Next-Generation Sequencing–Based Clonality Assessment of Ig Gene Rearrangements

Abstract

Ig 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. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice. Ig 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. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice. Ig gene (IG) clonality analysis is an important technique in the diagnosis of lymphoid diseases to aid in the distinction between benign and malignant B-cell lymphoid proliferations. The current gold standard technique for IG clonality analysis is the standardized and validated EuroClonality/BIOMED-2 multiplex PCR protocol, followed by GeneScan fragment length analysis.1van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. Garcia-Sanz R. van Krieken J.H. Droese J. Gonzalez D. Bastard C. White H.E. Spaargaren M. Gonzalez 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 (2372) Google Scholar, 2van Krieken J.H. Langerak A.W. Macintyre E.A. Kneba M. Hodges E. Sanz R.G. Morgan G.J. Parreira A. Molina T.J. Cabecadas J. Gaulard P. Jasani B. Garcia J.F. Ott M. Hannsmann M.L. Berger F. Hummel M. Davi F. Bruggemann M. Lavender F.L. Schuuring E. Evans P.A. White H. Salles G. Groenen P.J. Gameiro P. Pott C. Dongen J.J. Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936.Leukemia. 2007; 21: 201-206Crossref PubMed Scopus (238) Google Scholar, 3Langerak A.W. Groenen P.J. Bruggemann 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. Gameiro P. Garcia Sanz R. Gonzalez D. Grand D. Hakansson A. 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. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.Leukemia. 2012; 26: 2159-2171Crossref PubMed Scopus (276) Google Scholar The availability of next-generation sequencing (NGS) techniques opens up new possibilities for clonality analysis, allowing detection of small clones and accurate clonal comparison. Also, the large amount of data that is generated allows a mathematical/statistical approach to interpretation of the results of clonality analysis. Recently, the EuroClonality-NGS Working Group reported on a technically feasible method for NGS-based IG gene analysis of the IG heavy chain (IGH) and kappa light chain (IGK) genes.4Scheijen B. Meijers R.W.J. Rijntjes J. van der Klift M.Y. Mobs M. Steinhilber J. Reigl T. van den Brand M. Kotrova M. Ritter J.M. Catherwood M.A. Stamatopoulos K. Bruggemann M. Davi F. Darzentas N. Pott C. Fend F. Hummel M. Langerak A.W. Groenen P. EuroClonality-NGS Working Group Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS.Leukemia. 2019; 33: 2227-2240Crossref PubMed Scopus (34) Google Scholar, 5Bruggemann M. Kotrova 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 (77) Google Scholar, 6Knecht H. Reigl T. Kotrova M. Appelt F. Stewart P. Bystry V. Krejci A. Grioni A. Pal K. Stranska K. Plevova K. Rijntjes J. Songia S. Svaton M. Fronkova E. Bartram J. Scheijen B. Herrmann D. Garcia-Sanz R. Hancock J. Moppett J. van Dongen JJM Cazzaniga G. Davi F. Groenen P. Hummel M. Macintyre E.A. Stamatopoulos K. Trka J. Langerak A.W. Gonzalez D. Pott C. Bruggemann M. Darzentas N. EuroClonality-NGS Working Group 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 (39) Google Scholar Herein, we report the results of an international multicenter biological validation of this NGS IG clonality technique based on 209 specimens of reactive and neoplastic lymphoproliferations using the EuroClonality/BIOMED-2 protocol as benchmark technology. The B-cell neoplasms that were included in the study were mostly of germinal center or post-germinal center type and reflect the types of lymphoma that are most often tested for clonality in diagnostic practice, such as follicular lymphoma (FL) and marginal zone B-cell lymphoma. In addition, this provides insight in test performance of somatically hypermutated lymphoma subtypes, also including diffuse large B-cell lymphoma (DLBCL). For comparison, a small number of unmutated cases of chronic lymphocytic leukemia/small lymphocytic lymphoma was included. In addition, a large number of reactive lesions, both tonsils and reactive lymph nodes, was included to determine if small clones are detected in reactive lymphoproliferations. This is particularly important because of the high analytical sensitivity of NGS-based IG clonality testing by which clonal rearrangements for IGHV-IGHD-IGHJ, IGHD-IGHJ, and IGKV-IGKJ could be traced back at 2.5% dilutions,4Scheijen B. Meijers R.W.J. Rijntjes J. van der Klift M.Y. Mobs M. Steinhilber J. Reigl T. van den Brand M. Kotrova M. Ritter J.M. Catherwood M.A. Stamatopoulos K. Bruggemann M. Davi F. Darzentas N. Pott C. Fend F. Hummel M. Langerak A.W. Groenen P. EuroClonality-NGS Working Group Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS.Leukemia. 