Genomic Landscape of CXCR4 Mutations in Waldenstrom's Macroglobulinemia

Background. Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4^mut) as the most prevalent somatic mutations in Waldenstrom’s macroglobulinemia (WM). Interestingly, while MYD88 L265P mutation may be considered as a founder event because of it high frequency in WM. CXCR4 is a G-protein-coupled receptor that promotes migration and activation of several pathways including RAS, Akt and NFKB. CXCR4 S338X may have a prognostic impact with induction of mechanisms of resistance. Our aim was to screen CXCR4 mutation in a large cohort of WM at diagnosis and in the relapse setting, and to analyze the genomic landscape of WM using targeted next generation sequencing (NGS) and genome wide single nucleotide polymorphism array (SNPa).

Method. BM samples of 96 WM (mean age: 68 years) were analyzed. Tumoral DNA was extracted following CD19 B cell selection. Paired samples (tumor/T lymphocytes) were used as an intra-individual reference. In 22 pts, sequential tumor samples collected >6 months apart were analyzed. CXCR4 mutation was analyzed by sanger sequencing (SaS) and/or targeted NGS, along with MYD88L265P, CD79A, CD79B, N RAS, K RAS, PTEN, NOTCH1(exon34). Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was performed in 52 pts, and analyzed with UCSC Genome Browser HG18 assembly. CN-LOH (copy neutral- loss of heterozygosity) and CNA (copy number aberration) were mapped using console 3.02 software (Affymetrix). Flow cytometry was performed to assess CXCR4 and CD49d (VLA4) expression.

Results. We have first identified all CXCR4 mutations to be located in the C-terminal domain using NGS at a mean depth of 2000X to scan the coding exons of CXCR4 in a first group of 34 WM. We have next screened the C-terminal domain of CXCR4 in 96 WM using SaS; and confirmed CXCR4 mutations in 24% of WM (CXCR4^mut). Among the CXCR4 mutations observed, 10 and 13 were non-sense (CXCR4^NS) and frameshift mutations (CXCR4^FS), respectively. All mutations were heterozygous and led to a truncated receptor protein. The most frequent mutation was the C1013G (S338X) mutation (5/96) followed by C1013A (S338X) (4/96). No CN-LOH was observed at CXCR4 locus nor variation of copy gene number (gain or deletion) in our cohort using SNP array. Interestingly, CXCR4^mutwas associated to higher expression of CXCR4 protein by flow cytometry, independently of the type of CXCR4 mutation (n=45) (p=0.03); with no impact on the expression profile of the integrin VLA4 (CD49d) which directly interacts with CXCR4.

Finally we attempted to delineate the genomic signature related to presence of CXCR4^mut using targeted NGS for genes involved in RAS pathway (PTEN, K Ras, NRAS), TLR pathway (MYD88) and BCR pathway (CD79A, CD79b, CARD11). All CXCR4^mut had MYD88L265P mutation except one case. We have identified no mutational signature in RAS pathway related to CXCR4^mut. We then sought to understand whether the mutational profile might change overtime in WM using longitudinal samples. Variation of variant allele frequency of CXCR4 was observed in 3/22 cases. Interestingly, we have found that loss of CXCR4 mutation was concomitant to progression of BCR mutant clone, suggesting occurrence of CXCR4 mutation in a different clone from BCR pathway mutation and may be mutually exclusive to mutations in the BCR pathway in WM. Finally, the CXCR4^mutWM are characterized by a higher frequency of gain of chromosome 4 (p<0.01), deletion of 6q (p<0.05) and gain of chromosome X (p <0.05).

Conclusion. Our data has confirmed the high frequency of CXCR4 mutation in WM. We identified a genomic signature associated to their presence. Interestingly we found presence of CXCR4 and BCR pathway mutations to be mutually exclusive and to participate to clonal evolution over time. Although this remains to be validated in a larger cohort, it seems that WM is characterized with multiple alterations of molecules pertaining to several pathway leading to NFkB deregulation in WM rather than to a unique deregulation of the signaling pathway.