Genomic Landscape of CXCR4 Mutations in Waldenstrom ... · 30% of Waldenstrom macroglobulinemia, similar to germline€ mutationsfoundintheWHIM(warts,hypogammaglobulinemia, infections,

Biology of Human Tumors

Genomic Landscape of CXCR4 Mutations inWaldenstr€om MacroglobulinemiaSt�ephanie Poulain1,2,3, Christophe Roumier2,3, Aur�elie Venet-Caillault2, Martin Figeac4,Charles Herbaux3,5, Guillemette Marot6, Emmanuelle Doye2, Elisabeth Bertrand3,Sandrine Geffroy2, Fr�ed�eric Lepretre4, Olivier Nibourel2,3, Audrey Decambron1,Eileen Mary Boyle3,5, Aline Renneville2, Sabine Tricot1, Agn�es Daudignon1,Bruno Quesnel3,4, Patrick Duthilleul1, Claude Preudhomme2,3, and Xavier Leleu3,5

Abstract

Purpose: Whole-genome sequencing has revealed MYD88L265P and CXCR4 mutations (CXCR4mut) as the most prevalentsomatic mutations in Waldenstr€om macroglobulinemia. CXCR4mutation has proved to be of critical importance inWaldenstr€ommacroglobulinemia, in part due to its role as a mechanism ofresistance to several agents. We have therefore sought to unravelthe different aspects of CXCR4 mutations in Waldenstr€ommacroglobulinemia.

Experimental Design:We have scanned the two coding exonsof CXCR4 in Waldenstr€om macroglobulinemia using deep next-generation sequencing and Sanger sequencing in 98 patients withWaldenstr€om macroglobulinemia and correlated with SNP arraylandscape and mutational spectrum of eight candidate genesinvolved in TLR, RAS, and BCR pathway in an integrative study.

Results:We found all mutations to be heterozygous, somatic,and located in the C-terminal domain of CXCR4 in 25% of the

Waldenstr€om macroglobulinemia. CXCR4 mutations led to atruncated receptor protein associated with a higher expressionof CXCR4. CXCR4 mutations pertain to the same clone as toMYD88 L265P mutations but were mutually exclusive toCD79A/CD79B mutations (BCR pathway). We identified agenomic signature in CXCR4mut Waldenstr€om macroglobuline-mia traducing a more complex genome. CXCR4mutations werealso associated with gain of chromosome 4, gain of Xq, anddeletion 6q.

Conclusions:Our study panned out new CXCR4mutations inWaldenstr€ommacroglobulinemia and identified a specific signa-ture associated toCXCR4mut, characterizedwith complex genomicaberrations amongMYD88L265PWaldenstr€ommacroglobuline-mia. Our results suggest the existence of various genomic sub-groups in Waldenstr€om macroglobulinemia. Clin Cancer Res; 1–9.�2015 AACR.

IntroductionWhole-genome sequencing has revealed CXCR4 as the second

most frequent somatic mutation, identified in approximately30% of Waldenstr€om macroglobulinemia, similar to germlinemutations found in theWHIM (warts, hypogammaglobulinemia,infections, and myelokathexis) syndrome (1–3). MYD88 L265Pmutations remain the most frequent mutation reported inWaldenstr€om macroglobulinemia to date, in nearly 90% ofWaldenstr€om macroglobulinemia (4, 5). Interestingly, MYD88L265P may act as a founder mutation because of its high fre-

quency in Waldenstr€om macroglobulinemia; and thus it is sus-pected that a second hit may accelerate development and pro-gression of the malignant clones leading to full-blown inWaldenstr€om macroglobulinemia. Although the mechanism ofderegulation of CXCR4 axis is not fully understood inWaldenstr€om macroglobulinemia, it is possible that CXCR4mutation might represent one of these secondary events.

C-X-C chemokine receptor type 4 (CXCR4) is a G-protein–coupled receptor that plays an important role in lymphopoi-esis and cell trafficking (2, 6), along with its ligand, the stromalcell–derived factor-1 (CXCL12/SDF-1). The SDF1/CXCR4 axispromotes activation of several pathways including RAS, Akt,and NF-kB and interplays with BCR pathway (6–8). TheCXCR4 gene is located on the long arm of chromosome 2 atposition 21 and codes for a chemokine receptor that promotesmigration and survival of various B lymphoid malignancies(8–10).

CXCR4 mutations were identified using Sanger sequencing byTreon and colleagues in nearly 25% of Waldenstr€ommacroglob-ulinemia, and the CXCR4 C1013Gmutation was described as themost frequent recurrent CXCR4 mutation in 7% (11). However,the occurrence of potential subclonal ofCXCR4mutation and theproportion of CXCR4mutation over the other known mutationswas not available; in other words, how this mutation lies in thearchitecture of the MYD88-mutated clone. On contrary, Roccaroand colleagues have reported 30% incidence rate of the CXCR4

1Service d'H�ematologie-Immunologie-Cytog�en�etique, Centre Hospi-talier de Valenciennes, France. 2Laboratoire d'H�ematologie, Centre deBiologie et Pathologie, CHRU de Lille, France. 3INSERM UMR 1172,IRCL, Lille, France. 4IFR114, Plateforme de G�enomique, Lille, France.5Service des Maladies du Sang, Hopital Huriez, CHRU, Lille, France.6Universit�e de Lille, UDSL, EA2694 Biostatistics/Inria Lille Nord Eur-ope, MODAL, Lille, France.

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

Corresponding Author: Xavier Leleu, Hopital Huriez, CHRU, Rue Michel Polo-novski, Lille 59037, France. Phone: 33-3-20446883; Fax: 33-3-2044-4094;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-0646

�2015 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org OF1

Research. on August 14, 2020. © 2015 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 21, 2015; DOI: 10.1158/1078-0432.CCR-15-0646

http://clincancerres.aacrjournals.org/

C1013Gmutation using qPCR; however, they have solely studiedthis mutation (12).

