the light chain iglv3-21 deﬁnes a new poor prognostic … · and some igkv/iglv genes are used...

Precision Medicine and Imaging

The Light Chain IgLV3-21 Defines a New PoorPrognostic Subgroup in Chronic LymphocyticLeukemia: Results of a Multicenter StudyBasile Stamatopoulos1,2,3, Thomas Smith4, Emerence Crompot1, Karlien Pieters1,Ruth Clifford2,3, Marek Mraz5,6, Pauline Robbe2,3, Adam Burns2,7, Adele Timbs2,David Bruce2,7, Peter Hillmen8, Nathalie Meuleman9, Philippe Mineur10,Radu Firescu11, Marie Maerevoet9,12, Virginie De Wilde12, Andr�e Efira13,Jan Philipp�e14, Bruno Verhasselt14, Fritz Offner15, David Sims4, Andreas Heger4,H�el�ene Dreau2, and Anna Schuh2,7

Abstract

Purpose: Unmutated (UM) immunoglobulin heavy chainvariable region (IgHV) status or IgHV3-21 gene usage isassociated with poor prognosis in chronic lymphocytic leu-kemia (CLL) patients. Interestingly, IgHV3-21 is often co-expressed with light chain IgLV3-21, which is potentially ableto trigger cell-autonomous BCR-mediated signaling.However,this light chain has never been characterized independently ofthe heavy chain IgHV3-21.

Experimental Design:Weperformed total RNA sequencingin 32 patients and investigated IgLV3-21 prognostic impact interms of treatment-free survival (TFS) and overall survival(OS) in 3other independent cohorts for a total of 813patients.IgLV3-21 presence was tested by real-time PCR and confirmedby Sanger sequencing.

Results: Using total RNA sequencing to characterize32 patients with high-risk CLL, we found a high frequency(28%) of IgLV3-21 rearrangements. Gene set enrichment

analysis revealed that these patients express higher levelsof genes responsible for ribosome biogenesis and transla-tion initiation (P < 0.0001) as well as MYC target genes(P ¼ 0.0003). Patients with IgLV3-21 rearrangements dis-played a significantly shorter TFS and OS (P < 0.05),particularly those with IgHV mutation. In each of the threeindependent validation cohorts, we showed that IgLV3-21rearrangements—similar to UM IgHV status—conferredpoor prognosis compared with mutated IgHV (P < 0.0001).Importantly, we confirmed by multivariate analysis that thiswas independent of IgHV mutational status or subset #2stereotyped receptor (P < 0.0001).

Conclusions: We have demonstrated for the first timethat a light chain can affect CLL prognosis and that IgLV3-21 light chain usage defines a new subgroup of CLLpatients with poor prognosis. Clin Cancer Res; 24(20);5048–57. �2018 AACR.

IntroductionThe development and progression of chronic lymphocytic

leukemia (CLL) depends on stimuli received by the microenvi-ronment mediated by the B-cell receptor (BCR; ref. 1). Thisimmune receptor is composed of two heavy chains and two lightchains and has been studied extensively in CLL. In particular, thelevel of somatic hypermutations within the variable region of the

immunoglobulin heavy chain (IgHV) has been used as a prog-nostic biomarker for over 15 years (2, 3). Patients expressing anIgHV that has undergone somatic hypermutation and shows lessthan 98% identity with the closest germline are IgHV-mutated(M) andmore oftenhave an indolent disease course. Patientswithunmutated (UM) IgHV (more or equal 98% identity to germline)experience more rapid progression and shorter survival.

1Laboratory of Clinical Cell Therapy, Jules Bordet Institute, ULB-ResearchCancer Center (U-CRC), Universit�e Libre de Bruxelles (ULB), Brussels, Bel-gium. 2Molecular Diagnostic Centre, Oxford University Hospitals, Oxford,United Kingdom. 3Nuffield Department of Laboratory Sciences, University ofOxford, Oxford, United Kingdom. 4Computational Genomics Analysis andTraining Program, MRCWeatherall Institute of Molecular Medicine, Universityof Oxford, Oxford, United Kingdom. 5Department of Internal Medicine,Hematology and Oncology, University Hospital Brno, Brno, Czech Republic.6Central European Institute of Technology, Masaryk University, Brno, CzechRepublic. 7Department of Oncology, University of Oxford, Oxford, UnitedKingdom. 8St. James' Institute of Oncology, St James' University Hospital,Leeds, United Kingdom. 9Hematology Department, Jules Bordet Institute,Brussels, Belgium. 10Department of Hemato-Oncology, Grand Hôpital deCharleroi, Charleroi, Belgium. 11Hematology Department, CHU AmbroisePar�e, Mons, Belgium. 12Hematology Department, Hôpital Erasme (ULB),

Brussels, Belgium. 13Department of Hemato-oncology, Centre HospitalierUniversitaire Brugmann, Brussels, Belgium. 14Department of Clinical Chem-istry, Microbiology and Immunology, Ghent University Hospital, Ghent Uni-versity, Ghent, Belgium. 15Department of Internal Medicine, Hematology,Ghent University Hospital, Ghent, Belgium.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Basile Stamatopoulos, Jules Bordet Institute, ULBResearch Cancer Center (U-CRC), Universit�e Libre de Bruxelles (ULB), Routede Lennik 808, Bruxelles 1070, Belgium. Phone: 32-0-2-555-6621; Fax: 32-2-555-6624; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0133

�2018 American Association for Cancer Research.

