of April 19, 2017.This information is current as

ChallengesImmunohematology: Potential andClinical and Research Applications in High-Throughput Immunogenetics for

EuroClonality-NGS ConsortiumA. Groenen, Kostas Stamatopoulos and the Macintyre, Michael Hummel, Christiane Pott, Patricia J. T.Marie-Paule Lefranc, Mathieu Giraud, Elizabeth A. Gonzalez, Gianni Cazzaniga, Véronique Giudicelli,Nikos Darzentas, Jacques J. M. van Dongen, David Anton W. Langerak, Monika Brüggemann, Frédéric Davi,

ol.1602050http://www.jimmunol.org/content/early/2017/04/16/jimmun

published online 17 April 2017J Immunol 

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High-Throughput Immunogenetics for Clinical andResearch Applications in Immunohematology: Potentialand ChallengesAnton W. Langerak,* Monika Br€uggemann,† Frederic Davi,‡ Nikos Darzentas,x

Jacques J. M. van Dongen,* David Gonzalez,{ Gianni Cazzaniga,‖ Veronique Giudicelli,#

Marie-Paule Lefranc,# Mathieu Giraud,** Elizabeth A. Macintyre,†† Michael Hummel,‡‡

Christiane Pott,† Patricia J. T. A. Groenen,xx Kostas Stamatopoulos,{{ andthe EuroClonality-NGS Consortium1

Analysis and interpretation of Ig and TCR gene rear-rangements in the conventional, low-throughput wayhave their limitations in terms of resolution, coverage,and biases. With the advent of high-throughput, next-generation sequencing (NGS) technologies, a deeper anal-ysis of Ig and/or TCR (IG/TR) gene rearrangements isnow within reach, which impacts on all main applicationsof IG/TR immunogenetic analysis. To bridge the gener-ation gap from low- to high-throughput analysis, theEuroClonality-NGS Consortium has been formed, withthe main objectives to develop, standardize, and validatethe entire workflow of IG/TR NGS assays for 1) clon-ality assessment, 2) minimal residual disease detection,and 3) repertoire analysis. This concerns the preanalytical(sample preparation, target choice), analytical (amplifica-tion, NGS), and postanalytical (immunoinformatics) phases.Here we critically discuss pitfalls and challenges of IG/TRNGS methodology and its applications in hemato-oncologyand immunology. The Journal of Immunology, 2017,198: 000–000.

Specific Ag recognition of the adaptive immune systemis mediated by a remarkably diverse repertoire of Agreceptors—Igs on B lymphocytes (plus Abs secreted

by plasma cells) and TCRs on T lymphocytes—showing highaffinity for a particular Ag. Fundamental to Ig and/or TCR

(IG/TR) diversity is the combined effect of molecular [mainlyV(D)J recombination] and cellular diversification processesduring maturation of B and T lymphocytes (1–5).The result of Ag-independent B and T lymphocyte differ-

entiation and diversification that occurs in bone marrow andthymus is a broadly diverse, polyclonal repertoire of Ag-specificreceptors (naive or primary repertoire), whereas Ag-dependentmaturation in the periphery further shapes the IG/TR repertoirethrough selection processes (immunocompetent or antigen-experienced repertoire) (6, 7). IG/TR polyclonality is one endof a continuum of immune profiles (Fig. 1A). Upon Ag-specifictriggering and during inflammation certain IG/TR specificitiescan predominate and lead to one or more small clones (i.e., theoffspring of particular B or T lymphocytes) on top of the poly-clonal repertoire, thus reflecting a more oligoclonal immuneprofile. At the other end of the continuum, significant outgrowthof a single lymphocyte clone with particular Ag specificity wouldlead to a monoclonal immune profile, the hallmark of lymphoidmalignancies (Fig. 1A).In view of the extensive diversification mechanisms, the

probability that two independent B or T cell clones carry exactlythe same IG/TR gene rearrangement by chance alone is virtuallynegligible. IG/TR gene rearrangements thus form unique ge-netic markers that can be justifiably viewed as molecular sig-natures (DNA fingerprints), which have been instrumental forunderstanding both normal and pathologic immune responses(8–22). In addition, the spectrum of IG/TR repertoire diversity