In this study,we characterized the genomic landscapeofCXCR4somatic mutation in a large cohort of 98 patients withWaldenstr€om macroglobulinemia, combining deep sequencingof target genes allowing study of the clonal architecture in theWaldenstr€om macroglobulinemia cells, including subclonalanalysis, and to confirm the incidence rate of these mutationsand single polymorphism nucleotide array (SNPa) to decipherthe genomic landscape of CXCR4-mutated Waldenstr€ommacroglobulinemia.

Patients and MethodsPatients

Ninety-eight patients diagnosed with Waldenstr€om macro-globulinemia were included in this study (63males, 35 females).The diagnosis and treatment initiation criteria of Waldenstr€ommacroglobulinemia were as published (13, 14). Among thecohort with available clinical follow up (n ¼ 93), 68 patientswere treated. First-line treatmentwas initiated at diagnosis in 41%of the patients. Front-line therapy included chloraminophenealone in 12 cases, fludarabine alone in 3 cases, rituximab (R)alone in 3 cases or in association with chemotherapy in 51 cases(dexamethasone-R- cyclophosphamide in 19 cases, bortezomib-R-dexamethasone in 7, R-bendamustine in 5 cases). Patients wereuntreated at time of bone marrow collection and gave informedconsent prior to research sampling. No familial form ofWaldenstr€om macroglobulinemia was included.

Cell selectionResearch sampling consisted of bone marrow samples and

blood cells for all Waldenstr€ommacroglobulinemia. All sampleswere enriched using immunomagnetic beads (B cell isolation kitfor tumoral cells from bone marrow sample, and Pan T cellisolation kit, Miltenyi-Biotec), following Ficoll-paque gradientcentrifugation. The purity of samples was confirmed by multi-parametre flow cytometry using amarker combination (includingCD19, k, l, CD2, CD38, CD138, and CD27). More than 90% a

light-chain isotype–positive B cells was detected in all cases(mean, 95.8% of tumoral cells; ref. 15).

DNA sequencingDNA was extracted from isolated cells using Qui Amp kit

(Sigma-Aldrich Co.). MYD88 L265P, CD79A, and CD79B muta-tionswere analyzed as previously described (16) in 98 patients. C-terminal CXCR4 gene was amplified from gDNA by PCR aspreviously described (n ¼ 98 patients; ref. 5). All the exons ofCXCR4 gene were analyzed using targeted next-generationsequencing (NGS) in a cohort of 53 patients. NGS was analyzedusing the Ion Torrent PGM platform (Life Biotechnologies).Amplicons covering the regions of interest were designed withan amplicon length of 150 to 250 bp. Libraries were sequencedwith 200-bp read length on a 318 chip. Mutation calling wasperformed using the torrent suite variant caller under the lowstringency somatic settings (TS4.0). We have studied CXCR4mutations along with MYD88 L265P (exon 5), CD79A (ITAMdomain), CD79B (ITAM domain), CARD11 (exons 5–9), N-RAS(exons 2 and 3), K-RAS (exons 2 and 3), BRAF6 (exon 15), andPTEN (exon 5þ7) using NGS (n ¼ 53).

To quantify the mutated clone, we have analyzed the VAF(variant allele frequency) defined as the number of reads thatmapped each studied to this position, cover the variant base andshow the reference allele, divided by all fragments covering thesite. VAF was corrected by the percentage of tumoral cells in B cellselected sample. Mutation calls were considered positive whencalled by at least 20 variants reads. Complete sequence data of thecoding and splice site regions of CXCR4were generated at a meandepth coverage of 2,000� per nucleotide. NGS assay also alloweda better assessment of the percentage of CXCR4 mutant allelewhose sensitivity is of 1%.

Gene expression profilingGene expression profiling (GEP) was performed using U133A

arrays (Affymetrix) for seven of the patients with Waldenstr€ommacroglobulinemia. Total RNA was extracted from purified Btumoral cells population isolated from bone marrow using theTRIzolmethod. Expression data were normalized using the RobustMulti-array Average (RMA) algorithm. Differential gene expressionwas analyzed with the Bioconductor R package "limma," which iswell-known to improve variance modeling when the number ofreplicates is small. All probes with an adjusted P value under 0.05were considered differentially expressed. Their normalized expres-sion values were then represented in a heatmap, whose colors varyaccording to the standardized by row values of the input data. Inparallel, a gene set enrichment analysis (GSEA) was performed forseveral gene sets among them the C2 collection of the MSigDB(Supplementary Tables S1 and S2). The PGSEA package (17) wasused to calculate z scores for all gene sets, andP valueswereadjustedfor multiple testing (18).

Cytogenetic analysis, FISH, SNPa, and immunophenotypicstudies

Genome-wide detection of copy number alteration (CNA) andLOH was performed using the Genome-Wide Human SNP Array6.0 (Affymetrix). Conventional cytogenetic analysis was per-formed on DSP30 þ IL2 stimulated bone marrow cells. FISHwas performed to detect chromosomal aberrations: 6q23 dele-tion, 17p12, 11q22 deletions, 13q14 deletion, trisomy 12,

Translational Relevance

CXCR4mutation has proved to be of critical importance inWaldenstr€om macroglobulinemia, in part due to its role as amechanism of resistance to several agents. We have thereforesought to unravel the different aspects of CXCR4mutations inWaldenstr€om macroglobulinemia and to characterize thegenetic backgroundusing targeted next-generation sequencing(NGS) and SNP arrays. Mutational spectrum of eight candi-date genes involved inTLR, RAS, andBCRpathwaywas studiedin an integrative study. About 25% of Waldenstr€om macro-globulinemia displaysCXCR4mutation inC-terminal domainresponsible for higher CXCR4 expression. CXCR4 mutationspertain to the same clone as toMYD88 L265Pmutations, witha clear-cut genomic signature traducing a more complexgenome using SNP arrays but were mutually exclusive to BCRpathway mutations (CD79A/B mutations). CXCR4 mutationidentified a genomic subgroup ofMYD88L265PWaldenstr€ommacroglobulinemia.

Poulain et al.

Clin Cancer Res; 2016 Clinical Cancer ResearchOF2




trisomy 4 (n ¼ 90; ref. 15). Immunophenotypic expression ofCXCR4 (R et D systems, UK), CD49d, CD27, CD80, CD86,CD138 gating on CD19þ cells was performed along with CD38(Immunotech) and determination of the Matutes's score by flowcytometry. The ratio of mean fluorescence intensity (MFIR) wascalculated as the ratio of specific fluorescence with an isotypecontrol.

