group did not suggest using a fixed threshold for clonality calling; rather, the data should be interpreted in the context of histopathologic findings, clinical suspicions, and taking into consideration the degree of a sequence's dominance over the background. Using receiver operator characteristic analyses, the group also attempted to determine the optimal clonality cutoff for each of the four different target primer sets, using the ratio of sequencing reads of the dominant sequence/that of certain background sequences. However, the criteria may require further modification/refinement in the future to account for different patterns of results and for different tumor types. Leenders et al9Leenders A.M. Kroeze L.I. Rijntjes J. Luijks J. Hebeda K.M. Darzentas N. Langerak A.W. van den Brand M. Groenen P.J.T.A. Multiple immunoglobulin kappa rearrangements within a single clone unraveled by NGS-based clonality assessment.J Mol Diagn. 2021; 23: 1097-1104Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar used NGS-based clonality assessment to distinguish monoclonal from biclonal or oligoclonal lymphoproliferations. They showed three cases in which multiple IGK rearrangements (three to five) were found using the BIOMED-2 system applied to the IGK gene. The IGK locus differs from the other antigen receptor genes because after an unproductive rearrangement further DNA rearrangement frequently causes a deletion of parts of the gene. If the deletion event involves the κ deleting element (Kde) in the intron (intron-Kde), the initial IGK V-J rearrangement is preserved. Thus, if both IGK genes have unproductive rearrangements and both undergo intron-Kde–mediated deletion, it is possible to find evidence for four rearrangements in a monoclonal population of lymphocytes.10Langerak A.W. van Dongen J.J.M. Multiple clonal Ig/TCR products: implications for interpretation of clonality findings.J Hematop. 2012; 5: 35-43Crossref Scopus (29) Google Scholar This is not unusual. Furthermore, the IGK locus has a distal upstream cluster of V genes (IGKVD) in the opposite chromosomal orientation. These IGKV genes are also capable of rearranging with the IGKJ genes, causing an inversion of the intervening DNA and maintaining the presence of any preceding (or following) IGKVJ rearrangements using the proximal IGKV cluster.10Langerak A.W. van Dongen J.J.M. Multiple clonal Ig/TCR products: implications for interpretation of clonality findings.J Hematop. 2012; 5: 35-43Crossref Scopus (29) Google Scholar Theoretically, it is possible to have up to three detectable rearrangements per IGK locus, or six per cell. In one of the cases studied by Leenders et al,9Leenders A.M. Kroeze L.I. Rijntjes J. Luijks J. Hebeda K.M. Darzentas N. Langerak A.W. van den Brand M. Groenen P.J.T.A. Multiple immunoglobulin kappa rearrangements within a single clone unraveled by NGS-based clonality assessment.J Mol Diagn. 2021; 23: 1097-1104Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar rearrangements involving the upstream IGKVD cluster explained the presence of more than two (three) IGKVJ rearrangements per cell, in addition to two intron-Kde rearrangements. In the second case, two IGKVJ rearrangements were found as well as two deletion events, one of which was a IGKV-Kde deletion which removes any VJ rearrangement(s) on the same allele. In this case, one of the two IGKVJ rearrangements used the upstream IGKVD cluster. Thus, the two IGKVJ rearrangements were on the same allele. In the third case, the finding of three IGKVJ rearrangements by the BIOMED-2 system corresponded to only two rearrangements detected by NGS. This case was simply due to binding of one of the BIOMED-2 upstream IGKV primers to two different V genes. Thus, the application of NGS allowed the reclassification of three cases that had evidence for biclonality or oligoclonality to be consistent with an interpretation of monoclonality. For the past two decades, BIOMED-2–based clonality assays, using fragment analysis, have been the gold standard all over the world. With their previous publications4Brüggemann M. Kotrová M. Knecht H. Bartram J. Boudjogrha M. Bystry V. et al.Standardized next-generation sequencing of immunoglobulin and T-cell receptor gene recombinations for MRD marker identification in acute lymphoblastic leukaemia; a EuroClonality-NGS validation study.Leukemia. 