Genomic Comparison Of Clonal B-Cells In Waldenstrom Macroglobulinemia (WM) Versus IgM MGUS

It is hypothesized that similarly to multiple myeloma, also in WM there may be a continuum between IgM MGUS, smoldering (SWM) and symptomatic WM, rather than these entities being considered as separate. The very low frequency of MYD88 L265P initially reported in IgM MGUS suggested that this could be implicated in disease transformation. However, using sensitive ASO-PCR a significant proportion of patients of patients with IgM MGUS already harbors the MYD88 mutation. Thus, the molecular mechanisms driving the malignant transformation of WM remain largely unknown.

Here, we used high-sensitive 8-color multidimensional flow cytometry (MFC) to detect and sort the specific B-cell clone in BM samples (N=31) from a total of 22 newly-diagnosed WM patients (8 symptomatic, 14 SWM) as well as 9 patients with IgM MGUS. The later 9 cases had negative BM biopsy, but light-chain restricted clonal B-cells (typically CD22^low, CD25⁺, sIgM⁺, LAIR1^-) were identified by MFC (median 1.74%, range 0.2%-7.04%). MYD88 L265P was detected on FACS-sorted (purity ≥97%) clonal B-cells from 9/9, 13/14 and 7/8 IgM MGUS, SWM and WM patients, respectively.

We first compared the genomic profile of clonal B-cells through high density Cytoscan750K array. Overall, IgM MGUS, SWM and WM patients showed a median of 2, 1.5, and 3 copy number abnormalities (CNA)/case, respectively [defined by >25 consecutive imbalanced markers/segment, >100Kb genomic size and <50% overlapping variants with patient-paired control DNA (n=6), or unpaired DNA from BM normal B-cells from 20 healthy donors]. Whole chromosomal imbalances were detected in IgM MGUS (+18), SWM (+3, +12) and WM patients (+4, +12, +18, +19). Gain and deletion of chromosomal arms was also detected in the 3 disease stages: 3q+, 6q-, 8p-, 13q-, 17p-, 18q+ in IgM MGUS; 11q- in SWM; and 6q-, 17p-, 18q+, 22q- in WM. Thus, genomic imbalances typically observed in WM (3q+,6q- or 18q+) were already detectable in clonal B-cells from IgM MGUS patients. Trisomy 4 was not present, nor CNA at 4q33-34 (previously ascribed with increased susceptibility for IgM MGUS and WM). One minimal amplified region at 8q11.23 was noted in 6 of the 31 patients (19%). Median number of copy-number-neutral loss of heterozygosity (CNN-LOH) was also similar between IgM MGUS, SWM and WM (median of 3, 2, and 3 CNN-LOH/case, respectively). Of note, two IgM MGUS patients showed CNN-LOH in minimal deleted regions often detected in the aggressive forms of the disease such as 6q16.1and 6q25.3.

In accordance to the genomic profiles, preliminary analysis of gene expression profiles (GEP; HumanGene 1.0ST) between FACS-sorted clonal B-cells from IgM MGUS, SWM and WM patients showed virtually no deregulated genes (SAM Excel add-in with a FDR q-value<10^-5). Consequently, we grouped patients together (n=14) and compared them to normal BM B-cells from healthy donors. Moreover, taking into consideration the aberrant phenotypes of the Waldenström’s clone, a specific comparison was made between the GEP of clonal B-cells vs CD22⁺/CD25^- normal B-cells (n=6) as well as the small subset of normal BM B-cells that display the typical CD22^low/CD25⁺ WM phenotype (n=4). Clonal B-cells showed de-regulation of 776 genes (92 down- and 684 up-regulated) as compared to CD22⁺/CD25^- normal B-cells. By enrichment analysis (Ingenuity Pathways), top upstream regulators such as IFNg, the B-cell receptor (BCR) complex, and the synovial apoptosis inhibitor 1 (SYVN1) were activated in clonal B-cells, while the IL1 receptor antagonist (IL1RN) was inhibited. Well-known genes such as PRDM1, CD27, IL2Rα (CD25) or TRAF3 were also up-regulated in clonal B-cells. Noteworthy, up to 27 genes over-expressed by clonal B-cells were already up-regulated in normal BM CD22^low/CD25⁺ B-cells vs CD22⁺/CD25^- normal B-cells. Accordingly, when compared to the CD22^low/CD25⁺ normal B-cell counterpart, GEP of clonal B-cells was far less deregulated (185 genes being infra-expressed). In fact, genes such as IL1R2, TLR4, TNFRSF1A, IGF1R, FCER1G or TNFSF13B (target molecules of the NFKB and IL-6 pathways) were down-regulated in the WM clone vs CD22^low/CD25⁺ normal B-cells.

In summary, our results show that clonal B-cells from IgM MGUS patients already show a molecular profile that overlaps with that of WM, and suggest that the Waldenström’s clone may arise from normal CD22^low/CD25⁺ BM B-cells.