Genomic Landscape of CXCR4 Mutations in Waldenström Macroglobulinemia

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

Biology of Human Tumors Clinical Cancer Research Genomic Landscape of CXCR4 Mutations in Waldenstrom€ Macroglobulinemia Stephanie Poulain1,2,3, Christophe Roumier2,3, Aurelie Venet-Caillault2, Martin Figeac4, Charles Herbaux3,5, Guillemette Marot6, Emmanuelle Doye2, Elisabeth Bertrand3, Sandrine Geffroy2,Fred eric Lepretre4, Olivier Nibourel2,3, Audrey Decambron1, Eileen Mary Boyle3,5, Aline Renneville2, Sabine Tricot1,Agnes Daudignon1, Bruno Quesnel3,4, Patrick Duthilleul1, Claude Preudhomme2,3, and Xavier Leleu3,5

Abstract

Purpose: Whole-genome sequencing has revealed MYD88 Waldenstrom€ macroglobulinemia. CXCR4 mutations led to a mut L265P and CXCR4 mutations (CXCR4 ) as the most prevalent truncated receptor protein associated with a higher expression somatic mutations in Waldenstrom€ macroglobulinemia. CXCR4 of CXCR4. CXCR4 mutations pertain to the same clone as to mutation has proved to be of critical importance in Waldenstrom€ MYD88 L265P mutations but were mutually exclusive to macroglobulinemia, in part due to its role as a mechanism of CD79A/CD79B mutations (BCR pathway). We identified a mut resistance to several agents. We have therefore sought to unravel genomic signature in CXCR4 Waldenstrom€ macroglobuline- the different aspects of CXCR4 mutations in Waldenstrom€ mia traducing a more complex genome. CXCR4 mutations were macroglobulinemia. also associated with gain of chromosome 4, gain of Xq, and Experimental Design: We have scanned the two coding exons deletion 6q. of CXCR4 in Waldenstrom€ macroglobulinemia using deep next- Conclusions: Our study panned out new CXCR4 mutations in generation sequencing and Sanger sequencing in 98 patients with Waldenstrom€ macroglobulinemia and identified a specific signa- mut Waldenstrom€ macroglobulinemia and correlated with SNP array ture associated to CXCR4 , characterized with complex genomic landscape and mutational spectrum of eight candidate genes aberrations among MYD88L265P Waldenstrom€ macroglobuline- involved in TLR, RAS, and BCR pathway in an integrative study. mia. Our results suggest the existence of various genomic sub- Results: We found all mutations to be heterozygous, somatic, groups in Waldenstrom€ macroglobulinemia. Clin Cancer Res; 1–9. and located in the C-terminal domain of CXCR4 in 25% of the 2015 AACR.

Introduction quency in Waldenstrom€ macroglobulinemia; and thus it is sus- pected that a second hit may accelerate development and pro- Whole-genome sequencing has revealed CXCR4 as the second gression of the malignant clones leading to full-blown in most frequent somatic mutation, identified in approximately Waldenstrom€ macroglobulinemia. Although the mechanism of 30% of Waldenstrom€ macroglobulinemia, similar to germline deregulation of CXCR4 axis is not fully understood in mutations found in the WHIM (warts, hypogammaglobulinemia, Waldenstrom€ macroglobulinemia, it is possible that CXCR4 infections, and myelokathexis) syndrome (1–3). MYD88 L265P mutation might represent one of these secondary events. mutations remain the most frequent mutation reported in C-X-C chemokine receptor type 4 (CXCR4) is a G-protein– Waldenstrom€ macroglobulinemia to date, in nearly 90% of coupled receptor that plays an important role in lymphopoi- Waldenstrom€ macroglobulinemia (4, 5). Interestingly, MYD88 esis and cell trafficking (2, 6), along with its ligand, the stromal L265P may act as a founder mutation because of its high fre- cell–derived factor-1 (CXCL12/SDF-1). The SDF1/CXCR4 axis promotes activation of several pathways including RAS, Akt, and NF-kB and interplays with BCR pathway (6–8). The 1 Service d'Hematologie-Immunologie-Cytog en etique, Centre Hospi- CXCR4 gene is located on the long arm of chromosome 2 at talier de Valenciennes, France. 2Laboratoire d'Hematologie, Centre de Biologie et Pathologie, CHRU de Lille, France. 3INSERM UMR 1172, position 21 and codes for a chemokine receptor that promotes IRCL, Lille, France. 4IFR114, Plateforme de Genomique, Lille, France. migration and survival of various B lymphoid malignancies 5Service des Maladies du Sang, Hopital^ Huriez, CHRU, Lille, France. (8–10). 6 Universite de Lille, UDSL, EA2694 Biostatistics/Inria Lille Nord Eur- CXCR4 mutations were identified using Sanger sequencing by ope, MODAL, Lille, France. Treon and colleagues in nearly 25% of Waldenstrom€ macroglob- Note: Supplementary data for this article are available at Clinical Cancer ulinemia, and the CXCR4 C1013G mutation was described as the Research Online (http://clincancerres.aacrjournals.org/). most frequent recurrent CXCR4 mutation in 7% (11). However, Corresponding Author: Xavier Leleu, Hopital^ Huriez, CHRU, Rue Michel Polo- the occurrence of potential subclonal of CXCR4 mutation and the novski, Lille 59037, France. Phone: 33-3-20446883; Fax: 33-3-2044-4094; proportion of CXCR4 mutation over the other known mutations E-mail: [email protected] was not available; in other words, how this mutation lies in the doi: 10.1158/1078-0432.CCR-15-0646 architecture of the MYD88-mutated clone. On contrary, Roccaro 2015 American Association for Cancer Research. and colleagues have reported 30% incidence rate of the CXCR4