2019; 33: 2241-2253Crossref PubMed Scopus (79) Google Scholar, 5Knecht H. Reigl T. Kotrová M. Appelt F. Stewart P. Bystry V. Krejci A. Grioni A. Pal K. Stranska K. Plevova K. Rijntjes J. Songia S. Svatoň M. Froňková E. Bartram J. Scheijen B. Herrmann D. García-Sanz R. Hancock J. Moppett J. van Dongen J.J.M. Cazzaniga G. Davi F. Groenen P.J.T.A. Hummel M. Macintyre E.A. Stamatopoulos K. Trka J. Langerak A.W. Gonzalez D. Pott C. Brüggemann M. Darzentas N. Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS.Leukemia. 2019; 33: 2254-2265Crossref PubMed Scopus (40) Google Scholar, 6Scheijen B. Meijers R.W.J. Rijntjes J. van der Klift M.Y. Möbs M. Steinhilber J. Reigl T. van den Brand M. Kotrová M. Ritter J.M. Catherwood M.A. Stamatopoulos K. Brüggemann M. Davi F. Darzentas N. Pott C. Fend F. Hummel M. Langerak A.W. Groenen P.J.T.A. Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS.Leukemia. 2019; 33: 2227-2240Crossref PubMed Scopus (35) Google Scholar, 7Bystry V. Reigl T. Krejci A. Demko M. Hanakova B. Grioni A. Knecht H. Schlitt M. Dreger P. Sellner L. Herrmann D. Pingeon M. Boudjoghra M. Rijntjes J. Pott C. Langerak A.W. Groenen P.J.T.A. Davi F. Brüggemann M. Darzentas N. ARResT/interrogate: an interactive immunoprofiler for IG/TR NGS data.Bioinformatics. 2017; 33: 435-437PubMed Google Scholar and the two articles in this edition of The Journal of Molecular Diagnostics, the EuroClonality-NGS group has advanced toward the goal of standardizing the technical, interpretative, and bioinformatics aspects of NGS-based clonality testing, and demonstrated its superiority over BIOMED-2/GeneScan in certain situations. In addition, there are at least two commercially available NGS-based clonality assays designed on similar concepts, which have been clinically validated and used in studies on numerous patients. Both commercial assays also have existing published quantitative clonal interpretative criteria.11Arcila M.E. Yu W. Syed M. Kim H. Maciag L. Yao J. Ho C. Petrova K. Moung C. Salazar P. Rijo I. Baldi T. Zehir A. Landgren O. Park J. Roshal M. Dogan A. Nafa K. 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Application of the Euro Clonality next-generation sequencing-based marker screening approach to detect immunoglobulin heavy chain rearrangements in mantle cell lymphoma patients: first data from the Fondazione Italiana Linfomi MCL0208 trial.Br J Haematol. 2021; 194: 378-381Crossref PubMed Scopus (1) Google Scholar In the short-term, the choice of antigen-receptor gene rearrangement clonality testing method for individual laboratories will depend on multiple factors, including availability of NGS sequencing instruments and bioinformatics expertise, expectations concerning turnaround time, need for somatic hypermutation status and MRD testing, as well as financial and staffing considerations. We expect that as the costs of NGS decrease and the value of MRD studies increases, NGS will eventually become the first choice for analysis of antigen-receptor gene rearrangements. It is clear that because of the efforts of EuroClonality-NGS, their NGS-based clonality assay has comparable performance to the gold standard BIOMED-2/GeneScan approach, can add insights to immunogenetics, and can provide additional information that facilitates accurate interpretation in some cases. Finally, the necessary bioinformatics tools have been developed and are available to the laboratory community. This work brings us closer to widespread implementation of NGS for clonality testing. Next-Generation Sequencing–Based Clonality Assessment of Ig Gene Rearrangements: A Multicenter Validation Study by EuroClonality-NGSThe Journal of Molecular DiagnosticsVol. 23Issue 9PreviewIg gene (IG) clonality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)–based IG clonality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. Full-Text PDF Open AccessMultiple Immunoglobulin κ Gene Rearrangements within a Single Clone Unraveled by Next-Generation Sequencing–Based Clonality AssessmentThe Journal of Molecular DiagnosticsVol. 23Issue 9PreviewClonality assessment of the Ig heavy- and light-chain genes (IGH and IGK) using GeneScan analysis is an important supplemental assay in diagnostic testing for lymphoma. Occasionally cases with an IGK rearrangement pattern that cannot readily be assigned to a monoclonal lymphoma are encountered, whereas the occurrence of biclonal lymphomas is rare, and the result of the IGH locus of these cases is in line with monoclonality. Three such ambiguous cases were assessed for clonality using next-generation sequencing. Full-Text PDF Open Access
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