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Studying the nature of the BCR in CLL is crucial to under-stand how CLL clones are selected and how CLL cells receivesurvival signals. Indeed, BCR engagement rescues CLL cellsfrom apoptosis (4) and the response to IgM stimulation hasbeen linked to CLL prognosis (5, 6). These in vivo survivalstimulations could be due to an external antigen or an antigen-independent cell-autonomous signaling whereby a BCR inter-acts with intrinsic BCR structures (7). The fact that the immu-noglobulin gene repertoire in CLL is biased (8) and that 30% ofCLL patients express very similar (sometimes even identical)BCRs is in line with reports of antigen-driven selection andclonal expansion (9). Indeed, this phenomenon is unlikely tooccur by chance and suggests that similar epitopes or antigenscould stimulate and select CLL clones.

So far, studies of the BCR in CLL have focused mainly on theIgHV, but the role of the immunoglobulin light chain in influ-encing prognosis is largely unknown. As in the case of the heavychain repertoire, the light chain repertoire in CLL is also biasedand some IgKV/IgLV genes are used more often in CLL comparedwith normal B cells (10). The light chain IgLV3-21 (previouslynamed "VL2-14") represents the most abundant lambda lightchain in CLL (�21%) and is the third most abundant light chain(�7%, after IgKV3-20 and IgKV1-39) if both lambda and kappalight chains are considered (10). Interestingly, themajority of CLLpatients with the heavy chain IgHV3-21 also express the lightchain IgLV3-21. In addition, more than half of these patientsdisplay highly similar variable heavy complementarity determin-ing region 3 (VH CDR3) sequences and belong to the largeststereotyped receptor group (�3% of all CLL) named "subset #2"(11). Patients belonging to subset #2 (IgHV3-21/IgLV3-21) arecharacterized by a shorter overall survival, similar to UM IgHVpatients, despite the fact that two thirds of these patients areIgHV-mutated (12). Interestingly, non-subset #2/IgHV3-21patients differ from the aggressive subset #2 patient subgroup,and their prognosis is dependent on their IgHVmutational status,as in all other CLL cases (11).

The abnormal frequency of the light chain IgLV3-21 in aninitial cohort of 32 patients with high-risk CLL led us to furtherinvestigate its prognostic significance. In the present study, wetherefore correlated the presence of IgLV3-21 with treatment-

free survival (TFS) and/or overall survival (OS) in a total of813 patients from 4 different CLL cohorts: an initial cohort of32 high-risk patients (A), 2 cohorts of patients at diagnosis(B. n ¼ 270; C. n ¼ 289) and one cohort (D) of 224 CLLpatients from 2 Fludarabine-Cyclophosphamid-Rituximab(FCR)-based UK clinical trials.

Materials and MethodsPatients, sample collection and preparation, and DNA/RNAextraction

This study was conducted in accordance with the declarationof Helsinki, approved by the institutional review board (localEthics Committee of Jules Bordet Institute and John RadcliffeHospital) and was performed using samples collected fromCLL patients after written informed consent. All CLL patientshad a typical CD19þCD5þCD23þ phenotype and a Catovskyscore of 4/5 or 5/5. Additional details about patient popula-tions, sample collection, preparation and DNA/RNA extractioncan be found in Supplementary Text S1 and SupplementaryTables S1–S5.

Assessment of IgHV mutational status, sequence analysis,and prognostic factors

IgHV gene mutational analysis was performed by next-gener-ation deep sequencing and/or Sanger sequencing, as previouslydescribed (13), and the sequences were aligned with those in theinternational ImMunoGeneTics (IMGT) information systemdatabase (http://www.imgt.org/; ref. 14). Sequences with �2%deviation from any germline IgHV sequence were consideredunmutated (2). Amino acid sequences were generated from theIMGT website, protein sequences were aligned using ClustalOmega (15) and the consensus sequence was computedwith Weblogo (16). Patients were assigned to the major ste-reotyped subset using the "ARResT/AssignSubsets" onlinetool (17). Analysis of other prognostic factors, such as Binetstage, zeta-associated protein 70 (ZAP70), lipoprotein lipase(LPL), CD38 molecule (CD38) expression, cytogenetic abnor-malities, and lymphocyte doubling time (LDT), as well as targetresequencing for the analysis of recurrent mutations andgenome-wide SNP analyses, were performed as previouslydescribed (18–20). Additional details can be found in Supple-mentary Text S2.