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light-chain isotype–positive B cells was detected in all cases Translational Relevance (mean, 95.8% of tumoral cells; ref. 15). CXCR4 mutation has proved to be of critical importance in € Waldenstrom macroglobulinemia, in part due to its role as a DNA sequencing mechanism of resistance to several agents. We have therefore DNA was extracted from isolated cells using Qui Amp kit sought to unravel the different aspects of CXCR4 mutations in (Sigma-Aldrich Co.). MYD88 L265P, CD79A, and CD79B muta- € Waldenstrom macroglobulinemia and to characterize the tions were analyzed as previously described (16) in 98 patients. C- genetic background using targeted next-generation sequencing terminal CXCR4 gene was amplified from gDNA by PCR as (NGS) and SNP arrays. Mutational spectrum of eight candi- previously described (n ¼ 98 patients; ref. 5). All the exons of date genes involved in TLR, RAS, and BCR pathway was studied CXCR4 gene were analyzed using targeted next-generation € in an integrative study. About 25% of Waldenstrom macro- sequencing (NGS) in a cohort of 53 patients. NGS was analyzed CXCR4 globulinemia displays mutation in C-terminal domain using the Ion Torrent PGM platform (Life Biotechnologies). CXCR4 responsible for higher CXCR4 expression. mutations Amplicons covering the regions of interest were designed with MYD88 pertain to the same clone as to L265P mutations, with an amplicon length of 150 to 250 bp. Libraries were sequenced a clear-cut genomic signature traducing a more complex with 200-bp read length on a 318 chip. Mutation calling was genome using SNP arrays but were mutually exclusive to BCR performed using the torrent suite variant caller under the low CD79A B pathway mutations ( / mutations). CXCR4 mutation stringency somatic settings (TS4.0). We have studied CXCR4 fi € identi ed a genomic subgroup of MYD88L265P Waldenstrom mutations along with MYD88 L265P (exon 5), CD79A (ITAM macroglobulinemia. domain), CD79B (ITAM domain), CARD11 (exons 5–9), N-RAS (exons 2 and 3), K-RAS (exons 2 and 3), BRAF6 (exon 15), and PTEN (exon 5þ7) using NGS (n ¼ 53). To quantify the mutated clone, we have analyzed the VAF C1013G mutation using qPCR; however, they have solely studied (variant allele frequency) defined as the number of reads that this mutation (12). mapped each studied to this position, cover the variant base and In this study, we characterized the genomic landscape of CXCR4 show the reference allele, divided by all fragments covering the somatic mutation in a large cohort of 98 patients with site. VAF was corrected by the percentage of tumoral cells in B cell Waldenstrom€ macroglobulinemia, combining deep sequencing selected sample. Mutation calls were considered positive when of target genes allowing study of the clonal architecture in the called by at least 20 variants reads. Complete sequence data of the Waldenstrom€ macroglobulinemia cells, including subclonal coding and splice site regions of CXCR4 were generated at a mean analysis, and to confirm the incidence rate of these mutations depth coverage of 2,000 per nucleotide. NGS assay also allowed and single polymorphism nucleotide array (SNPa) to decipher a better assessment of the percentage of CXCR4 mutant allele the genomic landscape of CXCR4-mutated Waldenstrom€ whose sensitivity is of 1%. macroglobulinemia. Gene expression profiling Patients and Methods Gene expression profiling (GEP) was performed using U133A € Patients arrays (Affymetrix) for seven of the patients with Waldenstrom Ninety-eight patients diagnosed with Waldenstrom€ macro- macroglobulinemia. Total RNA was extracted from purified B globulinemia were included in this study (63 males, 35 females). tumoral cells population isolated from bone marrow using the The diagnosis and treatment initiation criteria of Waldenstrom€ TRIzol method. Expression data were normalized using the Robust macroglobulinemia were as published (13, 14). Among the Multi-array Average (RMA) algorithm. Differential gene expression cohort with available clinical follow up (n ¼ 93), 68 patients was analyzed with the Bioconductor R package "limma," which is were treated. First-line treatment was initiated at diagnosis in 41% well-known to improve variance modeling when the number of of the patients. Front-line therapy included chloraminophene replicates is small. All probes with an adjusted P value under 0.05 alone in 12 cases, fludarabine alone in 3 cases, rituximab (R) were considered differentially expressed. Their normalized expres- alone in 3 cases or in association with chemotherapy in 51 cases sion values were then represented in a heatmap, whose colors vary (dexamethasone-R- cyclophosphamide in 19 cases, bortezomib- according to the standardized by row values of the input data. In R-dexamethasone in 7, R-bendamustine in 5 cases). Patients were parallel, a gene set enrichment analysis (GSEA) was performed for untreated at time of bone marrow collection and gave informed several gene sets among them the C2 collection of the MSigDB consent prior to research sampling. No familial form of (Supplementary Tables S1 and S2). The PGSEA package (17) was Waldenstrom€ macroglobulinemia was included. used to calculate z scores for all gene sets, and P values were adjusted for multiple testing (18). Cell selection Research sampling consisted of bone marrow samples and Cytogenetic analysis, FISH, SNPa, and immunophenotypic blood cells for all Waldenstrom€ macroglobulinemia. All samples studies were enriched using immunomagnetic beads (B cell isolation kit Genome-wide detection of copy number alteration (CNA) and for tumoral cells from bone marrow sample, and Pan T cell LOH was performed using the Genome-Wide Human SNP Array isolation kit, Miltenyi-Biotec), following Ficoll-paque gradient 6.0 (Affymetrix). Conventional cytogenetic analysis was per- centrifugation. The purity of samples was confirmed by multi- formed on DSP30 þ IL2 stimulated bone marrow cells. FISH parametre flow cytometry using a marker combination (including was performed to detect chromosomal aberrations: 6q23 dele- CD19, k, l, CD2, CD38, CD138, and CD27). More than 90% a tion, 17p12, 11q22 deletions, 13q14 deletion, trisomy 12,