*Department of Immunology, Laboratory for Medical Immunology, Erasmus MC,University Medical Center, 3015 CN Rotterdam, the Netherlands; †Second MedicalDepartment, University Hospital Schleswig-Holstein, 24105 Kiel, Germany;‡Departement d’Hematologie, Assistance Publique – Hopitaux de Paris Hopital Pitie-Salpetriere and Universite Pierre et Marie Curie – Universite Paris IV, 75005 Paris,France; xMolecular Medicine Program, Central European Institute of Technology,Masaryk University, 625 00 Brno, Czech Republic; {Centre for Cancer Research andCell Biology, Queen’s University Belfast, Belfast BT9 7AE, United Kingdom; ‖CentroRicerca Tettamanti, Clinica Pediatrica Universita Milano-Bicocca, 20900 Monza, Italy;#UMR 9002 CNRS – Universite de Montpellier, 34396 Montpellier, France; **Centrede Recherche en Informatique Signal et Automatique de Lille, CNRS, Universite deLille, 59000 Lille, France; ††Departement d’Hematologie, Assistance Publique – Hopitauxde Paris Necker-Enfants Malades and Paris Descartes, 75015 Paris, France; ‡‡Institut f€urPathologie, Charite – Universitatsmedizin Berlin, D-10117 Berlin, Germany; xxDepartmentof Pathology, Radboud University Nijmegen Medical Center, 6525 GA Nijmegen,the Netherlands; and {{Institute of Applied Biosciences, Center for Research andTechnology Hellas, GR-57001 Thessaloniki, Greece

1All listed authors are members of the EuroClonality-NGS Consortium and have writtenthis article on behalf of the entire consortium.

ORCIDs: 0000-0002-2078-3220 (A.W.L.); 0000-0003-0580-5636 (D.G.); 0000-0003-2955-4528 (G.C.); 0000-0003-2741-8047 (M.G.); 0000-0003-0520-0493 (E.A.M.).

Received for publication December 15, 2016. Accepted for publication January 9, 2017.

See related articles in this issue: IJspeert et al. (J. Immunol. 198, 4156; DOI: https://doi.org/10.4049/jimmunol.1601921) and Boyer et al. (J. Immunol. 198, 4148; DOI:https://doi.org/10.4049/jimmunol.1601924).

This work was supported by EuroClonality.

Address correspondence and reprint requests to Dr. Anton W. Langerak, Laboratory forMedical Immunology, Department of Immunology, Erasmus MC, University MedicalCenter, Nb-1248a, Wytemaweg 80, 3015 CN Rotterdam, the Netherlands. E-mailaddress: a.langerak@erasmusmc.nl

Abbreviations used in this article: ALL, acute lymphoblastic leukemia; CLL, chroniclymphocytic leukemia; FL, follicular lymphoma; IG/TR, Ig and/or TCR; MRD, min-imal residual disease; NGS, next-generation sequencing; RepSeq, Repertoire Sequencing;RQ-PCR, real-time quantitative PCR; SHM, somatic hypermutation.

Copyright� 2017 by TheAmerican Association of Immunologists, Inc. 0022-1767/17/$30.00

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also translates into immunogenetic profiles that are especiallyuseful for clinical/diagnostic purposes for pathophysiologicalconditions, that is, clonotypic repertoire analysis, minimal re-sidual disease (MRD) detection and monitoring, and clonalityassessment (Fig. 1B) (23–26).

Limitations in low-throughput and potential of high-throughput IG/TR immunogenetics

Multiplex PCR assays for the detection of clonally rearrangedIG/TR genes have been designed, standardized, and validated bythe BIOMED-2/EuroClonality Consortium (euroclonality.org)(27), which also established guidelines for interpretation ofclonality results (28). These BIOMED-2 multiplex assays arenow widely used to obtain corroborative evidence for the dif-ferential diagnosis of reactive lesions versus malignant lympho-mas and for assessing clonal identity (25, 29–31), as well as foridentification of appropriate PCR targets for MRD detection.Highly sensitive (1024 to 1025) MRD analysis of follow-upsamples upon treatment is achieved by using IG/TR rearrange-ments for design of patient-specific oligonucleotides in real-timequantitative (RQ-)PCR. Efforts by the EuroMRD Consortium(euromrd.org) to standardize IG/TR-based RQ-PCR MRDdiagnostics have resulted in improved technical guidelines anddefinitions of MRD positivity and MRD quantitative range toexpress MRD levels in a reproducible way across multiple centersand therapeutic protocols (32). Such accurate MRD quantita-tion is relevant for use as surrogate markers for clinical outcomeand for actual monitoring in view of risk-based clinical decisionsor group stratification (33–40). Finally, IG/TR repertoire at thegenomic or transcript level has been studied in many immu-

nological conditions, although the true diversity of the IG/TRrepertoire is mostly only partly disclosed using low-throughputSanger-based sequencing. Nevertheless, clonal repertoire analysishas proven clinically relevant, as exemplified by chronic lym-phocytic leukemia (CLL) where identifying IGHV gene muta-tional status is established as one of the most robust prognosticmarkers in CLL (standardized by the European Research Ini-tiative on CLL [termed ERIC]; ericll.org) (41, 42).Even though many of the low-throughput IG/TR assays