Whole transcriptome sequencingWhole transcriptome sequencing libraries were prepared

using the TruSeq Stranded Total RNA Sample Preparation Kit(Illumina). Libraries underwent 2 � 76bp paired-end sequenc-ing on a HiSeq 2500 instrument (Illumina). Themedian numberof paired-end reads was 60.5 million (range, 49.7–79.7 million).Data processing and analyses were conducted using bioinfor-matics pipelines using the Computational Genomics Analysisand Training (CGAT) code collection (21) and CGAT Pipelinerepository (https://github.com/CGATOxford/CGATPipelines).Sequence data (Bam files) has been deposited at the EuropeanGenome-phenome Archive (EGA), which is hosted by the EBIand the CRG, under accession number EGAS00001002894.Further information about EGA can be found on https://ega-archive.org and "The European Genome-phenome Archive ofhuman data consented for biomedical research" (http://www.nature.com/ng/journal/v47/n7/full/ng.3312.html). Estimates of

Translational Relevance

The variable region of the immunoglobulin heavy chain(IgHV) is an important prognostic biomarker in chroniclymphocytic leukemia (CLL). Patients expressing a mutatedIgHV (less than 98% identity to the closest germline) have anindolent disease course, whereas patients with unmutatedIgHV (more or equal 98% identity to germline) experiencemore rapid progression and shorter survival. The majority ofCLL prognostic studies have focused only on the immuno-globulin heavy chain. In the present work, we demonstratefor the first time that the use of the light chain IgLV3-21 isassociated with a poor prognosis similar to that of unmutatedIgHV patients. This poor prognosis is independent of IgHVmutational status or the nature of the heavy chain and helps toidentify patients who will have an aggressive disease and arapid need for therapy.

The Light Chain IgLV3-21 Defines a Poor Prognosis in CLL

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the number of reads per transcript were obtained using kallisto(22) and all transcripts in Ensembl build 82 (hg38; http://sep2015.archive.ensembl.org/index.html). Gene-level read countswere obtained by aggregating across all isoforms per gene. Differ-ential expression and functional enrichment analyses are detailedin Supplementary Text S3.

Detection of the light chain IgLV3-21The estimated read counts for each variable gene region from

the RNA-Seq data were used to determine the light chain usage foreach patient. Sequences were confirmed by Sanger sequencingusing the primers listed in Supplementary Table S6. To screenadditional cohorts of patients for the presence of IgLV3-21, wedeveloped a rapid and simple method by real-time PCR (qPCR;Supplementary Text S4/Supplementary Fig. S1) thatwas validatedby Sanger Sequencing.

Survival and statistical analysesTFS, OS, and progression-free survival (PFS) distributions were

plotted using Kaplan–Meier estimates and were compared usingthe log-rank test. TFS and OS were calculated from the time ofdiagnosis until the date of first treatment and the date of death,respectively. The OS curves presented here include all deathevents, even if they were not CLL-related. When we consideredonly CLL-related deaths (CLL-OS), similar results were obtained(Supplementary Fig. S2). PFS was calculated from the end of first-line therapy to disease progression or death of the patient. Weused Fisher's exact test to describe associations between clinicalmarkers. Comparisons between 2 groups were assessed using anon-parametric Mann–Whitney test. Multivariate Cox regressionstepwise analysis was used to evaluate the effects of the differentprognostic variables on TFS and OS. All tests were two-sided. Aneffect was considered to be statistically significant if P < 0.05. Allanalyses were performed with GraphPad Prism 5.0 software orIBM SPSS 13.0 software.

ResultsIgLV3-21 light chain is overrepresented in a cohort ofhigh-risk CLL patients, independent of the presence of theheavy chain IgHV3-21, and is associated with poor prognosis(population A)

Using RNAseq data, we investigated the expression of theheavy and light chains in an initial cohort of 32 patients withhigh-risk CLL. All sequences were verified by Sanger sequencing(Supplementary Table S2). Surprisingly, 9 of the 32 (28%)patients expressed the light chain IgLV3-21, but only 1 of the9 (11%) patients expressed the heavy chain IgHV3-21. Theexpected frequency of IgLV3-21 is approximately 7% (10) inan unbiased CLL population. However, this population wascomposed of high-risk CLL patients in need of treatment. For 30of the 32 patients, TFS and OS data were available. IgLV3-21patients had a poorer prognosis than the other patients in thiscohort in terms of both TFS (P ¼ 0.0270) and OS (P ¼0.0287; Fig. 1A and B). The prognosis of IgLV3-21 patients wassignificantly different from that of M IgHV patients (TFS: P ¼0.0242; OS P ¼ 0.0098) but not from that of UM patients(TFS: P ¼ 0.0631; OS P ¼ 0.2851; Fig. 1C and D). However, thenumber of patients in this study was small, thus these resultsshould be confirmed in a larger cohort. It should be noted that,among the 9 IgLV3-21 patients, 4 (44%) were IgHV M.