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trisomy 4 (n ¼ 90; ref. 15). Immunophenotypic expression of We have identified 14 of 53 (26.4%) Waldenstrom€ macroglob- CXCR4 (R et D systems, UK), CD49d, CD27, CD80, CD86, ulinemia with CXCR4 mutations. We then confirmed the þ CD138 gating on CD19 cells was performed along with CD38 observed mutations using NGS using Sanger sequencing (SaS) (Immunotech) and determination of the Matutes's score by flow and extend to the entire cohort of 98 patients with Waldenstrom€ cytometry. The ratio of mean fluorescence intensity (MFIR) was macroglobulinemia (including the 53 done by NGS). We iden- calculated as the ratio of specific fluorescence with an isotype tified CXCR4 mutations (CXCR4mut) for a total of 24 of 98 control. (24.5%), and we have characterized several new mutations across The relationships between the clinical, biologic, and molecular the entire coding exons of CXCR4 (Fig. 1). parameters were determined using a nonparametric test (Mann– Overall, 17 different mutations were identified in CXCR4, Whitney), a t test, a c2, or Fisher exact tests when appropriate. including 12 that we have described for the first time in Correlations were tested through Spearman correlation coeffi- Waldenstrom€ macroglobulinemia. Interestingly, only one type cient. The differences between the results of the comparative tests of CXCR4 mutation was observed in either patient, and all were considered statistically significant if P < 0.05. All statistical mutations identified were located in the 45 amino acid intracy- analyses were done with the SPSS 15.0 software. Survival was toplasmic carboxy-terminal tail. These mutations were never studied using Kaplan–Meier test, comparisons were made with observed in the paired T lymphocytes and thus confirmed their log-rank test somatic feature. All mutations were heterozygous, and no UPDa (acquired uniparental disomy, LOH without variation of copy Results number) was observed at CXCR4 locus nor variation of copy gene number (gain or deletion) in our cohort, using SNP array. New CXCR4 mutations identified in Waldenstrom€ macroglobulinemia We have first used NGS (deep sequencing) to screen the entire High allelic frequency of CXCR4 mutation in Waldenstrom€ sequence of CXCR4 (n ¼ 53 Waldenstrom€ macroglobulinemia macroglobulinemia samples) with a greater sensitivity and to quantify the allelic NGS also allowed the quantification the allelic frequency of the frequency of the variant of CXCR4 mutations in B selected cells. variant of CXCR4 mutations. This quantification allows to

NH2

Extracellular domain

Transmembrane domain

Intracellular domain 315 322 318 Number Type of mutaon T Nucleode change Amino acid change R of paents CXCR4 1 Frameshi c.945_946ins C H315fs L 1 Frameshi c.952_953ins A T318fs* 326 1 Frameshi c.953_954delC T318fs T 327 1 Frameshi c.963-_964insC R322fs 1 Frameshi c.977_978ins C L326fs I 328 1 Frameshi c.979_985delAGATCCT K327fs 336 1 Frameshi c.982_983.del AT I328fs R G 1 Nonsense c.1000C

Figure 1. Somatic mutations in C terminus of CXCR4 identiﬁed by Sanger sequencing and targeted NGS in patients with Waldenstrom€ 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 schematic structure of CXCR4, sites of mutation are highlighted in blue (frameshift mutation) or gray (nonsense mutation). The S338 hotspot was represented in black.

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conclude for the percentage of mutation in the clonal population globulinemia tumor cells using flow cytometry. CXCR4 was for a given patient and thus to differentiate between clonal expressed in all cases (n ¼ 53), including the 12 CXCR4mut (dominant clonal) and subclonal. The mutation load of CXCR4 patients. The pattern varied widely using the MFIR to evaluate varied from 13.5% to 47.58% (mean, 35.2%) using the VAF (n ¼ the CXCR4 expression (MFI varied from 4.6 to 148.7). However, 14, CXCR4mut Waldenstrom€ macroglobulinemia in our series). there was a significant greater expression of CXCR4 protein in Interestingly, for 12 patients with Waldenstrom€ macroglobuline- Waldenstrom€ macroglobulinemia with CXCR4mut (P ¼ mia, the high VAF of CXCR4 mutation was suggested to be present 0.003; Fig. 3). This increased expression was observed indepen- in the dominant clone. However, we were also able to identify the dently of the type of mutation (data not shown). presence of CXCR4 mutations at the subclonal level in 4 patients mut using NGS, and for these 4 patients, the VAF varied from 13.5% to Phenotypic signature associated to CXCR4 genotype 26% (Fig. 2). CXCR4 directly interacts with CD49d in response to CXCL12 signaling in regulating migration and adhesion of Waldenstrom€ NS FS Nonsense (CXCR4 ) and frameshift (CXCR4 ) CXCR4 macroglobulinemia cells to the bone marrow microenvironment mutations lead to truncation of CXCR4 C-terminal (10). CD49d favors lymphocyte homing by cooperating with Among the CXCR4 mutations observed, 11 of 24 (45.8%) and CXCR4 in stromal cell adhesion and extracellular matrix (9). We NS 13 of 24 (54.2%) were nonsense (CXCR4 ) and frameshift then studied the impact of CXCR4 mutation on the expression of FS (CXCR4 ) mutations, respectively. The most frequent mutation the integrin VLA4 (CD49d) using flow cytometry (n ¼ 53). CD49d was the C1013G (S338X) mutation (5 of 98, 5.1%) followed by was expressed in all Waldenstrom€ macroglobulinemia cases; but C1013A (S338X; 3 of 98, 3%). The modelization of C1013G/ no difference in CD49d expression was observed according to CXCR4 and C1013A/CXCR4 variants predicts a stop codon in CXCR4 mutation. In contrast, a higher expression of CD49d was place of a serine at amino acid position 338 (S338X). We also observed in Waldenstrom€ macroglobulinemia with MYD88 found several other mutations to S338, suggesting a hotspot locus L265P mutation (P ¼ 0.048; Fig. 3). Similarly, we found no in CXCR4 gene. The S338 hotspot mutation was targeted in 50% difference in CD38 expression according to CXCR4 mutation, of mutated cases in Waldenstrom€ macroglobulinemia. Other despite of the known functional link between CXCR4, CD49d, CXCR4NS mutations were here described. Interestingly, the exact and CD38 (20). CD138 mediates cell adhesion to the extracellular same acquired R334X mutation in Waldenstrom€ macroglobuli- matrix and is a marker of plasmacytic differentiation (21). Inter- nemia was also previously described in the constitutional WHIM estingly, we found a significant lower expression of CD138 mut syndrome (19). (syndecan1) in the CXCR4 group (P ¼ 0.034). We found no difference in the CD27, CD23, CD10, K/L expression and the CXCR4mut CXCR4wild Increased expression of CXCR4 in Waldenstrom€ Matutes' score between and subgroups. mut macroglobulinemia with CXCR4 mut Because we observed that all mutations were located in the C- Genomic signature of CXCR4 Waldenstrom€ terminal tail of CXCR4 and that this C-terminal domain plays a macroglobulinemia critical role in the desensitization process that contributes to the We then sought to study whether a specific high-throughput mut regulation of the signal transduction and CXCR4 expression (2); SNPa signature was associated to CXCR4 Waldenstrom€ mac- we then sought to study the impact of CXCR4 mutation on the roglobulinemia in a series of 53 Waldenstrom€ macroglobuline- mut expression of CXCR4 on the membrane of Waldenstrom€ macro- mia including 12 CXCR4 . For this analysis, we have considered

