Clonal architecture of CXCR4 WHIM-like mutations in Waldenström Macroglobulinaemia

Abstract

CXCR4(WHIM) somatic mutations are distinctive to Waldenström Macroglobulinaemia (WM), and impact disease presentation and treatment outcome. The clonal architecture of CXCR4(WHIM) mutations remains to be delineated. We developed highly sensitive allele-specific polymerase chain reaction (AS-PCR) assays for detecting the most common CXCR4(WHIM) mutations (CXCR4(S338X C>A and C>G) ) in WM. The AS-PCR assays detected CXCR4(S338X) mutations in WM and IgM monoclonal gammopathy of unknown significance (MGUS) patients not revealed by Sanger sequencing. By combined AS-PCR and Sanger sequencing, CXCR4(WHIM) mutations were identified in 44/102 (43%), 21/62 (34%), 2/12 (17%) and 1/20 (5%) untreated WM, previously treated WM, IgM MGUS and marginal zone lymphoma patients, respectively, but no chronic lymphocytic leukaemia, multiple myeloma, non-IgM MGUS patients or healthy donors. Cancer cell fraction analysis in WM and IgM MGUS patients showed CXCR4(S338X) mutations were primarily subclonal, with highly variable clonal distribution (median 35·1%, range 1·2-97·5%). Combined AS-PCR and Sanger sequencing revealed multiple CXCR4(WHIM) mutations in many individual WM patients, including homozygous and compound heterozygous mutations validated by deep RNA sequencing. The findings show that CXCR4(WHIM) mutations are more common in WM than previously revealed, and are primarily subclonal, supporting their acquisition after MYD88(L265P) in WM oncogenesis. The presence of multiple CXCR4(WHIM) mutations within individual WM patients may be indicative of targeted CXCR4 genomic instability.

Keywords: CXCR4; IgM MGUS; MYD88 L265P; Marginal Zone Lymphoma; WHIM; Waldenström macroglobulinaemia.

Fig 1

Sanger tracings from CD19-selected cells derived from bone marrow aspirates of untreated WM patients showing compound heterozygous and homozygous CXCR4^WHIM mutations. (A) Sanger tracings showed the presence of multiple CXCR4^WHIM mutations in 3 Waldenström Macroglobulinaemia (WM) patients (WM1, WM2, WM3), and suggestive of homozygous CXCR4^WHIM mutations in another 3 WM patients (WM4, WM5, WM6). The estimated percentage of mutant and wild-type (WT) allele burden for MYD88 and CXCR4 are shown. (B) Representative Sanger tracings from cloning and sequencing studies for the 3 WM patients (WM1, WM2, WM3) with multiple CXCR4^WHIM mutations are shown, with mutant alleles shown in shaded areas. TA cloning and sequencing studies showed 3/44 and 12/44 clones to express CXCR4^{G332 fs} and CXCR4^S338X, respectively for WM1; 12/43 and 6/43 clones to express CXCR4^K333X and CXCR4^S338X, respectively for WM2; and 2/40 and 8/40 clones to express CXCR4^S338X and CXCR4^{S338 fs}, in WM3, respectively.

Fig 1

Sanger tracings from CD19-selected cells derived from bone marrow aspirates of untreated WM patients showing compound heterozygous and homozygous CXCR4^WHIM mutations. (A) Sanger tracings showed the presence of multiple CXCR4^WHIM mutations in 3 Waldenström Macroglobulinaemia (WM) patients (WM1, WM2, WM3), and suggestive of homozygous CXCR4^WHIM mutations in another 3 WM patients (WM4, WM5, WM6). The estimated percentage of mutant and wild-type (WT) allele burden for MYD88 and CXCR4 are shown. (B) Representative Sanger tracings from cloning and sequencing studies for the 3 WM patients (WM1, WM2, WM3) with multiple CXCR4^WHIM mutations are shown, with mutant alleles shown in shaded areas. TA cloning and sequencing studies showed 3/44 and 12/44 clones to express CXCR4^{G332 fs} and CXCR4^S338X, respectively for WM1; 12/43 and 6/43 clones to express CXCR4^K333X and CXCR4^S338X, respectively for WM2; and 2/40 and 8/40 clones to express CXCR4^S338X and CXCR4^{S338 fs}, in WM3, respectively.

Fig 2

Real-time AS-PCR results for CXCR4^{S338X C>A} or CXCR4^{S338X C>G} variants in samples from healthy donors, IgM MGUS, non-IgM MGUS, untreated and previously treated WM, CLL, MZL and MM patients. Violin plot representing allele-specific polymerase chain reaction (AS-PCR) differences in cycle threshold (ΔC_T) for samples evaluated for CXCR4^{S338X C>A} (A) and CXCR4^{S338X C>G} (B) variants. The span of grey area for each cohort represents the kernel density estimation of the sample distribution, and highlights the bimodal nature of the data. Box plots with interquartile ranges are shown in black with an overlay of the individual data points. Samples evaluated were from health donors (n = 32), as well as immunoglobulin M monoclonal gammopathy of unknown significance (IgM MGUS; n = 12), non-IgM MGUS (n = 7), untreated Waldenström Macroglobulinaemia (WM) (n = 102), previously treated WM (N = 62), marginal zone lymphoma (MZL; n = 20), chronic lymphocytic leukaemia (CLL; n = 32) and multiple myeloma (MM; n = 14) patients. Gray lines denote Δct cut-off values established for each AS-PCR assay.

Fig 3

Sanger tracings in CD19-selected cells from bone marrow aspirates of patients with CXCR4^WT and who demonstrated CXCR4^S338X mutations by allele-specific polymerase chain reaction assays. Sanger tracings show four patients with CXCR4^WT who demonstrated CXCR4^{S338X C>A} (WM1, WM2) and CXCR4^{S338X C>G} (WM3, WM4). Sanger tracings for two patients with CXCR4^{S338X C>A} (WM5) and CXCR4^{S338X C>G} (WM6) are shown for comparison.

Fig 4

Cancer cell fraction analysis for CXCR4^S338X expression in CD19-selected cells from bone marrow aspirates of WM patients. The clonal penetrance of CXCR4^S338X mutations in Waldenström Macroglobulinaemia (WM) was determined in 21 untreated WM and 2 immunoglobulin M monoclonal gammopathy of unknown significance (IgM MGUS) patients with known CXCR4^S338X and MYD88^L265P mutations. The cancer cell fraction for CXCR4^{S338X C>A and/or C>G} expression relative to MYD88^L265P is shown. CXCR4^{S338X C>G} was expressed in patients WM1-WM20, and IgM MGUS 1 and 2; CXCR4^{S338X C>A} was expressed in patients WM14–21, with both CXCR4^{S338X C>G and C>A} present in WM14–20.