The relationships between the clinical, biologic, andmolecularparameters were determined using a nonparametric test (Mann–Whitney), a t test, a c2, or Fisher exact tests when appropriate.Correlations were tested through Spearman correlation coeffi-cient. The differences between the results of the comparative testswere considered statistically significant if P < 0.05. All statisticalanalyses were done with the SPSS 15.0 software. Survival wasstudied using Kaplan–Meier test, comparisons were made withlog-rank test

ResultsNew CXCR4 mutations identified in Waldenstr€ommacroglobulinemia

We have first used NGS (deep sequencing) to screen the entiresequence of CXCR4 (n ¼ 53 Waldenstr€om macroglobulinemiasamples) with a greater sensitivity and to quantify the allelicfrequency of the variant of CXCR4 mutations in B selected cells.

We have identified 14 of 53 (26.4%) Waldenstr€om macroglob-ulinemia with CXCR4 mutations. We then confirmed theobserved mutations using NGS using Sanger sequencing (SaS)and extend to the entire cohort of 98 patients with Waldenstr€ommacroglobulinemia (including the 53 done by NGS). We iden-tified CXCR4 mutations (CXCR4mut) for a total of 24 of 98(24.5%), and we have characterized several newmutations acrossthe entire coding exons of CXCR4 (Fig. 1).

Overall, 17 different mutations were identified in CXCR4,including 12 that we have described for the first time inWaldenstr€om macroglobulinemia. Interestingly, only one typeof CXCR4 mutation was observed in either patient, and allmutations identified were located in the 45 amino acid intracy-toplasmic carboxy-terminal tail. These mutations were neverobserved in the paired T lymphocytes and thus confirmed theirsomatic feature. All mutations were heterozygous, and no UPDa(acquired uniparental disomy, LOH without variation of copynumber) was observed atCXCR4 locus nor variation of copy genenumber (gain or deletion) in our cohort, using SNP array.

High allelic frequency of CXCR4 mutation in Waldenstr€ommacroglobulinemia

NGS also allowed the quantification the allelic frequency of thevariant of CXCR4 mutations. This quantification allows to

COOH

Extracellular domain

Intracellular domain

S

I

LT

Number

of pa�ents

Type of muta�on

CXCR4 Nucleo�de change Amino acid change

1 Frameshi� c.945_946ins C H315fs1 Frameshi� c.952_953ins A T318fs*1 Frameshi� c.953_954delC T318fs1 Frameshi� c.963-_964insC R322fs1 Frameshi� c.977_978ins C L326fs1 Frameshi� c.979_985delAGATCCT K327fs1 Frameshi� c.982_983.del AT I328fs1 Nonsense c.1000C<T R334X*1 Nonsense c.1006G<T G336X3 Nonsense c.1013C<A S338X*5 Nonsense c.1013C<G S338X*1 Frameshi� c. 1012_1013delT S338fs1 Frameshi� c.1012_1015delTCAT S338fs2 Frameshi� c.1012_1013insT S338fs*1 Frameshi� c. 1017_1018del T S339fs

1 Frameshi� c. 1022_1023ins T S341fs

1 Nonsense c. 1031_1033delCT S344X*

R

R G338

336

339

341

344

326

334

327328

322

T 318

315

NH2

Transmembranedomain

Figure 1.Somatic mutations in C terminus of CXCR4 identified by Sanger sequencing and targeted NGS in patients with Waldenstr€om macroglobulinemia (WM).List of CXCR4 mutations observed in our study. n, number of patients. The mutations previously reported in WM by Treon et al. are noted with � . In the schematicstructure of CXCR4, sites of mutation are highlighted in blue (frameshift mutation) or gray (nonsense mutation). The S338 hotspot was represented in black.

CXCR4 Mutation in Waldenstr€om Macroglobulinemia

www.aacrjournals.org Clin Cancer Res; 2016 OF3




conclude for the percentage of mutation in the clonal populationfor a given patient and thus to differentiate between clonal(dominant clonal) and subclonal. The mutation load of CXCR4varied from 13.5% to 47.58% (mean, 35.2%) using the VAF (n¼14, CXCR4mut Waldenstr€om macroglobulinemia in our series).Interestingly, for 12 patients with Waldenstr€ommacroglobuline-mia, the high VAF ofCXCR4mutationwas suggested to be presentin the dominant clone. However, we were also able to identify thepresence of CXCR4mutations at the subclonal level in 4 patientsusing NGS, and for these 4 patients, the VAF varied from13.5% to26% (Fig. 2).

Nonsense (CXCR4NS) and frameshift (CXCR4FS) CXCR4mutations lead to truncation of CXCR4 C-terminal

Among the CXCR4mutations observed, 11 of 24 (45.8%) and13 of 24 (54.2%) were nonsense (CXCR4NS) and frameshift(CXCR4FS) mutations, respectively. The most frequent mutationwas the C1013G (S338X) mutation (5 of 98, 5.1%) followed byC1013A (S338X; 3 of 98, 3%). The modelization of C1013G/CXCR4 and C1013A/CXCR4 variants predicts a stop codon inplace of a serine at amino acid position 338 (S338X). We alsofound several othermutations to S338, suggesting a hotspot locusin CXCR4 gene. The S338 hotspot mutation was targeted in 50%of mutated cases in Waldenstr€om macroglobulinemia. OtherCXCR4NS mutations were here described. Interestingly, the exactsame acquired R334X mutation in Waldenstr€om macroglobuli-nemia was also previously described in the constitutional WHIMsyndrome (19).

Increased expression of CXCR4 in Waldenstr€ommacroglobulinemia with CXCR4mut

Because we observed that all mutations were located in the C-terminal tail of CXCR4 and that this C-terminal domain plays acritical role in the desensitization process that contributes to theregulation of the signal transduction and CXCR4 expression (2);we then sought to study the impact of CXCR4 mutation on theexpression of CXCR4 on the membrane of Waldenstr€om macro-

globulinemia tumor cells using flow cytometry. CXCR4 wasexpressed in all cases (n ¼ 53), including the 12 CXCR4mut

patients. The pattern varied widely using the MFIR to evaluatethe CXCR4 expression (MFI varied from 4.6 to 148.7). However,there was a significant greater expression of CXCR4 protein inWaldenstr€om macroglobulinemia with CXCR4mut (P ¼0.003; Fig. 3). This increased expression was observed indepen-dently of the type of mutation (data not shown).