100.00

80.00

60.00 Figure 2. Representation of VAF of CXCR4 and MYD88 mutation in each patient 40.00 using NGS. Each row represents a patient (x-axis). VAF of MYD88 L265P mutation is represented in light gray, CXCR4 VAF in black. Percentage 20.00 indicates allele frequencies determined by NGS corrected by the percentage of tumoral cells in the B selected samples 0.00 in each 53 patients (y-axis). , subclonal CXCR4 mutation in patients with Waldenstrom€ macroglobulinemia.

Variant allelicfrequency of CXCR4 variant

Variant allelicfrequency of MYD88L265P variant

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A B P = ns 150.00 P = 0.003 60.00

100.00 40.00

50.00 20.00 Expression of CXCR4 (MFIR) Expression of CD49d (MFIR) 0.00 0.00 CXCR4 CXCR4 CXCR4 CXCR4 wild mutated wild mutated C D P = ns P 150.00 = 0.048 60.00

100.00 40.00

50.00 20.00 Expression of CD49d (MFIR) Expression of CXCR4 (MFIR) 0.00 0.00 MYD88 MYD88 MYD88 MYD88 wild L265P wild L265P

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, not signiﬁcant.

"presence of one" as any genomic abnormality including gain, Mutational landscape of CXCR4mut Waldenstrom€ loss, and/or copy number without LOH (CN-LOH), identified macroglobulinemia using SNPa. In this first analysis, we have compared presence We next attempted to delineate the mutational spectrum and mut versus absence of genomic abnormality according to presence of intratumoral heterogeneity related to presence of CXCR4 for mut CXCR4 mutation. We found a relationship between CXCR4 genes involved in the RAS (this pathway may be activated by and a greater frequency of genomic aberrations in Waldenstrom€ CXCR4/protein G proteins), BCR and TLR pathways (MYD88 macroglobulinemia compared with CXCR4wild (91% vs. 68%, P ¼ exon 5), known to be interconnected with CXCR4 pathway, using 0.010). targeted NGS (6). We then evaluated the number of genomic abnormality as a When looking at the incidence rate of the CXCR4 mutations reflection of the genomic complexity on the basis of the number of with presence of MYD88 L265P mutation in the cohort of 98 mut SNP abnormalities. We found that CXCR4-mutated Waldenstrom€ patients, it appeared that 23 of 24 (95.8%) of the CXCR4 were macroglobulinemia had a greater mean number of abnormality MYD88 L265P mutated, except one Waldenstrom€ macroglobu- FS (5.8 vs. 2.8 per patient, P ¼ 0.046). No significant difference was linemia with a CXCR4 mutation. The high correlation between observed between Waldenstrom€ macroglobulinemia with clonal MYD88 L265P and CXCR4 mutational loads tends to suggest the or subclonal CXCR4 mutation. coexistence of the 2 mutations in the same clone (Fig. 2). How- Furthermore, we analyzed the relationship between CXCR4 ever, in four patients with Waldenstrom€ macroglobulinemia, a mutation and certain type of SNP aberration. We have observed lower VAF of CXCR4 in comparison to MYD88 L265P suggested a mut that Waldenstrom€ macroglobulinemia with CXCR4 had a subclonal CXCR4 mutation in the dominant clone harboring greater incidence rate of trisomy 4 (complete or partial), 58% MYD88 mutation. versus 12% respectively (P ¼ 0.002); a greater frequency of gain of Regarding the presence of mutations in the RAS pathway in Xq, including MYD88 pathway–based IRAK1 gene, and of dele- Waldenstrom€ macroglobulinemia, including PTEN, K-Ras, and tion 8p (P ¼ 0.002 and P ¼ 0.007, respectively; Fig. 4). Finally, we N-Ras, crucial regulators of RAS pathway, we have not identified also found a greater frequency of deletion 6q, including the NF-kB any mutation in our cohort (n ¼ 53). pathway–based TNFAIP3 gene (P ¼ 0.038). We found no rela- BCR pathway can be constitutively activated, either by signals tionship to deletion 7q, deletion 11q, deletion 17p, deletion from the microenvironment or by genetic aberration (22). Several 13q14, trisomy 18 in our study. No difference of SNPa feature mutations of key signaling molecules and regulators of the BCR was observed according to the type of CXCR4 mutation (nonsense pathway were reported overtime, particularly in the activated B- NS FS (CXCR4 ) versus frameshift (CXCR4 ) mutations. cell–like (ABC) diffuse large B-cell lymphoma (DLBCL) and in