IgLV3-21 patients display a distinct gene expression profileassociated with translation deregulation (Population A)

Differential expression analysis of IgLV3-21 CLL patients (com-pared with non- IgLV3-21 patients) revealed 1,712 differentiallyexpressed transcripts (41.4% upregulated and 58.6% downregu-lated; Supplementary Table S7) and 1,555 differentially expressedgenes (45.2% upregulated and 54.8% downregulated; Supple-mentary Table S8). Of the 1,555 differentially expressed genes,723 were protein coding and had a fold-change greater than1.5-fold (Supplementary Table S9). Hierarchical clustering basedon the 723 genes differentially expressed in IgLV3-21 patientsgrouped 8/9 IgLV3-21 patients together, demonstrating that thegene expression differences observed between IgLV3-21–positiveand negative patients were consistent (Fig. 1E).

We performed gene set enrichment analysis to identify geneontology (GO) categories and Biocarta, KEGG, and Broad/MITpathways that were enriched in the genes differentially expressedbetween our two classes of samples. Interestingly, when all differ-entially expressed genes (Supplementary Table S10) or only up-regulated genes (Supplementary Table S11) were considered, GOcategories/pathways relating to translation (ribosome, translationalreactome, peptide chain elongation, RNAmetabolism—P < 0.0001)and MYC target genes (P ¼ 0.0003) were significantly overrepre-sented (Fig. 1G–I). However, no pertinent categories/pathwayswere enriched within the downregulated genes (SupplementaryTable S12). To confirm the increased expression of genes withinthe identified enriched pathways, we selected 7 genes and vali-dated their increased expression by real-time PCR (Fig. 1F).

Validation of the poor prognosis associated with IgLV3-21usage in an independent cohort of 270CLLpatients at diagnosis(population B)

After obtaining results from the initial 32 patients, we inves-tigated the prognostic significance of IgLV3-21 expression in 2validation cohorts (populations B and C) composed of CLLsamples obtained at diagnosis. In population B, which wascomposed of 270 patients with amedian follow up of 83months(range, 1–397), we used real-time PCR to identify 30 (11%)IgLV3-21–expressing patients, including 7 (3%) patients fromsubset #2 (IgHV3-21/IgLV3-21). Patients with an IgLV3-21 lightchain had a significantly shorter median TFS (29 months) com-pared with other patients (82 months, P ¼ 0.0002; Fig. 2A).Similar results were observed for OS (137 vs. 231 months, P ¼0.0483; Fig. 2B). Upon subdividing patients into subset #2,other IgHV/IgLV3-21 þ non-subset #2, other IgHV UM, andother IgHV M, we found that the first 3 groups had similarmedian TFS (P < 0.0001) and OS (P < 0.05) rates that werestatistically shorter than those in IgHV M patients (Fig. 2C andD). Because the presence of IgHV3-21 did not influence prog-nosis, we pooled all patients with IgLV3-21. IgLV3-21 patientshad a median TFS of 29 months, which was not significantlydifferent from that of IgHV UM patients (36 months; P ¼0.6798) but was significantly worse than that of IgHV Mpatients (144 months, P < 0.0001; Fig. 2E). Similar resultswere obtained for OS (Fig. 2F). It should be noted that, amongthe 30 IgLV3-21 patients, 12 (40%) were IgHV M.

Multivariate analysis with classical prognostic factors inpopulation B (n ¼ 270)

To determine whether the presence of IgLV3-21 is anindependent prognostic factor, we performed a stepwise

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Figure 1.

IgLV3-21 is associated with poor prognosis and the expression of genes linked to translational deregulation as well as MYC target genes. TFS andoverall survival (OS) of IgLV3-21 patients compared with other patients (A and B) or to IgHV-mutated (M) or unmutated (UM) patients (C and D) in the initialpopulation of 30 high-risk CLL patients. IgLV3-21 patients display a poor prognosis that is significantly different from that of IgLV3-21 M patients but not fromthat of UM patients. The difference between curves was assessed by univariate analysis using the log-rank test. E, Heatmap of protein-coding genesdifferentially expressed between IgLV3-21 patients and other patients (FDR1.5-fold change). Patients are ordered according to the hierarchicalclustering shown above. F, Validation of selected differentially expressed genes by real-time PCR. Representative heatmaps show genes differentiallyexpressed between IgLV3-21 and other patients and are annotated to show enriched gene ontology (GO) terms or pathways: (G) Ribosome biogenesis(P < 0.001), (H) translational initiation (P < 0.0001), (I) MYC target gene hallmarks (P ¼ 0.0211).





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Figure 2.