2019; 33: 2227-2240Crossref PubMed Scopus (34) Google Scholar but which may also cause a false-positive (over)interpretation. DNA samples (n = 209) were collected from the archives of the Departments of Pathology at the University Hospital Tübingen (Tübingen, Germany), Charité-Universitätsmedizin Berlin (Berlin, Germany), Cambridge University Hospital (Cambridge, UK), Erasmus MC University Medical Center (Rotterdam, the Netherlands), and Radboud University Medical Center (Nijmegen, the Netherlands) and from the Hematology Department at the Pitié-Salpêtrière and Sorbonne University Hospital (Paris, France) in accordance with the Declaration of Helsinki. DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue (n = 150), frozen tissue (n = 41), or peripheral blood (n = 18) samples. BIOMED-2 multiplex PCRs and GeneScan analysis were performed according to standard procedures1van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. Garcia-Sanz R. van Krieken J.H. Droese J. Gonzalez D. Bastard C. White H.E. Spaargaren M. Gonzalez 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 (2372) Google Scholar in the participating centers that provided the samples. All conclusions per target, final molecular interpretation, GeneScan result files, and DNA samples were subsequently sent to the coordinating laboratory. For all cases with tissue available, central pathology review was performed by four hematopathologists (M.B., I.A., H.E., and F.F.) during a joint session at a multiheaded microscope. Cases were diagnosed as B-cell neoplasia (n = 124), reactive lymphoproliferation (n = 82), or inconclusive between neoplasia and a reactive condition (n = 3), as based on the available material. B-cell neoplasia samples were classified according to the 2017 revised fourth edition of the World Health Organization classification.7Swerdlow S.H. Campo E. Harris N.L. Jaffe E.S. Pileri S. Stein H. Thiele J. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues.Rev ed 4. International Agency for Research on Cancer, Lyon, France2017Google Scholar Supplemental Table S1 presents a more detailed specification of the included diagnoses. PCR library preparation and sequencing were performed as described previously.4Scheijen B. Meijers R.W.J. Rijntjes J. van der Klift M.Y. Mobs M. Steinhilber J. Reigl T. van den Brand M. Kotrova M. Ritter J.M. Catherwood M.A. Stamatopoulos K. Bruggemann M. Davi F. Darzentas N. Pott C. Fend F. Hummel M. Langerak A.W. Groenen P. EuroClonality-NGS Working Group Next-generation sequencing of immunoglobulin gene rearrangements for clonality assessment: a technical feasibility study by EuroClonality-NGS.Leukemia. 2019; 33: 2227-2240Crossref PubMed Scopus (34) Google Scholar Sequencing data were analyzed and visualized with the ARResT/Interrogate platform.8Bystry 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. Davi F. Bruggemann M. Darzentas N. EuroClonality N.G.S. ARResT/Interrogate: an interactive immunoprofiler for IG/TR NGS data.Bioinformatics. 2017; 33: 435-437PubMed Google Scholar From each sample, DNA was sent to two of the four participating laboratories that locally performed both the wet laboratory procedures and data interpretation. For each sample analyzed, an overall conclusion and a conclusion per target (IGHV-IGHJ, IGHD-IGHJ, IGKV-IGKJ, IGKV-KDE/intronRSS-KDE) was scored by the participating laboratory and subsequently submitted to the coordinating laboratory. The concordance between the laboratories (interlaboratory concordance) was scored for the overall interpretation and the conclusion per target. In case of a complete failure of the analysis, the analysis of the entire sample was repeated. In case of failure of a single target, this was scored as discordant due to failure of the target in a single laboratory. Data interpretation was performed at multiple levels. First, NGS results were compared between different laboratories. Second, results from the NGS-based clonality analysis were compared with the results from conventional EuroClonality/BIOMED-2 GeneScan clonality analysis. Third, the NGS-based conclusion was compared with the panel diagnosis. Technical scoring of the NGS-based IG clonality analysis was done per target. Like in the conventional analysis, no quantitative cutoff was used, but the data were interpreted according to the EuroClonality uniform scoring system for the technical description of the targets (described in the EuroClonality/BIOMED-2 guidelines3Langerak A.W. Groenen P.J. Bruggemann 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. Gameiro P. Garcia Sanz R. Gonzalez D. Grand D. Hakansson A. 