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mut pared with the two CXCR4 Waldenstrom€ macroglobulinemia (Fig. 5 and Supplementary Tables S1 and S2). We then sought to identify deregulated pathways using Ingenuity analysis software. We found CXCR4mut to be related to several functional network networks such as cell cycle and DNA replication or cell-to-cell signaling and interaction, cellular growth, and proliferation. Using GSEA for targeted gene sets previously associated with CXCR4 mutation (12), we also identiﬁed deregulation of protea- mut some pathway and BCR/B lymphocyte pathway in CXCR4 Waldenstrom€ macroglobulinemia (P ¼ 0.01 and P ¼ 0.04, respectively; Supplementary Table S1).

mut Clinical and biologic features of CXCR4 We thought to identify clinical–biologic characteristics of mut Waldenstrom€ macroglobulinemia according to CXCR4 features. The median age was 67 years (range, 36–92 years) in our mut series. We found that CXCR4 was associated to a higher IgM monoclonal component (r ¼ 0.309; P ¼ 0.006) and thrombocytopenia (r ¼0.226; P ¼ 0.048), markers of adverse prognosis in Waldenstrom€ macroglobulinemia. Indeed, 35% and 12% of patients had serum IgM level greater than 30 g/L (P ¼ 0.023) and Figure 4. 27% and 6.5% of patients had low platelet count lower than 100 Association network of genomic alterations in Waldenstrom€ g/L (P ¼ 0.018), respectively, in patients with CXCR4mut versus CXCR4 macroglobulinemia (WM). The heatmap represents co-occurrence of CXCR4wild. There was a trend for CXCR4mut patients to have more mutation with other genetic alterations analyzed in 53 patients with WM using € P ¼ SNPa, cytogenetic, and FISH. The co-occurrence of CXCR4 mutation with often IPSS Waldenstrom macroglobulinemia stage 3 ( 0.086). other genetic alterations was represented using MeV software (TM4 No association was found with regard to other markers of tumoral microarray software suite; refs. 31, 32). Each row corresponds to key locus or syndrome, such as adenopathy and splenomegaly, extramedul- regulator genes of the canonical and noncanonical NF-kB pathways targeted lary localization, or lymphocytosis (one would consider a marker by CNA, UPD, or mutation in WM. The columns represent individual patients of egression of tumoral cells from the bone marrow; Supplemen- color coded on the base of gene status. Each patient is represented by a tary Table S3). With a median follow-up of 6 years, 21 (22%) virtual column (black, mutation; CN LOH, deletion or gain of gene; gray, fi wild-type). patients in our series had died with a median OS (95% con dence interval) not reached and estimated at 63% at 10 years. No significant difference in type of treatment or number of treatment Waldenstrom€ macroglobulinemia (15, 23), including the immu- line was observed (data not shown). The estimated overall sur- noreceptor tyrosine–based activation motif (ITAM) signaling vival (OS) at 10 years according to CXCR4mut status was lower for modules of the CD79A and CD79B BCR coreceptors and the CXCR4mut patients, 50% versus 65.5% for patients with coiled-coil domain of CARD11 (22). Mutations of CD79B and CXCR4wild (P ¼ ns, Fig. 6). Interestingly, the difference in OS CD79A were observed in 12 of 98 (12.2%) of Waldenstrom€ between CXCR4mut versus CXCR4wild became significant in macroglobulinemia. Interestingly, we found no coexistence of mut patients with indolent Waldenstrom€ macroglobulinemia (esti- CXCR4 and CD79A/CD79B mutations, but in one patient who mut mated 5-year OS rate at 50% and 93%, respectively, P ¼ 0.019); had CXCR4 at the subclonal level. No mutation of CARD11 was the CXCR4mut patients required therapy more often and much observed in our studied group (n ¼ 53). One might conclude that earlier than the CXCR4wild. there is no unique somatic mutation in the BCR pathway, but instead a wide variety of molecules that could be altered through molecular alterations in the BCR pathway, likely mutually exclu- Discussion mut sive in the same clone, such as CXCR4 and CD79A/CD79B We have described 12 new CXCR4 mutations for a total of 17 mutations in the present situation in our study. patients with Waldenstrom€ macroglobulinemia so far, observed in 25% of patients with Waldenstrom€ macroglobulinemia, using Transcriptional signature related to presence of CXCR4 targeted deep sequencing. These data suggest a highly heteroge- mutation neous pattern of CXCR4 mutations in Waldenstrom€ macroglob- We finally studied the gene expression profile of Waldenstrom€ ulinemia and confirms using a more sensitive technique that was macroglobulinemia on 7 patients with Waldenstrom€ macroglob- reported by Treon and colleagues using Sanger (11). We have also ulinemia to understand whether the CXCR4 mutation impacted confirmed CXCR4 1013C>G, followed by 1013C>A, mutations to the gene expression and to identify a potential gene signature of be the most frequent CXCR4 mutations in Waldenstrom€ macro- mut Waldenstrom€ macroglobulinemia with CXCR4 . The differen- globulinemia, although in a lesser frequency as previously tial analysis allowed to identify a gene signature attached to the reported, 12.5% (for the 2 mutations using Sanger sequencing mut CXCR4 molecular profile, corresponding to 32 probes and 27 in CD19 selected cells) and 30% (only 1013C>A using allele- genes. We found several genes among these, such as MMP8, specific PCR in unselected tumoral samples) in Treon and col- CRISP3, or BRCC3 involved in cell death and survival or cell leagues and Roccaro and colleagues, respectively (11–12). Overall movement (ARG1, LYST). These probes were all underexpressed the high frequency of CXCR4 Whim-like mutation is a new wild in the five CXCR4 Waldenstrom€ macroglobulinemia com- hallmark of Waldenstrom€ macroglobulinemia (12, 24).

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Figure 5. Gene expression proﬁling. Heatmap of normalized expression values for differentially expressed probes. Colors vary according to the standardized by row values of the input data. The scale of the gene expression data is indicated. Some genes of interests are highlighted.