have thus been optimized and standardized to a high level,inherently they may occasionally provide suboptimal and evenmisleading results. Depending on the diagnostic application,the causes of concern as well as the issues at stake are different.However, fundamental to all is the enormous potential di-versity of IG/TR gene rearrangements necessitating the use ofmultiplex PCR assays with multiple primers that—even in themost optimal situation—are always a compromise. Indeed,PCR biases due to differential performance or misannealingof primers can lead to artificial asymmetries with regard togene frequencies, resulting in a false impression of repertoireskewing or even clonality status. The use of consensus primersin the amplification protocols, though practical, implies a lessthan complete coverage and thus a less comprehensive view ofrepertoire diversity. Moreover, consensus primers have atendency to miss clonal IG rearrangements in the presenceof somatic hypermutation (SHM). Both have merged as relevantproblems in studies of, especially, nonmalignant (i.e., oligo/polyclonal) repertoires, for example, in normal individualsas well as in settings of vaccination, autoimmunity, or im-mune reconstitution after allogenic hematopoietic stem celltransplantation or drug-induced lymphocyte depletion. SHMcan also be the cause for the inability to detect the clonalB cell population in B cell malignancies with high SHM load,for example, follicular lymphoma (FL), multiple myeloma,and others.Furthermore, the IG/TR low-throughput approaches have

their specific limitations in all diagnostic applications. Gene-Scan analysis/spectratyping and heteroduplex analysis, widelyused for evaluation of the clonality status (43–45), usuallyensure diagnostic accuracy with an analytical sensitivity ofmaximally 5%, dependent upon the type of lymphoma andthe context (e.g., specimen type and size, DNA quality andintegrity). This limited dynamic range of spectratyping ren-ders this approach suboptimal for determining low-level dis-semination or for monitoring purposes. RQ-PCR–basedquantification of clone-specific IG/TR gene rearrangements canbe highly informative for the detection of MRD; however,sensitivity varies, depending on the PCR protocol and therelative size of the background of normal (polyclonal) B andT lymphocytes. Also, oligoclonality at initial diagnosis andclonal evolution of IG/TR gene rearrangements between di-agnosis and relapse, both relevant for acute lymphoblasticleukemia (ALL) (46–48), or the occurrence of (ongoing) SHM(for some mature B cell malignancies) (49), may give false-negative results. Traditional repertoire analysis, conducted bySanger sequencing of (subcloned) rearranged IG/TR PCRamplicons or by single cell strategies, is limited in depth by thestrain on laboratory manpower and resources required, ham-pering study of the dynamics of immune responses (e.g., invaccination) and clonal evolution (e.g., intraclonal diversifi-cation of Ig genes in B cell malignancies). Moreover, using

FIGURE 1. IG/TR repertoire diversity translating into clinical applications

in low-throughput methodology and high-throughput immunogenetics. (A)

Continuum of IG/TR repertoire diversity ranging from true polyclonality (far

left end) through oligoclonality and low level clonality (middle) to clear

monoclonality (far right end). (B) Schematic representation of how IG/TR

repertoire diversity translates into low-throughput methodology-based de-

tection of repertoire, MRD, and clonality testing (left to right). (C) Schematic

representation of how high-throughput methodology discloses the full IG/TR

sequence information of the entire cell population, thus allowing much more

information for repertoire analysis, MRD monitoring, and clonality assess-

ment to be drawn.

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multiplex assays that do not encompass the entire V domainencoding region may lead to 1) ambiguities in IG/TR gene andallele identification, and 2) incomplete appreciation of the trueimpact of SHM (41, 42).With the introduction of next-generation sequencing (NGS)

in immunogenetics, also collectively termed Repertoire Se-quencing (RepSeq) analysis (50–59), a deeper analysis of IG/TR gene rearrangements is now within reach, which couldhave a profound impact on the three main applications ofimmunogenetic analysis (clonality assessment, MRD detec-tion and monitoring, and repertoire analysis; Table I). Basedon the wealth of IG/TR sequences generated, much moreinformation on the entire IG/TR repertoire diversity of thecells can thus be disclosed (Fig. 1C).However, there are still many challenges toward routine

clinical application that need to be overcome (Table II; see alsonext paragraph for more details concerning the main appli-cations). These are all subjects of study in the EuroClonality-NGS Consortium (euroclonalityngs.org; coordinated by asteering group chaired by A. W. Langerak), which consists ofseveral EuroClonality laboratories experienced in the design ofassays for detecting IG/TR rearrangements, supplementedby laboratories with expertise in IG/TR gene-based MRDstudies (from the EuroMRD network) or IG/TR repertoirestudies and immunoinformatics (from the ERIC network).

Challenges in RepSeq data analysis for different applications

Clonality assessment. For NGS-based clonality assessment thefollowing issues are at stake.