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. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.Leukemia. 2012; 26: 2159-2171Crossref PubMed Scopus (276) Google Scholar). The categories for the technical description per target were as follows: clonal (optional with more detailed descriptions: weak clonal rearrangement or presence of background), polyclonal (optional: irregular polyclonal), no specific products, or multiple products. Especially for the category multiple products, a duplicate measurement, coming from the other laboratory, was essential to see whether the same dominant clonotypes were identified between the two laboratories. In case of a discordant NGS-based result, a third NGS-based analysis was performed, and the majority result was used for comparison with GeneScan results. If the three NGS-based analyses resulted in three different results, the target was excluded from NGS-based versus GeneScan comparison. The final molecular conclusion was based on the integration of the technical evaluations of the different targets, which was done according to the EuroClonality/BIOMED-2 guidelines,3Langerak A.W. Groenen P.J. Bruggemann 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. Gameiro P. Garcia Sanz R. Gonzalez D. Grand D. Hakansson A. 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. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.Leukemia. 2012; 26: 2159-2171Crossref PubMed Scopus (276) Google Scholar resulting in five main categories: clonal, multiple clones/oligoclonal, polyclonal, no specific product, or pseudoclonal. More detailed molecular interpretations were provided as well.3Langerak A.W. Groenen P.J. Bruggemann 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. Gameiro P. Garcia Sanz R. Gonzalez D. Grand D. Hakansson A. 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. EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations.Leukemia. 2012; 26: 2159-2171Crossref PubMed Scopus (276) Google Scholar For the overall analysis of concordance, interpretations of particular molecular conclusions were grouped as follows: clonal, clonal with (some) polyclonal background, clonal (bi-allelic), or clonal (biclonal) were all categorized as clonal; polyclonal, polyclonal irregular, multiple products, or oligoclonal were categorized as not clonal; very small clones (ie, <5%) with a high polyclonal background or polyclonal profiles with a clone of unknown significance were categorized as polyclonal. For NGS-based IG clonality testing, a panel of primer sets targeting the IG heavy [framework (FR) 3] and light chain (κ) genes (ie, IGHV-IGHJ FR3, IGHD-IGHJ, IGKV-IGKJ, IGKV-KDE/intronRSS-KDE) was applied. The median number of reads per case was 44,832 for IGHV-IGHJ, 17,663 for IGHD-IGHJ, 66,606 for IGKV-IGKJ, and 9869 for IGKV-KDE/intronRSS-KDE. The overall molecular conclusion of NGS-based IG clonality analysis was concordant between the laboratories in 207 of 209 cases (99% interlaboratory concordance) (Table 1). In two cases, the two laboratories reached a discordant overall molecular conclusion. One case (EC-105) concerned a grade 3B FL with transformation to DLBCL in a lymph node, in which clonal IGKV-IGKJ and IGKV-KDE/intronRSS-KDE products were detected in one of the two laboratories, whereas the other laboratory detected a polyclonal result (Supplemental Figure S1A). Results for IGHV-IGHJ and IGHD-IGHJ were polyclonal in both laboratories. Subsequent analysis of the IGK targets by a third laboratory also gave a polyclonal result, resulting in a final polyclonal score. The other case (EC-207) concerned a peripheral blood analysis of a patient with Sjögren syndrome in whom a clonal product for IGHD-IGHJ was detected in only one laboratory. All other targets gave a polyclonal result in both laboratories. Repeated analysis of IGHD-IGHJ gave a polyclonal result (Supplemental Figure S1B), again resulting in a final score of polyclonality.Table 1IG Clonality Analysis Concordance AssessmentInterlaboratory concordanceValue, N (%)Interlaboratory concordance for NGS-based analysis, overall molecular conclusion Concordant207 (99) Discordant2 (1)Interlaboratory concordance for NGS-based analysis, per target conclusion IGHV-IGHJConcordant193 (92)Discordant16 (8)Different sequencing result∗Clonal