Interestingly, all mutations, either nonsense or frameshift, cooperation between these pathways (11). An important dataset occurred in the C-terminus of CXCR4. It was suggested that the obtained in our series came from the quantification of the allelic truncation of the distal amino acid region of the C-terminus frequency of the variant (VAF) of CXCR4 mutations into tumoral known to regulate signaling of CXCR4 by CXCL12, might dereg- cells using NGS that may identify minor subclones of less than 1% ulate the CXCR4/SDF1 axis signaling pathway, and thus partic- ipate in the pathogenesis of Waldenstrom€ macroglobulinemia (2, 11, 12, 25). One might propose that the type of mutation does not matter much in Waldenstrom€ macroglobulinemia, on the contrary to the loss of C-terminus domain of CXCR4 protein which modified function might play a role in Waldenstrom€ macroglobulinemia pathogenesis (2). Functional studies may confirm this hypothesis in the near future. We found a greater CXCR4 expression on tumor cells of Waldenstrom€ macroglobulinemia carrying CXCR4 mutation, independently of the type of mutation, similar to that was described in WHIM syndrome. It is suggested that the increase of CXCR4 receptor expression would induce an altered migration profile in response to SDF1 (25), resulting in an increased egression of tumor cells as demonstrated in mouse model (12). In our study, we also found a decreased expression of CD138, a cell surface syndecan-1 that mediates cancer cell adhesion to the extracellular matrix, and alteration of expression of several genes involved in matrix degradation such as MMP8 or cell migration on gene expression profiling. Taking together, CXCR4 mutation might alter the ability of the tumor cells to interact with the bone marrow microenvironment in Waldenstrom€ macroglobulinemia, as shown in CXCR4 S338X cells (12). € Figure 6. Nearly all patients with Waldenstrom macroglobulinemia with OS according to presence or absence of CXCR4 mutation in Waldenstrom€ CXCR4 mutations harbored the MYD88 L265P mutation, as macroglobulinemia. The solid line indicates the CXCR4wild patients, the initially described by Treon and colleagues suggesting a potential dashed line CXCR4mut patients. ns, not significant.

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in our study. Targeted deep sequencing allows to conclude for the of Waldenstrom€ macroglobulinemia to determine associated risk percentage of mutations in the clonal population for a given of progression, relapse, and drug resistance. patient and thus to differentiate between clonal (dominant clon- Overall, Waldenstrom€ macroglobulinemia with CXCR4 muta- al) and subclonal mutations. Combining analysis of VAF of tion had a specific clinicobiologic and genomic signature associ- several genes, CXCR4 mutation was expressed in the same clone ated to features characterized with adverse prognosis in as MYD88 L265P, as we have identified a high allelic frequency of Waldenstrom€ macroglobulinemia, including high IgM M-spike CXCR4 mutation MYD88 L265P in Waldenstrom€ macroglobuli- and thrombocytopenia (part of the IPSS Waldenstrom€ macro- nemia tumor cells; but for 4 patients who had CXCR4 mutation globulinemia score adverse features) and greater frequency of present only at the subclonal level. Overall, these data may suggest complex genomic aberrations pattern using SNP array. Our data the following hypothesis, where MYD88 L265P mutation is a therefore may suggest a worse prognosis for CXCR4 mutation founder mutation in Waldenstrom€ macroglobulinemia, a first subgroup of asymptomatic Waldenstrom€ macroglobulinemia, genetic hit that would promote NF-kB and JAK/STAT3 signaling but we did not observe significant impact on overall survival in (4, 26). Direct inhibition of MYD88 L265P signaling overcomes our cohort as previously described (11). Further studies are CXCL12-trigered survival effects in CXCR4-mutated cells, sup- needed to explore the prognosis value of CXCR4 mutation in porting a primary role for MYD88 signaling in Waldenstrom€ Waldenstrom€ macroglobulinemia in clinical trials. Interestingly, macroglobulinemia (25). CXCR4 could then be one of the sec- the potential clinical impact of Waldenstrom€ macroglobulinemia ondary events in some patients who showed subclonal mutation with CXCR4 mutation has already been suggested by Roccaro and of CXCR4 compared with MYD88 L265P mutation present in the colleagues and Treon and colleagues on the basis of their in vitro main clone. However, longitudinal analysis studies are needed to studies showing drug resistance in CXCR4-mutated Waldenstrom€ explore the dynamic of clonal architecture to identify driver or macroglobulinemia cells exposed to BTK, mTOR, and PI3K inhi- additional