Assessing the clonal relationship between lesions. AlthoughGeneScan analysis/spectratyping takes (identical) size of thegenerated IG/TR amplicons as the basis for clonality assess-ment, NGS also provides the individual sequences to create so-called clonotypes, a sequence-based compilation of identicalrearrangements. This allows a quantitative consideration of theindividual IG/TR rearrangements, and also helps to confirmthe clonal relationship of the amplified rearrangements inmultiple lesions from different locations or in multiple lesionsover time (Fig. 2). NGS will also enable rapid identificationand evaluation of bone marrow involvement in lymphomapatients. In addition, intraclonal diversity in mature B cellmalignancies that undergo continuous SHM processes canbe evaluated via NGS.

Complementarity of IG/TR targets to reduce the risk of missing

clones.NGS-based clonality assessment still relies on an initialmultiplex PCR step, which will be hampered by the samelimitations as all other PCR-based IG/TR studies, that is,polymorphisms and especially SHM that prevent primerannealing. Additionally, the diverse amplification efficiencyleading to unequal amplification of different rearrangements inmultiplex PCR potentially prevents precise quantification.NGS-based clonality assessment thus also requires multiplecomplementary IG/TR targets to ensure a high detection rate;for B cell malignancies a combination of IGH V-D-J and IGKV-J (and preferably also IGHD-J), and for T cell malignanciesa combination of TRB and TRG assays. Costs for NGS-basedclonality assays are affordable—also when compared with thetraditional approaches by GeneScan analysis. This can beaccomplished by multiplexing of targets and samples for thesequencing step following initial separate PCRs. In consid-ering costs, it is worth noting that the required depth of NGS

clonality approaches is not as deep as for MRD purposes, thusallowing running more patient samples in parallel for thevarious clonality targets. Thus, NGS-based clonality assess-ment has the potential to replace the classical GeneScan ap-proach.

Capture-based detection of clonal IG/TR rearrangements and

translocations. An alternative approach is to use a hybridiza-tion capture or pull-down methodology to enrich for IG/TRloci DNA using small probes or baits, potentially allowing forless biased sequencing of all target regions. In addition, thisapproach is suitable for parallel identification of chromosomaltranslocations involving IG/TR gene loci, present in lym-phoma and leukemia samples, in the same workflow (60, 61).Finally, hybridization capture approaches allow the analysis ofall the IG/TR targets—including unproductive rearrange-ments—in a single assay, saving precious material, and alsofacilitates the use of a given target as a reference or baseline fornormalization against the others, that is, comparing the levelof total rearranged B lymphocytes versus T lymphocytes, orclonal B lymphocytes out of total B lymphocytes.

Reappraisal of the meaning of clonality. The depth of resolutionprovided by NGS-based clonality assessment will almostcertainly require a reappraisal of the term clonality. In par-ticular, defining clonality simply based on the fact that a certainpercentage of identical IG/TR rearrangement sequences can befound does not accurately address the clinical meaning andimplications of the term. On the other hand, the futureamassment of a large body of NGS-based IG/TR sequence dataobtained in the context of clonality assessment will certainlyreveal the true extent of repertoire skewing in healthy indi-viduals, in patients with reactive lesions or other immuno-logical conditions such as postinfection or vaccination, or inoncology patients receiving immunotherapeutics. Therefore, amajor challenge in the quantitative NGS era will be to evaluateand possibly revise the current definitions as to what constitutesclonality and what is the border, if any, to accurately distin-guish reactive from malignant lymphoproliferations in aclinicopathological context. One of the challenges is to definehow and where borders should be drawn in the spectrum ofmono-, oligo-, and polyclonality in patients with lympho-proliferations associated with viruses (e.g., HIV, EBV, orhepatitis C virus) and with lymphoproliferations suspiciousfor malignant lymphoma. Now for the first time, to ourknowledge, the question can be addressed whether clonal

Table I. Potential impact of IG/TR RepSeq analysis on different clinicalapplications

Application Impact

Clonality testing Accurate evaluation of IG/TR clonal relationship inmultiple lesions (true relapse or dissemination versus

new or secondary malignancy)Assessment of low-level dissemination of malignant cloneAssessment of intraclonal diversity of malignant clone

MRD monitoring More easy and unbiased identification of IG/TR targetsSensitive monitoring of malignant clone

Evaluation of oligoclonal IG/TR heterogeneity atdiagnosis and clonal evolution of resistant clone

Repertoire analysis Assessment of higher depth/coverage of IG/TRrearrangements

Assessment of higher number of IG/TR clonotypes

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sizes and numerical cut-offs are of (any) biological or clinicalvalue.