Validation of the poor prognosis associated with IgLV3-21 usage in population B. TFS and overall survival (OS) of IgLV3-21 patients compared withother patients (A and B) in an unbiased CLL population of 270 CLL patients. C (TFS) and D (OS) show the prognosis of IgLV3-21 patients compared withIgHV-mutated (M), unmutated (UM), or subset #2 (IgHV3-21/IgLV3-21) patients. IgLV3-21 patients display a poor prognosis that is significantly differentfrom IgHV M patients but not from UM or subset #2 patients. Tables under the figures provide the median TFS/OS, the number of patients persubgroup, and the paired P values. E (TFS) and F (OS) show the prognosis of all IgLV3-21 patients compared with IgHV-mutated (M) and unmutated (UM)patients. The difference between curves was assessed by univariate analysis using the log-rank test.






multivariate analysis designed to identify the optimal set ofindependent variables to predict TFS/OS, including the fol-lowing classical prognostic factors: Binet stages, IgHV muta-tional status, ZAP70 (Zeta-associated protein), LPL (lipopro-tein lipase), CD38 and the use of subset #2 stereotypedreceptor. The stepwise analysis identified the presence ofIgLV3-21 as a prognostic factor for TFS prediction with anon-significant P value (P ¼ 0.0717; Supplementary TableS13A–S13B). However, when repeating the same analysis forIgHV M patients only (n ¼ 166), the presence of IgLV3-21 wasidentified as an independent prognostic factor for both TFSand OS prediction with a significant P value (P < 0.001),suggesting that IgLV3-21 may have a significant and indepen-dent prognostic impact in patients with mutated IgHV (Sup-plementary Table S13 C–S13D).

Validation of the poor prognosis associated with IgLV3-21usage in an independent cohort of 289CLLpatients at diagnosis(population C) and 224 CLL patients from a clinical trial(population D)

In population C, which was composed of 289 patients witha median follow up of 78 months (range, 0.3–288), weidentified 33 (11%) IgLV3-21–positive patients by real-timePCR including 9 (3%) patients from subset #2 (IgHV3-21/IgLV3-21). The prognostic impact of IgLV3-21 light chainpresence that was identified in populations A and B wasvalidated in population C (Supplementary Fig. S3). IgLV3-21-expressers had a median TFS of 31 months, which wasnot significantly different from that of IgHV UM patients (21months; P ¼ 0.9458) but was significantly worse than thatof IgHV M patients (128 months, P < 0.0001; SupplementaryFig. S3E). Similar results were obtained for OS (SupplementaryFig. S3F). It should be noted that, of the 33 IgLV3-21 expres-sers, 16 (48%) were IgHV M. Similar results were obtainedfrom clinical trial population D (see Supplementary Fig. S4and Supplementary Text S5).

Multivariate analysis with recurrent mutations and cytogeneticabnormalities in population D

Recurrent mutations and cytogenetic data were available for181 patients in population D. Multivariate analysis for TFSprediction was performed, including recurrent mutations inCLL (TP53, ATM, NOTCH1, SF3B1, MYD88, SAMHD1), com-mon cytogenetic abnormalities [del(17p), del(11q), del(6q),del(13q) trisomy 12], the use of subset #2 stereotyped receptorand the occurrence of IgLV3-21. This analysis selected thepresence of IgLV3-21 as an independent prognostic factor.The analysis also selected del(13q), TP53 mutation and IgHVstatus as independent prognostic factors, although only IgHVstatus had a p-value < 0.05 in the final model (IgLV3-21: P ¼0.0603) of TFS prediction (Supplementary Table S14 A).However, when we performed the same analysis in M patientsonly (n ¼ 69), the presence of IgLV3-21 (P ¼ 0.0415) wasselected as an independent predictor of poor prognosis (Sup-plementary Table S14 B). Interestingly, association analysisshowed that the presence of the IgLV3-21 chain was signifi-cantly associated with the presence of SF3B1 (P < 0.0001) andATM (P ¼ 0.0245) mutations, stereotyped receptor fromsubset #2 (P < 0.0001), and the absence of both NOTCH1mutation (P ¼ 0.0367) and trisomy 12 (P ¼ 0.0033; Supple-mentary Table S15).

Analysis of IgLV3-21 as a PFS predictor after FCR-basedtreatment (population D)

Although IgLV3-21 is a significant predictor of TFS andOS (Figs. 1 and 2; Supplementary Fig. S3), we did not observea significant difference in terms of PFS after FCR-based treat-ment between IgLV3-21 and other patients (Fig. 3A). However,IgLV3-21 patients have a shorter PFS compared with IgHV-mutated patients (Fig. 3B) but we could not exclude that thisdifference is due to a high proportion of IgHV-unmutatedpatients carrying IgLV3-21 light chain (Fig. 3D). Indeed, wedid not observe a significant difference in term of PFSbetween IgLV3-21/other M IgHV and the other M IgHV patients(P ¼ 0.1247). A multivariate Cox analysis for PFS predictionincluding IgLV3-21 usage, identified presence of subset #2 re-arrangement, IgHV mutational status and classical cytogeneticabnormalities [del(13q), trisomy 12, del(11q), del(17p)]selected IgHV mutational status (HR, 3.1; P < 0.0001) anddeletion 17p (HR, 7.3; P < 0.0001) as independent predictorsof PFS, whereas IgLV3-21 was not considered (SupplementaryTable S16).