Phenotypic signature associated to CXCR4mut genotypeCXCR4 directly interacts with CD49d in response to CXCL12

signaling in regulating migration and adhesion of Waldenstr€ommacroglobulinemia cells to the bone marrow microenvironment(10). CD49d favors lymphocyte homing by cooperating withCXCR4 in stromal cell adhesion and extracellular matrix (9). Wethen studied the impact of CXCR4mutation on the expression ofthe integrin VLA4 (CD49d) usingflowcytometry (n¼53).CD49dwas expressed in all Waldenstr€om macroglobulinemia cases; butno difference in CD49d expression was observed according toCXCR4 mutation. In contrast, a higher expression of CD49d wasobserved in Waldenstr€om macroglobulinemia with MYD88L265P mutation (P ¼ 0.048; Fig. 3). Similarly, we found nodifference in CD38 expression according to CXCR4 mutation,despite of the known functional link between CXCR4, CD49d,andCD38 (20). CD138mediates cell adhesion to the extracellularmatrix and is a marker of plasmacytic differentiation (21). Inter-estingly, we found a significant lower expression of CD138(syndecan1) in the CXCR4mut group (P ¼ 0.034). We found nodifference in the CD27, CD23, CD10, K/L expression and theMatutes' score between CXCR4mut and CXCR4wild subgroups.

Genomic signature of CXCR4mut Waldenstr€ommacroglobulinemia

We then sought to study whether a specific high-throughputSNPa signature was associated to CXCR4mut Waldenstr€om mac-roglobulinemia in a series of 53 Waldenstr€om macroglobuline-mia including 12CXCR4mut. For this analysis, we have considered

0.00

20.00

40.00

60.00

80.00

100.00

Variant allelic of CXCR4frequency variant

Variant allelic of MYD88L265P variant frequency

Figure 2.Representation of VAF of CXCR4 andMYD88 mutation in each patientusing NGS. Each row represents apatient (x-axis). VAF of MYD88 L265Pmutation is represented in light gray,CXCR4 VAF in black. Percentageindicates allele frequencies determinedby NGS corrected by the percentage oftumoral cells in the B selected samplesin each 53 patients (y-axis). �, subclonalCXCR4 mutation in patients withWaldenstr€om macroglobulinemia.

Poulain et al.





"presence of one" as any genomic abnormality including gain,loss, and/or copy number without LOH (CN-LOH), identifiedusing SNPa. In this first analysis, we have compared presenceversus absence of genomic abnormality according to presence ofCXCR4 mutation. We found a relationship between CXCR4mut

and a greater frequency of genomic aberrations in Waldenstr€ommacroglobulinemia comparedwithCXCR4wild (91%vs. 68%,P¼0.010).

We then evaluated the number of genomic abnormality as areflectionof the genomic complexity on the basis of the number ofSNP abnormalities.We found thatCXCR4-mutatedWaldenstr€ommacroglobulinemia had a greater mean number of abnormality(5.8 vs. 2.8 per patient, P ¼ 0.046). No significant difference wasobserved between Waldenstr€om macroglobulinemia with clonalor subclonal CXCR4 mutation.

Furthermore, we analyzed the relationship between CXCR4mutation and certain type of SNP aberration. We have observedthat Waldenstr€om macroglobulinemia with CXCR4mut had agreater incidence rate of trisomy 4 (complete or partial), 58%versus 12% respectively (P¼ 0.002); a greater frequency of gain ofXq, including MYD88 pathway–based IRAK1 gene, and of dele-tion 8p (P¼ 0.002 and P¼ 0.007, respectively; Fig. 4). Finally, wealso found a greater frequency of deletion 6q, including theNF-kBpathway–based TNFAIP3 gene (P ¼ 0.038). We found no rela-tionship to deletion 7q, deletion 11q, deletion 17p, deletion13q14, trisomy 18 in our study. No difference of SNPa featurewas observed according to the type ofCXCR4mutation (nonsense(CXCR4NS) versus frameshift (CXCR4FS) mutations.

Mutational landscape of CXCR4mut Waldenstr€ommacroglobulinemia

We next attempted to delineate the mutational spectrum andintratumoral heterogeneity related to presence of CXCR4mut forgenes involved in the RAS (this pathway may be activated byCXCR4/protein G proteins), BCR and TLR pathways (MYD88exon 5), known to be interconnected with CXCR4 pathway, usingtargeted NGS (6).

When looking at the incidence rate of the CXCR4 mutationswith presence of MYD88 L265P mutation in the cohort of 98patients, it appeared that 23 of 24 (95.8%) of the CXCR4mut wereMYD88 L265P mutated, except one Waldenstr€om macroglobu-linemia with a CXCR4FS mutation. The high correlation betweenMYD88 L265P and CXCR4mutational loads tends to suggest thecoexistence of the 2 mutations in the same clone (Fig. 2). How-ever, in four patients with Waldenstr€om macroglobulinemia, alower VAF ofCXCR4 in comparison toMYD88 L265P suggested asubclonal CXCR4 mutation in the dominant clone harboringMYD88 mutation.

Regarding the presence of mutations in the RAS pathway inWaldenstr€om macroglobulinemia, including PTEN, K-Ras, andN-Ras, crucial regulators of RAS pathway, we have not identifiedany mutation in our cohort (n ¼ 53).