mutations in Waldenstrom€ macroglobulinemia that bitors but not proteasome inhibitors (29, 30). This study, along could contribute to clinical progression or chemoresistance. with other, thus led us to propose that it is primetime for a Indeed, it was shown that a diminished clinical activity of ibru- systematic evaluation of CXCR4 mutation as part as the pretreat- tinib was observed in patients with Waldenstrom€ macroglobuli- ment package of Waldenstrom€ macroglobulinemia, to identify nemia with CXCR4 mutations (25;30). patients with CXCR4 mutation that might benefit more from In addition, we also found absence of co-occurrence of CD79A/ proteasome inhibitors as compared with other options, such a CD79B and CXCR4 mutations in Waldenstrom€ macroglobuline- BTK inhibitors. mia.These data may suggest that these types of activating mutations In conclusion, approximately 25% of Waldenstrom€ macro- of BCR and CXCR4 pathway were mutually exclusive in MYD88 globulinemia harbor CXCR4 mutation, leading to altered CXCR4 L265P Waldenstrom€ macroglobulinemia (6). A cooperation protein at the C-terminal end, irrespective of the pleiotropic between CXCR4 and TLR signaling activated by MYD88L265P pattern of mutations. The study of CXCR4 mutations showed mutation was described in B cells (6). The functional role of existence of intraclonal (variation in co-expression of MYD88 and mutations in the BCR pathway is not fully described in CXCR4 mutations) and interclonal (BCR and CXCR4 mutations Waldenstrom€ macroglobulinemia, but CD79B mutation was in MYD88L265P Waldenstrom€ macroglobulinemia) heterogene- shown to alter the BCR response in diffuse large B lymphoma ity when analyzing the molecular landscape of CXCR4 mutation (15, 23). We might thus suggest the existence of two subgroups of in Waldenstrom€ macroglobulinemia. Patients with CXCR4 muta- MYD88L265P Waldenstrom€ macroglobulinemia, one withCXCR4 tion displayed a specific clinical–biologic –genomic and transcrip- mutations and one with BCR pathway mutations that may involve tomic signature, possibly related to specific physiopathologic in cooperation with the aforementioned driver mutation which mechanism related to CXCR4 mutation. This dataset further primarily affect TLR signaling. These data further may advance the suggests the genomic heterogeneity of Waldenstrom€ macroglob- concept that a complex rather than a unique deregulation of the ulinemia, beyond the current indolent versus symptomatic TLR/NF-kB pathway characterizes MYD88 L265P Waldenstrom€ classification. macroglobulinemia with others interconnected pathways. We have observed that CXCR4 mutation was more frequently Disclosure of Potential Conflicts of Interest associated to a complex genomic signature, including gain of No potential conflicts of interest were disclosed. chromosome 4, deletion 6q, and gain of chromosome X using SNPa. High-throughput genomic studies have identified multiple Authors' Contributions mechanisms of genetic changes in Waldenstrom€ macroglobuli- Conception and design: S. Poulain, M. Figeac, F. Lepretre, X. Leleu nemia including several recurrent CNA such as deletion 6q, Development of methodology: S. Poulain, A. Venet-Caillault, M. Figeac, deletion 13q, or gain of chromosome 4 and CN-LOH (15, 27). S. Geffroy, X. Leleu This genomic pattern segregates further Waldenstrom€ macroglob- Acquisition of data (provided animals, acquired and managed patients, ulinemia among the others B- cell tumors. The prognostic role of provided facilities, etc.): S. Poulain, C. Roumier, M. Figeac, C. Herbaux, G. Marot, E. Bertrand, A. Decambron, E.M. Boyle, A. Renneville, S. Tricot, these genomic alterations is not fully understood, but the pres- B. Quesnel, X. Leleu ence of more than 3 SNPa abnormalities was associated with Analysis and interpretation of data (e.g., statistical analysis, biostatistics, symptomatic status (15, 27, 28). Taking together, this finding computational analysis): S. Poulain, C. Roumier, M. Figeac, G. Marot, E. Doye, suggests an inter- and intraclonal heterogeneity and the potential B. Quesnel, X. Leleu selective advantage of specific combinations of genetic lesions in Writing, review, and/or revision of the manuscript: S. Poulain, C. Roumier, Waldenstrom€ macroglobulinemia, in particular in the subset of C. Preudhomme, X. Leleu CXCR4mut € Administrative, technical, or material support (i.e., reporting or organizing Waldenstrom macroglobulinemia. The understanding data, constructing databases): S. Poulain, C. Roumier, E. Doye, F. Lepretre, of these genomic abnormalities, including gene mutations and O. Nibourel, B. Quesnel, X. Leleu CNA, described herein might help deciphering the pathogenesis Study supervision: S. Poulain, E. Doye, P. Duthilleul, X. Leleu