versus non-clonal.11Failure to sequence target in one laboratory5 IGHD-IGHJConcordant181 (87)Discordant28 (13)Different sequencing result∗Clonal versus non-clonal.12Failure to sequence target in one laboratory15Different clonotypes, low template amount1 IGKV-IGKJConcordant201 (96)Discordant8 (4)Different sequencing result∗Clonal versus non-clonal.8Failure to sequence target in one laboratory0 IGKV-KDE/intronRSS-KDEConcordant196 (94)Discordant13 (6)Different sequencing result∗Clonal versus non-clonal.8Failure to sequence target in one laboratory5Concordance for NGS-based vs GeneScan analysis, overall molecular conclusion Concordant205 (98.1) Discordant3 (1.4) Not evaluable1 (0.5)Concordance for NGS-based vs GeneScan analysis, per target conclusion IGHV-IGHJConcordant173 (83)Discordant34 (16)Clonal result in NGS, not in GS28Clonal result in GS, not in NGS6Not evaluable2 (1) IGHD-IGHJConcordant170 (81)Discordant23 (11)Clonal result in NGS, not in GS9Clonal result in GS, not in NGS6Interpretable result in NGS, not in GS8Not evaluable16 (8) IGKV-IGKJConcordant186 (89)Discordant22 (11)Clonal result in NGS, not in GS19Clonal result in GS, not in NGS3Not evaluable1 (0.5) IGKV-KDE/intronRSS-KDEConcordant190 (91)Discordant17 (8)Clonal result in NGS, not in GS7Clonal result in GS, not in NGS0Interpretable result in NGS, not in GS10Not evaluable2 (1)The interlaboratory concordance was in line with the interlaborabory concordance evaluated by GeneScanning, as reported in the general testing phase, being >80% for IGHD-IGHJ and approximately 90% for IGK rearrangements.1van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. Garcia-Sanz R. van Krieken J.H. Droese J. Gonzalez D. Bastard C. White H.E. Spaargaren M. Gonzalez 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 (2372) Google ScholarIG, Ig gene; IGH, IG heavy chain gene, IGK, IG kappa light chain gene, NGS, next-generation sequencing.∗ Clonal versus non-clonal. Open table in a new tab The interlaboratory concordance was in line with the interlaborabory concordance evaluated by GeneScanning, as reported in the general testing phase, being >80% for IGHD-IGHJ and approximately 90% for IGK rearrangements.1van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. Garcia-Sanz R. van Krieken J.H. Droese J. Gonzalez D. Bastard C. White H.E. Spaargaren M. Gonzalez 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 (2372) Google Scholar IG, Ig gene; IGH, IG heavy chain gene, IGK, IG kappa light chain gene, NGS, next-generation sequencing. When comparing the NGS-based clonality results per target, a high level of concordance (87% to 96%) was shown (Table 1). Discordances were observed in 8% of the target comparisons, which were solved by repetitive testing. This discordance rate per target is in line with the laboratory discordances reported in the general testing of the BIOMED-2 primers by GeneScanning.1van Dongen J.J. Langerak A.W. Bruggemann M. Evans P.A. Hummel M. Lavender F.L. Delabesse E. Davi F. Schuuring E. Garcia-Sanz R. van Krieken J.H. Droese J. Gonzalez D. Bastard C. White H.E. Spaargaren M. Gonzalez 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 (2372) Google Scholar Because of the complementarity of the different targets and the concordance of the other targets, the discordance of a target did not affect the overall conclusion. Although carryover of clonal sequences from sample to sample is a potential risk, signs of slight carryover were only occasionally noticed in a small subset of PCRs, and this never influenced the final scoring of the targets. Results from conventional GeneScan clonality analysis were retrieved from the initial diagnostic workup or, if unavailable, performed specifically for this study. Comparison of the overall molecular conclusion was possible for all but one case (EC-204) in which insufficient DNA was left to perform GeneScan analysis, leaving 208 cases for further analysis. The comparison was restricted to analysis of those targets that were shared between GeneScan and NGS-based clonality (ie, IGHV-IGHJ framework 3; IGHD-IGHJ; and GeneScan and NGS-based IG clonality analysis resulted in the same overall molecular conclusion in of 208 cases (98%) (Table 1). The three discordant cases were all and one In one case (Supplemental Table a clonal result was with GeneScan for IGHD-IGHJ but not with In the other two cases and DLBCL clonal IGHV-IGHJ and IGK results were with NGS-based analysis, whereas GeneScan could not detect clonal rearrangements 1).

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