BCR pathway can be constitutively activated, either by signalsfrom themicroenvironment or by genetic aberration (22). Severalmutations of key signaling molecules and regulators of the BCRpathway were reported overtime, particularly in the activated B-cell–like (ABC) diffuse large B-cell lymphoma (DLBCL) and in

MYD88wild

CXCR4wild

CXCR4mutated

MYD88wild

MYD88L265P

MYD88L265P

CXCR4wild

CXCR4mutated

Expr

essi

onof

CXCR

4(M

FIR)

Expr

essi

onof

CXCR

4(M

FIR)

Expr

essi

onof

CD49

d(M

FIR)

Expr

essi

onof

CD49

d(M

FIR)

P = 0.003

P = 0.048

P = ns

P = ns

A

C D

B

0.00 0.00

20.00

40.00

60.00

0.00

20.00

40.00

60.00

50.00

100.00

150.00

0.00

50.00

100.00

150.00

Figure 3.Expression of CXCR4 and CD49d according to MYD88 and CXCR4 mutation status. Boxplots are shown with an overlay of the individual data points. ns, notsignificant.






Waldenstr€ommacroglobulinemia (15, 23), including the immu-noreceptor tyrosine–based activation motif (ITAM) signalingmodules of the CD79A and CD79B BCR coreceptors and thecoiled-coil domain of CARD11 (22). Mutations of CD79B andCD79A were observed in 12 of 98 (12.2%) of Waldenstr€ommacroglobulinemia. Interestingly, we found no coexistence ofCXCR4mut and CD79A/CD79Bmutations, but in one patient whohadCXCR4mut at the subclonal level.NomutationofCARD11wasobserved in our studied group (n¼ 53). Onemight conclude thatthere is no unique somatic mutation in the BCR pathway, butinstead a wide variety of molecules that could be altered throughmolecular alterations in the BCR pathway, likely mutually exclu-sive in the same clone, such as CXCR4mut and CD79A/CD79Bmutations in the present situation in our study.

Transcriptional signature related to presence of CXCR4mutation

We finally studied the gene expression profile of Waldenstr€ommacroglobulinemia on 7 patients with Waldenstr€ommacroglob-ulinemia to understand whether the CXCR4 mutation impactedthe gene expression and to identify a potential gene signature ofWaldenstr€om macroglobulinemia with CXCR4mut. The differen-tial analysis allowed to identify a gene signature attached to theCXCR4mut molecular profile, corresponding to 32 probes and 27genes. We found several genes among these, such as MMP8,CRISP3, or BRCC3 involved in cell death and survival or cellmovement (ARG1, LYST). These probes were all underexpressedin the five CXCR4wild Waldenstr€om macroglobulinemia com-

pared with the two CXCR4mut Waldenstr€om macroglobulinemia(Fig. 5 and Supplementary Tables S1 and S2). We then sought toidentify deregulated pathways using Ingenuity analysis software.We found CXCR4mut to be related to several functional networknetworks such as cell cycle and DNA replication or cell-to-cellsignaling and interaction, cellular growth, and proliferation.Using GSEA for targeted gene sets previously associated withCXCR4mutation (12), we also identified deregulation of protea-some pathway and BCR/B lymphocyte pathway in CXCR4mut

Waldenstr€om macroglobulinemia (P ¼ 0.01 and P ¼ 0.04,respectively; Supplementary Table S1).

Clinical and biologic features of CXCR4mut

We thought to identify clinical–biologic characteristics ofWaldenstr€om macroglobulinemia according to CXCR4mut fea-tures. The median age was 67 years (range, 36–92 years) in ourseries. We found that CXCR4mut was associated to a higher IgMmonoclonal component (r ¼ 0.309; P ¼ 0.006) and thrombo-cytopenia (r¼�0.226; P¼ 0.048), markers of adverse prognosisin Waldenstr€om macroglobulinemia. Indeed, 35% and 12% ofpatients had serum IgM level greater than 30 g/L (P¼ 0.023) and27% and 6.5% of patients had low platelet count lower than 100g/L (P ¼ 0.018), respectively, in patients with CXCR4mut versusCXCR4wild. There was a trend for CXCR4mut patients to havemoreoften IPSSWaldenstr€ommacroglobulinemia stage 3 (P¼ 0.086).No associationwas foundwith regard to othermarkers of tumoralsyndrome, such as adenopathy and splenomegaly, extramedul-lary localization, or lymphocytosis (one would consider a markerof egression of tumoral cells from the bone marrow; Supplemen-tary Table S3). With a median follow-up of 6 years, 21 (22%)patients in our series had diedwith amedianOS (95% confidenceinterval) not reached and estimated at 63% at 10 years. Nosignificant difference in type of treatment or number of treatmentline was observed (data not shown). The estimated overall sur-vival (OS) at 10 years according to CXCR4mut status was lower forCXCR4mut patients, 50% versus 65.5% for patients withCXCR4wild (P ¼ ns, Fig. 6). Interestingly, the difference in OSbetween CXCR4mut versus CXCR4wild became significant inpatients with indolent Waldenstr€om macroglobulinemia (esti-mated 5-year OS rate at 50% and 93%, respectively, P ¼ 0.019);the CXCR4mut patients required therapy more often and muchearlier than the CXCR4wild.

DiscussionWe have described 12 new CXCR4 mutations for a total of 17

patients with Waldenstr€om macroglobulinemia so far, observedin 25% of patients with Waldenstr€ommacroglobulinemia, usingtargeted deep sequencing. These data suggest a highly heteroge-neous pattern of CXCR4 mutations in Waldenstr€om macroglob-ulinemia and confirms using a more sensitive technique that wasreported by Treon and colleagues using Sanger (11).We have alsoconfirmedCXCR4 1013C>G, followed by 1013C>A,mutations tobe the most frequent CXCR4 mutations in Waldenstr€om macro-globulinemia, although in a lesser frequency as previouslyreported, 12.5% (for the 2 mutations using Sanger sequencingin CD19 selected cells) and 30% (only 1013C>A using allele-specific PCR in unselected tumoral samples) in Treon and col-leagues andRoccaro and colleagues, respectively (11–12).Overallthe high frequency of CXCR4 Whim-like mutation is a newhallmark of Waldenstr€om macroglobulinemia (12, 24).