MRD detection. Despite recent developments offering proof ofprinciple that NGS-based MRD assessment using IG/TRgenes is workable in lymphoid leukemias and lymphomasand potentially even more sensitive than alternative options(RQ-PCR, multicolor flow cytometry) (54, 55, 62–65), sev-eral issues remain to be addressed, which will be the topic ofthe sections below.

Identification of the correct index clone. The standard way toidentify a leukemia/lymphoma-associated index sequence is toperform IG/TR multiplex PCR followed by amplicon sequenc-ing and clonotype selection based on a frequency threshold$5% of all analyzed sequences (Fig. 3A). This procedure is errorprone, because—depending on the clinical setting—IG/TR generearrangements of unrelated B and T lymphocyte clones canaccount for a considerable fraction of amplified sequences andmight be misinterpreted as leukemia/lymphoma-specific rear-rangements (53), in particular if the true malignant IG/TR re-arrangement is missed by the applied primer set, for example,due to harboring extensive SHM rates (66).

Correct MRD quantification. Published studies on IG/TRamplicon sequencing frequently quantify the MRD level bycounting the number of index sequences and dividing themby the total number of sequenced amplicons. Given thatthe applied multiplex PCR assays only amplify rearranged IG/TR genes, although cells with the respective IG/TR genein germline configuration are not targeted, this might leadto considerable errors in MRD quantification, particularlyin situations with a low number of polyclonal backgroundB lymphocytes. For example, when IGH NGS is performed ina B cell malignancy after anti–B cell treatment resulting in asubtotal depletion of polyclonal B lymphocytes, preferentialsequencing of IGH-rearranged B lymphocytes might lead toa considerable overestimation of MRD (Fig. 3B). Therefore,adequate internal controls have to be included, for whichdifferent approaches have been proposed: 1) different plas-mids containing known IGH gene rearrangements (62); 2)synthetic control templates spiked at limiting dilution intoeach sample to compute the average number of reads for eachsequenced spiked synthetic template (67); 3) spike-in of defined

Table II. Challenges of IG/TR RepSeq analysis for different clinicalapplications

Application Challenge

Clonality testing Multiplexing and complementarity of IG/TR targetsDefining amount of starting material in the context of

specimen type, DNA integrity, and estimatedneoplastic cell load

Defining value of clonal size, numerical cut-off values,and limits of detection

Reappraisal/redefinition of the meaning of clonalityData analysis pipeline including visualization

MRD monitoring Multiplexing and complementarity of IG/TR targetsAmount and type of starting materialUse of internal controls (e.g., spike-in)

Definition of limits of detection (quantifiable, sensitivity)Correct for disproportional PCR amplification of

rearrangementsData analysis pipeline including visualization

Repertoire analysis Multiplexing and complete coverage of genesEqual amplification and thus representation of genesSequence information of entire V gene (IG loci)

Error correction prior to accurately defining mutations,polymorphisms

Data analysis pipeline including visualization

FIGURE 2. NGS-based clonality assessment. (A) IGK NGS analysis in a tonsil sample showing high reproducibility of the diversity in three different lab-

oratories. Data presented as IGKV gene (x-axis) versus frequency (y-axis), highlighting different IGKJ genes in different colors. (B) Clonal identity in two lymph

node biopsies (1 y time difference) as evidenced from identical clonotypes in the two consecutive samples. Data presented as CDR3 aa length (x-axis) versusfrequency (y-axis), highlighting clonotype sequences in different colors. Visualization using ARResT/Interrogate (92).

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amounts of buffy coat (68). Again, in mature B cell malignancieslike FL, where clonal heterogeneity is caused by ongoing SHM,one has to follow the presence of evolved clonotypes to assess thecorrect MRD level during treatment or at relapse.Detection of clonal heterogeneity and clonal evolution. Inlymphoid malignancies different degrees and ways of IG/TRheterogeneity have been documented. In ALL ongoing generearrangements lead to IG/TR oligoclonality (e.g., IGHD-J toIGH V-D-J changes, and VH substitutions of complete VH-DH-JH rearrangements). Using an IGH NGS approach, IGHoligoclonality was computationally identified in the vast ma-jority of childhood B cell precursor–ALL cases by comparingthe IGH D-J stem of different complete IGH gene rear-rangements with the IGH D-J stem of the index sequence;∼10% of cases showed .1000 related sequences (53).Whether such potentially related sequences should also betracked in follow-up samples has not yet been prospectivelyanalyzed. In contrast, in mature B cell malignancies, IGHclonal heterogeneity is mainly the result of ongoing SHM,leading to intraclonal diversity (69). This might lead to adecrease of amplification efficacy of the respective rearrange-ment in NGS and thereby to a low or even false-negativeMRD result. This can at least partly be compensated by re-lying on additional index sequences from other IG targets, ifpresent.Validation, quality control, and standardized interpretation of