The poor prognosis associated with IgLV3-21 is independent ofIgHV3-21 usage, IgHV mutational status, and classicalcytogenetic abnormalities and can add prognostic informationto the CLL-IPI.

For the following analysis, we pooled all patients from thedifferent populations in order to increase the number ofpatients for subgroup analysis. To investigate whether the poorprognosis of IgLV3-21-expressing patients could be due toIgHV mutational status, we divided the population into 5 sub-groups: M IgHV, UM IgHV, subset #2 (IgHV3-21/IgLV3-21), MIgHV/IgLV3-21, and UM IgHV/IgLV3-21. As presented in Fig. 4,all patients with the IgLV3-21 light chain have a comparablemedian TFS and OS to that of UM patients (P < 0.0001),independent of their mutational status or the presence of astereotyped heavy chain IgHV3-21 assigned to subset #2. Aspresented in Supplementary Table S17 (multivariate Cox anal-ysis including IgLV3-21 usage, subset #2 usage, IgHV muta-tional status and classical cytogenetic abnormalities), IgLV3-21was selected as an independent prognostic factor for TFS (HR,1.5; P ¼ 0.0004, n ¼ 685) and OS (HR, 1.97; P ¼ 0.0031; n ¼471) prediction. More interestingly, when only IgHV-mutatedpatients were considered, patients with IgLV3-21 had a 2.4times greater risk of undergoing treatment (P < 0.0001, n ¼333) and an 8.7 times greater risk of death (n ¼ 250, P <0.0001) compared with patients with hypermutated IgHV andno IgLV3-21 usage. The additional prognostic informationadded by IgLV3-21 usage to the CLL international prognosticindex (CLL-IPI) is presented in Supplementary Fig. S5. IgLV3-21 is able to identify patients with poor prognosis in the "low"subgroup and in the "intermediate" subgroup (at least for TFS).In the "high" and "very high" subgroups, IgLV3-21 did not addany prognostic information.

Immunogenetic features of IgLV3-21 patientsTo further characterize the BCR of patients carrying an

IgLV3-21 rearrangement, we pooled all available IgLV3-21sequences from the present study (n ¼ 128) with thosepreviously published by Stamatopoulos and colleagues(10; n ¼ 20) and compared these HV/LV sequences to twocontrol groups [patients with k (n ¼ 174) and other l






(n ¼ 76) light chains; ref. 10]. Data are available in Supple-mentary Text S6, Supplementary Fig. S6 and SupplementaryTable S18.

DiscussionOver the past decade, clinical and biological evidence has

established the BCR as one of the key drivers of clonal selectionand survival of CLL cells (23–26). So far, the majority of studiesinvestigating the prognostic significance of the BCR havefocused on the heavy chain (2, 3), although there is emergingevidence that the light chains might also play a crucial role inBCR function in CLL (27).

In the current study, we present a comprehensive character-ization of gene expression, prognosis, and immunogenic fea-tures of patients carrying the IgLV3-21 rearrangement and revealits association with short TFS and OS in 4 separate cohorts ofover 800 patients. Importantly, we showed that IgLV3-21patients had a poor prognosis irrespective of the presence orabsence of a BCR assigned to subset #2. In IgHV-mutatedpatients, IgLV3-21 usage is independent of other classical prog-nostic factors, cytogenetic abnormalities and recurrent drivermutations and defines a poor risk group within this goodprognosis subgroup. In addition, we highlight that IgLV3-21usage could indicate poor prognosis in patients classified as lowrisk by the CLL-IPI.

Figure 3.

Association of IgLV3-21 usage and PFS after FCR-based treatment. PFS of IgLV3-21 patients compared with other patients (A) in a clinical trialpopulation of 224 CLL patients. B, PFS of IgLV3-21 patients compared with IgHV-mutated (M) and unmutated (UM) patients. C, PFS of IgLV3-21/otherIgHV patients compared with IgHV-mutated (M), unmutated (UM), and IgLV3-21/IgHV3-21 patients. D, PFS of M IgHV/IgLV3-21 and UM IgHV/IgLV3-21,subset #2 (IgHV3-21/IgLV3-21) patients compared with IgHV-mutated (M) and unmutated (UM) patients. The difference between curves was assessed byunivariate analysis using the log-rank test.






Based on an initial cohort of 32 patients with high-risk CLL, wefound a higher frequency (28% vs. 7%) of IgLV3-21 expresserscompared with all CLL patients (10). Interestingly, the frequencyof IgLV3-21 was 28% (population A) and 25% (population D) inhigh risk cohorts with a short median TFS of 24 and 30 months,respectively, whereas it fell to 11% for an unbiased cohortobtained at diagnosis (populations B and C), which is close towhat has been reported previously (10).