Figure 4.Association network of genomic alterations in Waldenstr€ommacroglobulinemia (WM). The heatmap represents co-occurrence of CXCR4mutationwith other genetic alterations analyzed in 53patientswithWMusingSNPa, cytogenetic, and FISH. The co-occurrence of CXCR4 mutation withother genetic alterations was represented using MeV software (TM4microarray software suite; refs. 31, 32). Each row corresponds to key locus orregulator genes of the canonical and noncanonical NF-kB pathways targetedby CNA, UPD, or mutation in WM. The columns represent individual patientscolor coded on the base of gene status. Each patient is represented by avirtual column (black, mutation; CN LOH, deletion or gain of gene; gray,wild-type).

Poulain et al.





Interestingly, all mutations, either nonsense or frameshift,occurred in the C-terminus of CXCR4. It was suggested that thetruncation of the distal amino acid region of the C-terminusknown to regulate signaling of CXCR4 by CXCL12, might dereg-ulate the CXCR4/SDF1 axis signaling pathway, and thus partic-ipate in the pathogenesis of Waldenstr€om macroglobulinemia(2, 11, 12, 25). Onemight propose that the type ofmutation doesnot matter much in Waldenstr€om macroglobulinemia, on thecontrary to the loss of C-terminus domain of CXCR4 proteinwhich modified function might play a role in Waldenstr€ommacroglobulinemia pathogenesis (2). Functional studies mayconfirm this hypothesis in the near future.

We found a greater CXCR4 expression on tumor cells ofWaldenstr€om macroglobulinemia carrying CXCR4 mutation,independently of the type of mutation, similar to that wasdescribed in WHIM syndrome. It is suggested that the increaseof CXCR4 receptor expression would induce an altered migrationprofile in response to SDF1 (25), resulting in an increased egres-sion of tumor cells as demonstrated in mouse model (12). In ourstudy, we also found a decreased expression of CD138, a cellsurface syndecan-1 that mediates cancer cell adhesion to theextracellular matrix, and alteration of expression of several genesinvolved inmatrix degradation such asMMP8or cellmigrationongene expression profiling. Taking together, CXCR4 mutationmight alter the ability of the tumor cells to interact with the bonemarrowmicroenvironment inWaldenstr€ommacroglobulinemia,as shown in CXCR4 S338X cells (12).

Nearly all patients withWaldenstr€ommacroglobulinemia withCXCR4 mutations harbored the MYD88 L265P mutation, asinitially described by Treon and colleagues suggesting a potential

cooperation between these pathways (11). An important datasetobtained in our series came from the quantification of the allelicfrequency of the variant (VAF) of CXCR4mutations into tumoralcells usingNGS thatmay identifyminor subclones of less than 1%

Figure 6.OS according to presence or absence of CXCR4 mutation in Waldenstr€ommacroglobulinemia. The solid line indicates the CXCR4wild patients, thedashed line CXCR4mut patients. ns, not significant.

Figure 5.Gene expression profiling. Heatmap ofnormalized expression values fordifferentially expressed probes. Colorsvary according to the standardized byrow values of the input data. The scaleof the gene expression data isindicated. Some genes of interests arehighlighted.






in our study. Targeted deep sequencing allows to conclude for thepercentage of mutations in the clonal population for a givenpatient and thus to differentiate between clonal (dominant clon-al) and subclonal mutations. Combining analysis of VAF ofseveral genes, CXCR4 mutation was expressed in the same cloneasMYD88 L265P, as we have identified a high allelic frequency ofCXCR4 mutation MYD88 L265P in Waldenstr€om macroglobuli-nemia tumor cells; but for 4 patients who had CXCR4 mutationpresent only at the subclonal level.Overall, these datamay suggestthe following hypothesis, where MYD88 L265P mutation is afounder mutation in Waldenstr€om macroglobulinemia, a firstgenetic hit that would promote NF-kB and JAK/STAT3 signaling(4, 26). Direct inhibition of MYD88 L265P signaling overcomesCXCL12-trigered survival effects in CXCR4-mutated cells, sup-porting a primary role for MYD88 signaling in Waldenstr€ommacroglobulinemia (25). CXCR4 could then be one of the sec-ondary events in some patients who showed subclonal mutationof CXCR4 compared withMYD88 L265P mutation present in themain clone. However, longitudinal analysis studies are needed toexplore the dynamic of clonal architecture to identify driver oradditional mutations in Waldenstr€om macroglobulinemia thatcould contribute to clinical progression or chemoresistance.Indeed, it was shown that a diminished clinical activity of ibru-tinib was observed in patients with Waldenstr€om macroglobuli-nemia with CXCR4 mutations (25;30).

In addition, we also found absence of co-occurrence of CD79A/CD79B and CXCR4 mutations in Waldenstr€om macroglobuline-mia.Thesedatamay suggest that these typesof activatingmutationsof BCR and CXCR4 pathway were mutually exclusive in MYD88L265P Waldenstr€om macroglobulinemia (6). A cooperationbetween CXCR4 and TLR signaling activated by MYD88L265Pmutation was described in B cells (6). The functional role ofmutations in the BCR pathway is not fully described inWaldenstr€om macroglobulinemia, but CD79B mutation wasshown to alter the BCR response in diffuse large B lymphoma(15, 23). We might thus suggest the existence of two subgroups ofMYD88L265PWaldenstr€ommacroglobulinemia, onewithCXCR4mutations and one with BCR pathwaymutations that may involvein cooperation with the aforementioned driver mutation whichprimarily affect TLR signaling. These data further may advance theconcept that a complex rather than a unique deregulation of theTLR/NF-kB pathway characterizes MYD88 L265P Waldenstr€ommacroglobulinemia with others interconnected pathways.

We have observed that CXCR4 mutation was more frequentlyassociated to a complex genomic signature, including gain ofchromosome 4, deletion 6q, and gain of chromosome X usingSNPa. High-throughput genomic studies have identifiedmultiplemechanisms of genetic changes in Waldenstr€om macroglobuli-nemia including several recurrent CNA such as deletion 6q,deletion 13q, or gain of chromosome 4 and CN-LOH (15, 27).This genomic pattern segregates furtherWaldenstr€ommacroglob-ulinemia among the others B- cell tumors. The prognostic role ofthese genomic alterations is not fully understood, but the pres-ence of more than 3 SNPa abnormalities was associated withsymptomatic status (15, 27, 28). Taking together, this findingsuggests an inter- and intraclonal heterogeneity and the potentialselective advantage of specific combinations of genetic lesions inWaldenstr€om macroglobulinemia, in particular in the subset ofCXCR4mut Waldenstr€ommacroglobulinemia. The understandingof these genomic abnormalities, including gene mutations andCNA, described herein might help deciphering the pathogenesis

of Waldenstr€ommacroglobulinemia to determine associated riskof progression, relapse, and drug resistance.

