Somatic Variants Associated with MDS/AML Are Present in MYD88 Mutated Waldenstrom's Macroglobulinemia

Background: The Rapid Heme Panel (RHP) is a targeted amplicon-based custom next-generation sequencing used as a clinical assay to identify mutations in 95 genes related to hematological malignancies that include acute leukemias (AML, ALL), myelodyplastic syndromes (MDS), and B-cell lymphoproliferative disorders. Clonal hematopoiesis of indeterminate potential (CHIP) has recently been shown to be an age-related precursor to MDS when blast or dysplasia criteria are not met for a formal MDS diagnosis. The RHP is used to identify the top somatic mutated genes closely associated with CHIP or MDS, and includes MYD88 and CXCR4 variant testing for Waldenstrom's Macroglobulinemia (WM). Materials and Methods: Sample processing and sequencing of bone marrow (BM) samples from WM patients was used for isolation of genomic DNA for RHP. Amplicons were designed to cover 250bp and 95 genes. Analyses were performed by identifying and separating somatic variants from those seen as germline. Variants with read percentage depth of 50% were presumed to be of germline origin. Variants with read percentages greater than the mutated MYD88 read percentage depth were identified as potential CHIP or MDS clonal variants. These variants were validated using Sanger sequencing using CD19-selected and CD19-depleted BM mononuclear cells. Mutant variants were annotated against Ensembl Variant Effect Predictor (VEP) database. Results: Three-hundred thirty-six WM patients were included in this analysis. The median age for these patients was 66.7 (range 31-93 years). The median BM disease involvement for these patients was 35%. RHP identified the MYD88 L265P mutation in 267 (80%) patients. The remaining patients were either unmutated (n=34), or lacked MYD88 reads (n=35). Therefore the percentage of patients with MYD88 L265P among those with MYD88 reads was 88.7%. CXCR4 C-terminal domain mutations were also observed in 55/267 (20.6%) WM patients with MYD88 L265P mutations. In addition to MYD88 and CXCR4, variants in 66 other genes contained within the RHP profile were identified that included variants in genes associated with MDS/AML in 9 (3.3%) patients. The median age for these patients was 64 (range 44-80 years) and included variants at: EP300^S1400N, a histone acetyltransferase regulating transcription (n=1); TET2^T1270fs, a methylcytosine dioxygenase that converts methylcytosine to 5-hydroxymethylcytosine (n=1); and DNMT3A^V338fs, DNMT3A^Y584fs, DNMT3A^G587fs, and DNMT3A^L754R, a DNA methyltransferase that represses transcription (n=4). Variants in DNMT3B^R97C, DNMT3B^E39Q, and DNMT3B^A384T (n=3). Sanger sequencing of BM mononuclear cells confirmed the somatic, non-CD19 origin for DNMT3A and TET2. Four of 9 patients were previously untreated, and two received alkylator based therapy previously. None exhibited BM morphological evidence of MDS or AML. Conclusions: Infrequently variants related to MDS/AML are present in BM of patients with WM, including previously untreated patients. These variants occur in the absence of BM morphological evidence of MDS, and may be consistent with CHIP.