NGS-based MRD results. Technical guidelines, as developedfor RQ-PCR based MRD analysis, are currently lacking forNGS-basedMRD; moreover, data interpretation regarding thedefinition of MRD positivity/negativity is very heterogeneousin the published literature. Considering the potential highersensitivity of NGS, minimal technical requirements havetherefore to be defined including the theoretical sensitivity for asingle sample analyzed for MRD. This is particularly relevantwith respect to the prognostic impact of certain MRDthresholds; the sensitivity of NGS could indeed be higher thanother methods, but is in fact a function of the DNA amount orthe number of cells analyzed in a single sample. Furthermore, aclear definition of MRD positivity/negativity is needed based

on technical assay performance, the number of good qualityreads, and the total number of cells analyzed. Given thecomplexity of NGS-based MRD assessment, successful par-ticipation in external quality control rounds should be aprerequisite for laboratories generating MRD data for clinicaldecision making. Notably, quality control rounds for RQ-PCRand NGS-based MRD are being organized for experiencedlaboratories by the EuroMRD Consortium twice yearly.

Repertoire analysis. NGS-based repertoire analysis requires op-timization at the level of sequence methodology as well as at thelevel of data analysis.Methodological challenges and possible solutions. Consideringthat Ag receptor gene repertoires may vary considerably be-tween different lymphocyte subpopulations (70–72), sortedcells should be used whenever possible, especially when ana-lyzing complex repertoires of nonclonal lymphoid pop-ulations. In order to cover representative diversity and drawmeaningful conclusions from experimental data, it is impor-tant to ensure adequate sampling, including biological as wellas sequencing replicates. One microgram of DNA corre-sponds to only 150,000 cells or up to 300,000 copies of agiven IG/TR locus, which represents a minor proportion ofthe total repertoire. Given that IG/TR transcript levels mayvary considerably between cell populations (for instance morethan 100-fold difference in IG transcript levels between naiveB cells and plasma cells), mRNA use complicates the quan-tification of clonal expansions and may lead to erroneousconclusions about clonal architecture. Nevertheless, startingwith mRNA allows the use of 59 RACE/template switchingprotocols that reduce amplification biases, but often at a costof shorter sequence length (70–74). Within the EuroClonality-NGS Consortium, gDNA was chosen as the template in mul-tiplex PCR with 59 IGH primers annealing to the peptide leaderregion to amplify the complete VH sequence. This is importantfor 1) accurate identification of the rearranged germline V geneand allele, and 2) robust determination of the SHM load in theIG repertoire. Optimization of primer sets was performed usinga collection of plasmids containing most functional V genes to

FIGURE 3. Clonotype frequencies and accurate MRD quantification. (A) IGH clonotype frequency in the peripheral blood of 12 cases of follicular lymphoma

at diagnosis (left) and in normal buffy coat (right). A threshold of 5% is generally used to identify lymphoma/leukemia-related clonotypes at diagnosis. A

significant number of clonotypes .2 and ,5% is seen in buffy coat. Dashed lines indicate the 5% threshold for index clone selection and 2 and 1% thresholds.

(B) Correct MRD quantification is dependent on the background level of polyclonal B lymphocytes. MRD levels may greatly differ following chemotherapy

versus B cell depletion therapy, yet might give rise to the same relative frequency of index sequences, thus necessitating the use of internal references for accurately

calculating MRD levels.

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ensure proper recognition of the targets and minimize ampli-fication biases. Errors introduced during the amplification andsequencing phases complicate data interpretation, particularly inthe case of IG gene repertoire analysis, where discriminatingbetween SHM versus error may prove challenging. Among theerror correction algorithms that have been proposed (75–77),the most promising approach relies on molecular barcodingof each individual template molecule to improve data qualityand sequencing accuracy (78–80). Barcoding strategies havealso been developed on an mRNA template but suffer fromlimited sequencing depth (79). Reliable repertoire analysisby amplification-based NGS strategies is dependent on com-parable amplification efficiency, which can be difficult to con-trol in highly multiplexed strategies. Preferential amplificationcan be detected by comparing repertoires during normal devel-opment, as demonstrated for TRD rearrangements during thymicdevelopment (Fig. 4A). IG/TR loci that undergo deletion, such asIGK and TRD, also present particular challenges for repertoireanalysis.

Finally, Ag receptors are heterodimers and sequence in-formation of both chains is necessary for thorough repertoireanalysis. Although, in lymphoid tumors, the chain pair of theclonotypic Ag receptor can be determined fairly easily from thedominant clonotypes, it is virtually impossible to do so inpolyclonal populations with a vastly heterogeneous sequencecomposition. NGS technologies are evolving to investigatepaired chain repertoires; methods to circumvent this probleminclude 1) two-dimensional barcoding system tagging H chainand L chain V genes of the BcR Ig within individual cells (81),and 2) partitioning single cells in emulsion-based dropletsfollowed by a linkage RT-PCR producing a composite BcR IgH chain and L chain V region product (51, 82–84).