Overall, Waldenstr€om macroglobulinemia with CXCR4muta-tion had a specific clinicobiologic and genomic signature associ-ated to features characterized with adverse prognosis inWaldenstr€om macroglobulinemia, including high IgM M-spikeand thrombocytopenia (part of the IPSS Waldenstr€om macro-globulinemia score adverse features) and greater frequency ofcomplex genomic aberrations pattern using SNP array. Our datatherefore may suggest a worse prognosis for CXCR4 mutationsubgroup of asymptomatic Waldenstr€om macroglobulinemia,but we did not observe significant impact on overall survival inour cohort as previously described (11). Further studies areneeded to explore the prognosis value of CXCR4 mutation inWaldenstr€om macroglobulinemia in clinical trials. Interestingly,the potential clinical impact ofWaldenstr€ommacroglobulinemiawithCXCR4mutation has already been suggested by Roccaro andcolleagues and Treon and colleagues on the basis of their in vitrostudies showing drug resistance inCXCR4-mutatedWaldenstr€ommacroglobulinemia cells exposed to BTK, mTOR, and PI3K inhi-bitors but not proteasome inhibitors (29, 30). This study, alongwith other, thus led us to propose that it is primetime for asystematic evaluation of CXCR4 mutation as part as the pretreat-ment package of Waldenstr€om macroglobulinemia, to identifypatients with CXCR4 mutation that might benefit more fromproteasome inhibitors as compared with other options, such aBTK inhibitors.

In conclusion, approximately 25% of Waldenstr€om macro-globulinemia harbor CXCR4mutation, leading to altered CXCR4protein at the C-terminal end, irrespective of the pleiotropicpattern of mutations. The study of CXCR4 mutations showedexistence of intraclonal (variation in co-expression ofMYD88 andCXCR4 mutations) and interclonal (BCR and CXCR4 mutationsin MYD88L265P Waldenstr€ommacroglobulinemia) heterogene-ity when analyzing the molecular landscape of CXCR4 mutationinWaldenstr€ommacroglobulinemia. Patients withCXCR4muta-tion displayed a specific clinical–biologic–genomic and transcrip-tomic signature, possibly related to specific physiopathologicmechanism related to CXCR4 mutation. This dataset furthersuggests the genomic heterogeneity of Waldenstr€om macroglob-ulinemia, beyond the current indolent versus symptomaticclassification.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Poulain, M. Figeac, F. Lepretre, X. LeleuDevelopment of methodology: S. Poulain, A. Venet-Caillault, M. Figeac,S. Geffroy, X. LeleuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Poulain, C. Roumier, M. Figeac, C. Herbaux,G. Marot, E. Bertrand, A. Decambron, E.M. Boyle, A. Renneville, S. Tricot,B. Quesnel, X. LeleuAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Poulain, C. Roumier, M. Figeac, G. Marot, E. Doye,B. Quesnel, X. LeleuWriting, review, and/or revision of the manuscript: S. Poulain, C. Roumier,C. Preudhomme, X. LeleuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Poulain, C. Roumier, E. Doye, F. Lepretre,O. Nibourel, B. Quesnel, X. LeleuStudy supervision: S. Poulain, E. Doye, P. Duthilleul, X. Leleu

Poulain et al.





AcknowledgmentsThe authors thank Pauline Vandycke, Val�erie Grandi�eres, Claudine Delsaut,

Axelle S�eghir, and Sabine Ranwez for their excellent technical assistance.

Grant SupportThis work was supported by the Comit�e Septentrion de la Ligue contre le

Cancer et la Fondation Francaise pour la Recherche contre le My�elome et lesGammapathies (FFRMG).

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 March 17, 2015; revised August 5, 2015; accepted August 31, 2015;published OnlineFirst October 21, 2015.

References1. Hunter ZR, Xu L, YangG, ZhouY, Liu X, Cao Y, et al. The genomic landscape

of Waldenstr€om macroglobulinemia is characterized by highly recurringMYD88 and WHIM-like CXCR4 mutations, and small somatic deletionsassociated with B-cell lymphomagenesis. Blood 2014;123:1637–46.

2. Al Ustwani O, Kurzrock R, Wetzler M. Genetics on a WHIM. Br J Haematol2014 Jan;164:15–23.

3. Hernandez PA, Gorlin RJ, Lukens JN, Taniuchi S, Bohinjec J, Francois F,et al. Mutations in the chemokine receptor gene CXCR4 are associated withWHIM syndrome, a combined immunodeficiency disease. Nat Genet2003;34:70–4.

4. Treon SP, Xu L, Yang G, Zhou Y, Liu X, Cao Y, et al. MYD88 L265P somaticmutation in Waldenstrom's macroglobulinemia. N Engl J Med 2012;367:826–33.

5. Poulain S, Boyle EM, Roumier C, Demarquette H, Wemeau M, Geffroy S,et al. MYD88 L265P mutation contributes to the diagnosis of Bing Neelsyndrome. Br J Haematol 2014;167:506–13.

6. Chatterjee S, Behnam Azad B, Nimmagadda S. The intricate role of CXCR4in cancer. Adv Cancer Re 2014;124:31–82.

7. Conley-LaCombMK, Saliganan A, Kandagatla P, Chen YQ, CherML, ChinniSR. PTEN lossmediated Akt activation promotes prostate tumor growth andmetastasis via CXCL12/CXCR4 signaling. Mol Cancer 2013;12:85.

8. Nie Y, Waite J, Brewer F, Sunshine MJ, Littman DR, Zou YR. The role ofCXCR4 in maintaining peripheral B cell compartments and humoralimmunity. J Exp Med 2004;200:1145–56.

9. Burger JA, Gribben JG. The microenvironment in chronic lymphocyticleukemia (CLL) and other B cell malignancies: insight into disease biologyand new targeted therapies. Semin Cancer Bio 2014;24:71–81.