Gene-expression profiles obtained from total RNA sequencingrevealed genes that were differentially expressed between IgLV3-21-expressers and other patients. Specifically, genes involved intranslational regulation (including ribosome biogenesis and RNAprocessing) and MYC target genes were overrepresented in thosepatients. These data are compatible with the data presented byYeomans and colleagues (28), who reported that the engagementof BCR inCLL induced a global increase of translation aswell as anincrease inMYC-specificmRNA translation. In addition, several oftheoverexpressed genes inpatients carrying IgLV3-21have roles intranslation (NPM1, RLP18, RPS6, MRPL44) and were alsoreported by Pozzo and colleagues (29) to be overexpressed inNOTCH1-mutated patients and to be modulated by NOTCH1signaling via MYC transcription. In the present study, NOTCH1mutation was significantly associated with the absence of IgLV3-21, suggesting that the increase in NPM1 and other ribosomalproteins is not linked to NOTCH1 pathway deregulation. Alter-natively, the association of mutated SF3B1 with IgLV3-21 couldlead to splicing dysregulation affecting multiple cellular func-

tions, including DNA damage response, telomere maintenance,and Notch signaling (30). However, half of IgLV3-21 patientswere SF3B1-wildtype (population D), indicating that our resultswere at least partially independent of SF3B1. The overexpressionof NPM1 and other ribosomal proteins is compatible with anenhanced biosynthetic pathway leading to a growth advantage inIgLV3-21–expressing cells and subsequently to a poor prognosisof these patients.

In the initial cohort of 32 patients with high-risk CLL, weobserved a shorter median TFS and OS for patients with IgLV3-21. These results, which were obtained using a small cohort, werevalidated in three other independent cohorts of 270, 289, and 224patients collected in different centers across Europe. Although themajority of IgHV3-21 cases (B: 80%, C: 83%, D: 86%) wereassociated with the light chain IgLV3-21, as previously reported(31), the inverse was not necessarily true. In each of these 3cohorts, IgLV3-21 patients had a median TFS/OS that was notsignificantly different from that of IgHV UM or subset #2 patientsbut was shorter than that of M patients. However, IgLV3-21 wasnot found as a predictive marker of FCR-based treatmentresponse, at least in the present cohort (population D). Themedian follow-up after the end of the first treatment of thispopulation was 70 months (range, 0.2–95.2). These resultsshould be confirmed in an independent cohort and with a longerfollow-up.

It is well established that the heavy and the light chain reper-toire is biased inCLL (8–10).Whenwe analyzed theHV repertoire

Figure 4.

The poor prognosis associated with IgLV3-21 is independent of IgHV3-21 usage and IgHV mutational status. Data for patients from populations A, B, C, andD were pooled for TFS analysis (n ¼ 813), and data for patients from populations A, B, and C were pooled for OS analysis (n ¼ 589). Panels A (TFS)and B (OS) show the prognosis of M IgHV/IgLV3-21 and UM IgHV/IgLV3-21 subset #2 (IgHV3-21/IgLV3-21) patients compared with IgHV-mutated (M)and unmutated (UM) patients. IgLV3-21 patients display a poor prognosis that is significantly different from that of IgHV M patients but not from that ofUM or subset #2 patients independent of their heavy chain mutational status. The table under the figure shows the median TFS/OS, the number ofpatients per subgroup, and the paired P values. The difference between curves was assessed by univariate analysis using the log-rank test.






of IgLV3-21, we observed that this light chain is also predomi-nantly associated with particular heavy chains, because theIgHV3-21, IgHV3-48, and IgHV3-23 families represent 50% ofall IgHVs. Interestingly, 90% of IgLV3-21 light chains wereunmutated and the corresponding heavy chains were less fre-quently mutated compared with other control groups. This con-firms that the BCR, including its light chain, is globally conserved,as illustrated by the consensus sequence. In accordance withprevious studies, we show that some amino acid changes arerecurrent, such as the S!G replacement in the CDR3 of the lightchain, which occurs in 30% of patients (10, 27). It has beensuggested that the introduction of a glycine from the germlinesequence at this position likely facilitates BCR-BCR interactions(32). However, at least in our patients, this did not have prog-nostic significance.

Using the ARResT/AssignSubsets tool (17), we observedthat, except in subset #2 patients (representing 27% of theIgLV3-21 patients), the other heavy chain was rarely assigned toa major stereotyped subset. In the other 104 IgLV3-21 patients,only 3 patients displayed stereotyped IgHV (subset #5, #5and #7H). However, recent work shows that immunoglobulinB-cell receptors of unrelated clonotypes may recognize com-mon epitopes that are not predictable from the molecularfeatures of the immunoglobulin B-cell receptor, includingCDR3 composition and length (33). It is thus premature toexclude the stereotypy hypothesis based only on amino acidsequences.

The biological reasons underlying the poor prognosis ofIgLV3-21 patients are still unclear. Two hypotheses based onBCR stimulation could explain the poor prognosis of thesepatients: canonical (auto-)antigen binding (34) or cell-auton-omous antigen-independent signaling (7). It is now well-accepted that CLL selection is an antigen-driven process andthat BCR is a key player in CLL cell survival (35). Regarding theextremely conserved sequence of the IgLV3-21, we could spec-ulate that this particular light chain may recognize a commonexternal antigen (viruses or other pathogens) or an autoantigen(apoptotic bodies).