NGS-based repertoire data analysis in malignant and

nonmalignant lymphocytes. To test whether NGS could re-place the conventional Sanger sequencing for accurate assessment

of the SHM status in CLL, both approaches need to be compared.Following a pilot study showing identical results in the vastmajority of CLL cases (93 out of 95, 98%), validation of thetechnique is currently ongoing in several EuroClonality-NGSlaboratories. Of note, satellite clones differing from the pre-dominant clone by nucleotide substitutions and more rarely byindels are often observed, which as mentioned earlier, couldrepresent true SHM or artifacts (Fig. 4B). Furthermore, addi-tional unrelated clonal VDJ sequences are detected at a lowerfrequency in a number of cases; whether these represent minorindependent CLL clones or derive from other concomitantlymphoproliferation(s) still remains elusive.

NGS profiling of complex repertoires may also proveinstrumental in dissecting normal and pathological immuneresponses (e.g., infection, immune reconstitution, vaccina-tion, allergy, or autoimmunity). Importantly, the prospectivelarge-scale accumulation of immune receptor sequence datain various pathological contexts may help unveil immuno-genetic signatures that are distinctive in certain entities, andprovide valuable insight regarding pathogenic mechanisms ofdiseases and effect of immunomodulating therapies, immunesurveillance defects, or even normal immune system con-stitution.

Our recent NGS study of the T cell repertoire in CLLdocumented oligoclonal expansions, with T cell clones beingpersistent and further expanding over time and other T cellclones being shared by different patients, hence appearing tobe disease specific. Altogether, these findings revealed an as yetunappreciated extent of TCR skewing, with implications forfuture interventions into CLL microenvironment interactionsand interdependencies (85).

Computational RepSeq analysis

In all cases, molecular analysis of IG/TR genes eventuallyconcerns the in silico analysis of the nucleotide sequences of

FIGURE 4. NGS-based repertoire analysis. (A) TRD repertoire during human thymocyte development. Different types of TRD rearrangements are present at

different frequencies during human thymic development. (B) Additional clonotypic sequences as detected by NGS in different CLL samples. Each of the two

dominant clonotypic sequences (a productive IGHV3-11 rearrangement and a nonproductive IGHV3-47 rearrangement) is surrounded by minor satellite se-

quences that differ from the main one by unique point mutations. Whether they are true SHM or PCR/sequence artifacts is currently unknown. Visualization

using Vidjil software (91).

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their rearrangements. Although NGS enables us to exploreimmune repertoires and responses in their immense variabilityand complexity, it also naturally produces vast amounts ofcomplex data that render these steps highly nontrivial.Raw NGS data processing is a critical but often neglected

first step. It can involve statistical demultiplexing to ensureminimal digital contamination (i.e., misassignment of reads tosamples due to noisy barcodes) while maximizing read re-covery and thus depth (86), prefiltering of capture data toremove non-IG/TR sequences and thus aid computationalefficiency, paired-end and multilane joining to maximizequality and information content, and primer annotation andtrimming. This last component has many important func-tions: assay development, assessment of run quality andamplicon (and thus junctional) completeness, removal of theartificial primer sequences before immunogenetic annotation,amplification bias correction when combined with a controlsample (e.g., highlighting misbehaving primers), and com-putational efficiency.Processed reads are then immunogenetically annotated (e.g.,

involved V and J genes and alleles, and exact CDR3 sequence).Standardization is important here, and although the use ofdifferent underlying algorithms is arguably inevitable, it isimportant to at least safeguard the consistent use of germlinesequences, rules for CDR3 identification and translation(including C104 and W/F 118 anchor positions), and no-menclature of V, D, and J genes and alleles. At this stage, andespecially for clinical applications, it is also desirable to identifyincomplete and special/uncommon rearrangements. Theeventual classification of reads into all these rearrangementtypes is in fact necessary when the experimental design,(i.e., when the amplification of specific rearrangements takesplace in separate tubes) requires the normalization of abun-dances. It also allows classifying reads with no signs of anyrearrangement as unknown and excluding them from the totalsample read count used as the basis for relative abundances.This supervised abundance calculation is critical in clinicalapplications when thresholds are used for diagnosis or prog-nosis or therapy decisions.Basic immunogenetic annotation can be used to construct