10. NgoHT, Leleu X, Lee J, Jia X,MelhemM, Runnels J, et al. SDF-1/CXCR4 andVLA-4 interaction regulates homing in Waldenstrom macroglobulinemia.Blood 2008;112:150–8.

11. Treon SP, Cao Y, Xu L, Yang G, Liu X, Hunter ZR. Somatic mutations inMYD88 and CXCR4 are determinants of clinical presentation and overallsurvival in Waldenstrom macroglobulinemia. Blood 2014;123:2791–6.

12. Roccaro AM, Sacco A, Jimenez C, Maiso P, Moschetta M, Mishima Y, et al.C1013G/CXCR4 acts as a driver mutation of tumor progression andmodulator of drug resistance in lymphoplasmacytic lymphoma. Blood2014,123:4120–31

13. Owen RG, Pratt G, Auer RL, Flatley R, Kyriakou C, Lunn MP, et al. Guide-lines on the diagnosis and management of Waldenstrom macroglobuli-naemia. Br J Haemato 2014;165:316–33.

14. DimopoulosMA,Kastritis E,OwenRG,KyleRA, LandgrenO,Morra E, et al.Treatment recommendations for patients with Waldenstrom macroglob-ulinemia (WM) and related disorders: IWWM-7 consensus. Blood2014;124:1404–11.

15. Poulain S, Roumier C, Galiegue-Zouitina S, Daudignon A, Herbaux C,Aiijou R, et al. Genome wide SNP array identified multiple mechanisms ofgenetic changes in Waldenstrom macroglobulinemia. Am J Hematol2013;88:948–54.

16. Poulain S, Roumier C, Decambron A, Renneville A, Herbaux C, Bertrand E,et al. MYD88 L265Pmutation inWaldenstrommacroglobulinemia. Blood2013;121:4504–11.

17. Kim SY, VolskyDJ. PAGE: parametric analysis of gene set enrichment. BMCBioinformatics 2005;6:144.

18. Hochberg Ba. Controlling the false discovery rate: a practical andpowerful approach to multiple testing. J Royal Stat Soc, Series B 571995:289–300

19. Taniuchi S,MasudaM, Fujii Y, Izawa K, KaneganeH, Kobayashi Y. The roleof a mutation of the CXCR4 gene in WHIM syndrome. Haematologica2005;90:1271–2.

20. Dal BoM, Tissino E, Benedetti D, Caldana C, Bomben R, Del Poeta G, et al.Microenvironmental interactions in chronic lymphocytic leukemia: themaster role of CD49d. Semin Hematol 2014;51:168–76.

21. Teng YH, Aquino RS, Park PW. Molecular functions of syndecan-1 indisease. Matrix Biol 2012;31:3–16.

22. Lim KH, Yang Y, Staudt LM. Pathogenetic importance and therapeuticimplications of NF-kappaB in lymphoid malignancies. Immunol Rev2012;246:359–78.

23. Davis RE, Ngo VN, Lenz G, Tolar P, Young RM, Romesser PB, et al. Chronicactive B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature2010;463:88–92.

24. MartinezN, AlmarazC, Vaque JP, Varela I, Derdak S, Beltran S, et al.Whole-exome sequencing in splenic marginal zone lymphoma reveals mutationsin genes involved in marginal zone differentiation. Leukemia 2014;28:1334–40.

25. Cao Y, Hunter ZR, Liu X, Xu L, Yang G, Chen J, et al. CXCR4 WHIM-likeframeshift and nonsense mutations promote ibrutinib resistance but donot supplantMYD88(L265P) -directed survival signalling inWaldenstrommacroglobulinaemia cells. Br J Haematol 2015;168:701–7.

26. Wang JQ, Jeelall YS, Beutler B,HorikawaK,GoodnowCC.Consequences ofthe recurrent MYD88(L265P) somatic mutation for B cell tolerance. J ExpMed 2014;211:413–26.

27. Braggio E, PhilipsbornC,Novak A,Hodge L, Ansell S, Fonseca R.Molecularpathogenesis of Waldenstrom's macroglobulinemia. Haematologica2012;97:1281–90.

28. Nguyen-Khac F, Lambert J, Chapiro E, Grelier A, Mould S, Barin C, et al.Chromosomal aberrations and their prognostic value in a series of 174untreated patients with Waldenstrom's macroglobulinemia. Haematolo-gica 2013;98:649–54.

29. Roccaro AM, Sacco A, Jimenez C, Maiso P, Moschetta M, Mishima Y, et al.C1013G/CXCR4 acts as a driver mutation of tumor progression andmodulator of drug resistance in lymphoplasmacytic lymphoma. Blood2014;123:4120–31.

30. Cao Y, Hunter ZR, Liu X, Xu L, YangG, Chen J, et al. TheWHIM-like CXCR4(S338X) somaticmutation activates AKT andERK, and promotes resistanceto ibrutinib and other agents used in the treatment of Waldenstrom'sMacroglobulinemia. Leukemia 2015;29:169–76.

31. Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA,et al. TM4 microarray software suite. Methods Enzymol 2006;411:134–93.

32. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, et al. TM4: a free,open-source system for microarray data management and analysis. Bio-techniques 2003;34:374–8.






Published OnlineFirst October 21, 2015.Clin Cancer Res Stéphanie Poulain, Christophe Roumier, Aurélie Venet-Caillault, et al. Macroglobulinemia

Mutations in WaldenströmCXCR4Genomic Landscape of

Updated version

10.1158/1078-0432.CCR-15-0646doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2015/10/21/1078-0432.CCR-15-0646.DC1Access the most recent supplemental material at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/early/2016/01/08/1078-0432.CCR-15-0646To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-15-0646

http://clincancerres.aacrjournals.org/content/suppl/2015/10/21/1078-0432.CCR-15-0646.DC1

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/early/2016/01/08/1078-0432.CCR-15-0646


Documents

Genomic Landscape of CXCR4 Mutations in Waldenstrom ... · 30% of Waldenstrom macroglobulinemia, similar to germline€ mutationsfoundintheWHIM(warts,hypogammaglobulinemia, infections,