On the other hand, the BCR stimulation leading to CLL cellsurvival and thus a poor prognosis for IgLV3-21 patients couldbe the result of cell-autonomous antigen-independent signal-ing, as described by D€uhren-von Minden and colleagues (7),who were the first to suggest that CLL cells could be activatedby the triggering of BCR internal epitopes. In line with thishypothesis, Minici and colleagues (32) demonstrated theimportance of the light chain IgLV3-21 in BCR self-recognitionand autonomous signaling by studying the crystal structure ofhomotypic B-cell receptor interactions in subset #2 patients.Together, these results suggest that the IgLV3-21 light chainmight play a pivotal role in homotypic interactions and suggestthat CLL cells expressing IgLV3-21 might undergo continuousand autonomous constitutive stimulation promoting CLL cellactivation, proliferation, and survival.

In line with these two hypotheses, we observed a decrease inCXCR4 cell surface expression in IgLV3-21 patients (Supple-mentary Fig. S7). CXCR4 is known to be downregulated afterIgM stimulation (36) and in cells receiving stimulus from astromal microenvironment (37). Interestingly, all IgLV3-21patients (with UM or M IgHV or with IgHV3-21) presenteda low level of CXCR4 surface expression, which was signifi-cantly different from patients with M IgHV-expressing another

light chain. These results suggest that IgLV3-21 patients under-go an in vivo BCR stimulation (through interactions withtheir microenvironment or through autonomous signaling),which downregulates CXCR4 and provides a survival signal tothe cells.

In conclusion, we have demonstrated that the presence ofthe IgLV3-21 light chain independently defines a new subgroupin CLL characterized by a distinct gene expression profile linkedto translational upregulation, increased MYC target geneexpression and a prognosis similar to IgHV UM patients,irrespective of whether the IgLV3-21 rearrangement occurs inIgHV-mutated or unmutated patients. Therefore, similar to itsheavy chain counterpart, the light chain IgLV3-21 might provea valuable marker to differentiate high-risk from low-riskIgHV M CLL patients.

Disclosure of Potential Conflicts of InterestP. Hillmen reports receiving commercial research grants from AbbVie,

Gilead, Janssen, Pharmacyclics, and Roche Pharmaceuticals, and speakersbureau honoraria from AbbVie, Acerta Pharma, Janssen, and Roche Pharma-ceuticals. A. Schuh is a consultant/advisory board member for AbbVie, Gilead,Janssen, and Roche. No potential conflicts of interest were disclosed by theother authors.

DisclaimerThe views expressed are those of the authors and not necessarily those of

the UK's National Health Service, National Institute of Health Research, orDepartment of Health.

Authors' ContributionsConception and design: B. Stamatopoulos, P. Hillmen, A. SchuhDevelopment of methodology: B. Stamatopoulos, E. Crompot, B. VerhasseltAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Mraz, P. Robbe, A. Burns, A. Timbs, D. Bruce,P. Hillmen, N. Meuleman, P. Mineur, R. Firescu, M. Maerevoet, V. De Wilde,A. Efira, J. Philipp�e, B. Verhasselt, F. Offner, H. Dreau, A. SchuhAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): B. Stamatopoulos, T. Smith, R. Clifford, P. Robbe,A. Burns, F. Offner, D. Sims, A. Heger, A. SchuhWriting, review, and/or revision of the manuscript: B. Stamatopoulos,T. Smith, D. Bruce, P. Hillmen, R. Firescu, M. Maerevoet, J. Philipp�e, F. Offner,D. Sims, A. SchuhAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Pieters, P. Robbe, R. Firescu, J. Philipp�e,H. DreauStudy supervision: P. Hillmen, D. Sims, A. Schuh

AcknowledgmentsThis work was supported by a T�el�evie grant provided by the F.R.S.-FNRS

(Fonds National de la Recherche Scientifique) of Belgium, the "David andAlice Van Buuren Fund," the "Fonds IRIS-Recherche," "Les Amis de l'InstitutJules Bordet," "Wallonie-Bruxelles International World" (WBI.World), theBekales Fundation and the foundation Lambeau-Marteaux, and the NationalInstitute for Health Research (NIHR) Oxford Biomedical Research Centre(BRC). M.Mraz was supported by theMinistry of Health of the Czech Republic,grant 16-29622A.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 12, 2018; revised March 7, 2018; accepted June 22, 2018;published first June 26, 2018.






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2018;24:5048-5057. Published OnlineFirst June 26, 2018.Clin Cancer Res Basile Stamatopoulos, Thomas Smith, Emerence Crompot, et al. in Chronic Lymphocytic Leukemia: Results of a Multicenter StudyThe Light Chain IgLV3-21 Defines a New Poor Prognostic Subgroup

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the light chain iglv3-21 deﬁnes a new poor prognostic … · and some igkv/iglv genes are used...

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