clonotypes (73). The exact definition of a clonotype has beendiverse in the literature and has depended on the underlyingquestion, experiment, and data. In general, a clonotype can beconsidered as a distinct rearrangement event, in which case itcan and has been used to report repertoires in an expression-independent manner.Eventually, mining these inherently complex data for in-

formation requires additional functionalities, such as filteringand visualization, within an interactive graphical environmentthat provides flexibility and enables expert user input. How-ever, this interactivity needs to be bidirectional and includefeedback and guidance to the user toward unambiguous andconsistent conclusions.Taken together and properly standardized and validated,

these key elements (i.e., comprehensive and consistent an-notation of sequences; true representation of the repertoire;meaningful clonotype definition and quantitation; end-user-friendly but unambiguous visualization) can form a consis-tent computational platform usable by both researchers andclinicians. Within EuroClonality-NGS complementaryimmunoinformatics expertise is available that covers these key

elements with a collection of dedicated computational re-sources and tools in the form of IMGT (imgt.org) (87–90),Vidjil (vidjil.org) (91), and most recently ARResT/Interrogate(bat.infspire.org/arrest/) (92).

Specific challenges for MRD detection. A true computationalchallenge for clonotype assessment in the MRD setting is thepronounced ongoing SHM of the IG locus in FL leading toclonal evolution and heterogeneity during treatment course andat relapse. Here, the acceptance criteria for the numbers ofmismatches in relation to the diagnostic clonotype have torespect a continuous mutation process of FL cells, and the clonalrelationship of heterogeneous sequences has therefore to becarefully defined to assess a correct MRD value over time.Overall, high reproducibility and a standardized NGS-basedquantification are particularly important for the comparabil-ity of NGS-basedMRD in the setting of prospective multicentertrials, as is the goal of EuroClonality-NGS.

Specific challenges for repertoire analysis. In addition to raw dataprocessing, V(D)J gene assignment, and clonotype identifi-cation, Ag receptor gene repertoire studies require additionalsequence analysis extending from diversity profiles to clonalarchitecture, CDR3 length and characteristics, clonal dy-namics (if temporal samples are analyzed), and clonotypecomparisons between different lymphoid populations/individuals/disease entities. The complexity of the datagenerated would argue for concomitant analysis via inde-pendent existing pipelines (87–92) or newly published ones(93, 94).

ConclusionsRepSeq analysis has quickly found its way into hematology andimmunology research. Implementation of this high-throughputtechnology into routine clinical applications requires stan-dardization, validation, and application-specific challenges thatshould be covered in a network of laboratories with specialiststhat bring immunobiological knowledge, technical experienceon NGSmethodology, and immunoinformatics expertise. Onlythen will RepSeq analysis be fully exploited for its high potentialin diagnostic and translational research, with the full benefitfor patients. The EuroClonality-NGS Consortium was formedto bridge the immunogenetics generation gap from low-throughput IG/TR analysis to high-throughput RepSeq anal-ysis.Main objectives of the EuroClonality-NGSConsortium areto develop, standardize, and validate IG/TR NGS assays for thedifferent clinical applications in a platform-independent way.Even though several such assays have already been published,there still is a need for optimization in assay developmentwith the aim to ensure better coverage of all the genes and alsoto evaluate other types of rearrangements (partial IGH D–Jrearrangements, IGK locus rearrangements including thoseinvolving the k-deleting element, partial TRB D–J rearrange-ments, IG/TR translocations, etc.).One of the most important aspects in the implementation of

RepSeq analysis in both research and routine diagnostic practiceconcerns standardization of the entire NGS workflow, whichpertains not only to the analytical phase, but also to the pre-analytical (e.g., sample preparation and target choice) and thepostanalytical phases (e.g., immunoinformatics pipeline, datavisualization, and interpretation). Another very important aspect

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of implementing RepSeq analysis for different applications re-lates to validation of the technology against standard method-ologies (GeneScan analysis, Sanger sequencing, RQ-PCR, andmultiparameter flow cytometry) via large-scale, multilaboratorytesting of clinical samples in the context of clinical trials.Eventually, this should provide robust tools and methodology,which will allow exploiting the full potential of this powerful newtechnology in diagnostic patient care. An additional confoundingfactor, at least for diagnostic applications of RepSeq analysis, isthe lack of expertise and guidelines for implementation, evalu-ation, and clinical translation of the results. Both validationstudies and guideline development are among the key activities ofthe EuroClonality-NGS Consortium.

AcknowledgmentsWe thank all members of the EuroClonality-NGS Consortium for their input in

the discussions, Jos Rijntjes and Florian Thonier for preparing figures, Marieke

Bitter for artwork and general support in management of the EuroClonality-

NGS Consortium, and Bibi van Bodegom for secretarial assistance.

DisclosuresThe EuroClonality network receives royalty income from InVivoScribe Tech-

nologies.

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