OF8 Clin Cancer Res; 2016 Clinical Cancer Research

CXCR4 Mutation in Waldenstrom€ Macroglobulinemia

Acknowledgments The costs of publication of this article were defrayed in part by the The authors thank Pauline Vandycke, Valerie Grandieres, Claudine Delsaut, payment of page charges. This article must therefore be hereby marked advertisement Axelle Seghir, and Sabine Ranwez for their excellent technical assistance. in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant Support This work was supported by the Comite Septentrion de la Ligue contre le Cancer et la Fondation Francaise¸ pour la Recherche contre le Myelome et les Received March 17, 2015; revised August 5, 2015; accepted August 31, 2015; Gammapathies (FFRMG). published OnlineFirst October 21, 2015.

References 1. Hunter ZR, Xu L, Yang G, Zhou Y, Liu X, Cao Y, et al. The genomic landscape 17. Kim SY, Volsky DJ. PAGE: parametric analysis of gene set enrichment. BMC of Waldenstrom€ macroglobulinemia is characterized by highly recurring Bioinformatics 2005;6:144. MYD88 and WHIM-like CXCR4 mutations, and small somatic deletions 18. Hochberg Ba. Controlling the false discovery rate: a practical and associated with B-cell lymphomagenesis. Blood 2014;123:1637–46. powerful approach to multiple testing. J Royal Stat Soc, Series B 57 2. Al Ustwani O, Kurzrock R, Wetzler M. Genetics on a WHIM. Br J Haematol 1995:289–300 2014 Jan;164:15–23. 19. Taniuchi S, Masuda M, Fujii Y, Izawa K, Kanegane H, Kobayashi Y. The role 3. Hernandez PA, Gorlin RJ, Lukens JN, Taniuchi S, Bohinjec J, Francois F, of a mutation of the CXCR4 gene in WHIM syndrome. Haematologica et al. Mutations in the chemokine receptor gene CXCR4 are associated with 2005;90:1271–2. WHIM syndrome, a combined immunodeﬁciency disease. Nat Genet 20. Dal Bo M, Tissino E, Benedetti D, Caldana C, Bomben R, Del Poeta G, et al. 2003;34:70–4. Microenvironmental interactions in chronic lymphocytic leukemia: the 4. Treon SP, Xu L, Yang G, Zhou Y, Liu X, Cao Y, et al. MYD88 L265P somatic master role of CD49d. Semin Hematol 2014;51:168–76. mutation in Waldenstrom's macroglobulinemia. N Engl J Med 2012;367: 21. Teng YH, Aquino RS, Park PW. Molecular functions of syndecan-1 in 826–33. disease. Matrix Biol 2012;31:3–16. 5. Poulain S, Boyle EM, Roumier C, Demarquette H, Wemeau M, Geffroy S, 22. Lim KH, Yang Y, Staudt LM. Pathogenetic importance and therapeutic et al. MYD88 L265P mutation contributes to the diagnosis of Bing Neel implications of NF-kappaB in lymphoid malignancies. Immunol Rev syndrome. Br J Haematol 2014;167:506–13. 2012;246:359–78. 6. Chatterjee S, Behnam Azad B, Nimmagadda S. The intricate role of CXCR4 23. Davis RE, Ngo VN, Lenz G, Tolar P, Young RM, Romesser PB, et al. Chronic in cancer. Adv Cancer Re 2014;124:31–82. active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature 7. Conley-LaComb MK, Saliganan A, Kandagatla P, Chen YQ, Cher ML, Chinni 2010;463:88–92. SR. PTEN loss mediated Akt activation promotes prostate tumor growth and 24. Martinez N, Almaraz C, Vaque JP, Varela I, Derdak S, Beltran S, et al. Whole- metastasis via CXCL12/CXCR4 signaling. Mol Cancer 2013;12:85. exome sequencing in splenic marginal zone lymphoma reveals mutations 8. Nie Y, Waite J, Brewer F, Sunshine MJ, Littman DR, Zou YR. The role of in genes involved in marginal zone differentiation. Leukemia 2014;28: CXCR4 in maintaining peripheral B cell compartments and humoral 1334–40. immunity. J Exp Med 2004;200:1145–56. 25. Cao Y, Hunter ZR, Liu X, Xu L, Yang G, Chen J, et al. CXCR4 WHIM-like 9. Burger JA, Gribben JG. The microenvironment in chronic lymphocytic frameshift and nonsense mutations promote ibrutinib resistance but do leukemia (CLL) and other B cell malignancies: insight into disease biology not supplant MYD88(L265P) -directed survival signalling in Waldenstrom and new targeted therapies. Semin Cancer Bio 2014;24:71–81. macroglobulinaemia cells. Br J Haematol 2015;168:701–7. 10. Ngo HT, Leleu X, Lee J, Jia X, Melhem M, Runnels J, et al. SDF-1/CXCR4 and 26. Wang JQ, Jeelall YS, Beutler B, Horikawa K, Goodnow CC. Consequences of VLA-4 interaction regulates homing in Waldenstrom macroglobulinemia. the recurrent MYD88(L265P) somatic mutation for B cell tolerance. J Exp Blood 2008;112:150–8. Med 2014;211:413–26. 11. Treon SP, Cao Y, Xu L, Yang G, Liu X, Hunter ZR. Somatic mutations in 27. Braggio E, Philipsborn C, Novak A, Hodge L, Ansell S, Fonseca R. Molecular MYD88 and CXCR4 are determinants of clinical presentation and overall pathogenesis of Waldenstrom's macroglobulinemia. Haematologica survival in Waldenstrom macroglobulinemia. Blood 2014;123:2791–6. 2012;97:1281–90. 12. Roccaro AM, Sacco A, Jimenez C, Maiso P, Moschetta M, Mishima Y, et al. 28. Nguyen-Khac F, Lambert J, Chapiro E, Grelier A, Mould S, Barin C, et al. C1013G/CXCR4 acts as a driver mutation of tumor progression and Chromosomal aberrations and their prognostic value in a series of 174 modulator of drug resistance in lymphoplasmacytic lymphoma. Blood untreated patients with Waldenstrom's macroglobulinemia. Haematolo- 2014,123:4120–31 gica 2013;98:649–54. 13. Owen RG, Pratt G, Auer RL, Flatley R, Kyriakou C, Lunn MP, et al. Guide- 29. Roccaro AM, Sacco A, Jimenez C, Maiso P, Moschetta M, Mishima Y, et al. lines on the diagnosis and management of Waldenstrom macroglobuli- C1013G/CXCR4 acts as a driver mutation of tumor progression and naemia. Br J Haemato 2014;165:316–33. modulator of drug resistance in lymphoplasmacytic lymphoma. Blood 14. Dimopoulos MA, Kastritis E, Owen RG, Kyle RA, Landgren O, Morra E, et al. 2014;123:4120–31. Treatment recommendations for patients with Waldenstrom macroglob- 30. Cao Y, Hunter ZR, Liu X, Xu L, Yang G, Chen J, et al. The WHIM-like CXCR4 ulinemia (WM) and related disorders: IWWM-7 consensus. Blood (S338X) somatic mutation activates AKT and ERK, and promotes resistance 2014;124:1404–11. to ibrutinib and other agents used in the treatment of Waldenstrom's 15. Poulain S, Roumier C, Galiegue-Zouitina S, Daudignon A, Herbaux C, Macroglobulinemia. Leukemia 2015;29:169–76. Aiijou R, et al. Genome wide SNP array identiﬁed multiple mechanisms of 31. Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, genetic changes in Waldenstrom macroglobulinemia. Am J Hematol et al. TM4 microarray software suite. Methods Enzymol 2006;411: 2013;88:948–54. 134–93. 16. Poulain S, Roumier C, Decambron A, Renneville A, Herbaux C, Bertrand E, 32. Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, et al. TM4: a free, et al. MYD88 L265P mutation in Waldenstrom macroglobulinemia. Blood open-source system for microarray data management and analysis. Bio- 2013;121:4504–11. techniques 2003;34:374–8.

www.aacrjournals.org Clin Cancer Res; 2016 OF9

Genomic Landscape of CXCR4 Mutations in Waldenström Macroglobulinemia

Stéphanie Poulain, Christophe Roumier, Aurélie Venet-Caillault, et al.

Clin Cancer Res Published OnlineFirst October 21, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-15-0646

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2015/10/21/1078-0432.CCR-15-0646.DC1

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