Clonotypic Analysis Of Immunoglobulin Heavy Chain (IgH) Sequences In Patients With Waldenström’s Macroglobulinemia. Correlation With MYD88 L265P Somatic Mutation Status, Clinical Features and Outcome

Waldenström’s Macroglobulinemia (WM) cells express a unique clonotypic rearrangement of heavy chain immunoglobulin (IgH) genes in each individual patient. Recent studies demonstrate high frequency of the MYD88 L265P somatic mutation in patients with WM, implying a new pathway for the pathogenesis of this disease. In this study we characterized clonal IgH genes rearrangements, IGHV gene use, somatic hypemutations (SHM) and presence of the ΜΥD88 L265P mutation in a cohort of WM patients and we investigated any eventual correlation with patients’ clinical features.

36 patients with WM were studied (median age 64y, 19 males and 17 females, symptomatic 72%, asymptomatic 28%) of whom 45%, 39% and 16% were stage IPSSWM 1, 2 and 3, respectively. Median time to treatment initiation was 13 months and median overall survival was 61 months. DNA was extracted from patients’ bone marrow/blood samples and clonal IgH VDJ rearrangements were amplified by PCR using the Biomed-2 Concerted Action protocol. Monoclonal VDJ rearranged PCR fragments were sequenced and analyzed using the IMGT database (www.imgt.org). Thirty-one patients’ DNA samples were also analyzed for the presence of the ΜΥD88 L265P mutation by using a mutation specific PCR assay.

The most frequent clonal rearrangements observed in our patients were in the IGVH3 family (73% of cases); the IGVH3-23 gene and the IGVH3-74 gene were used in 24.4% and 18,9% of cases, respectively. Somatic hypermutation (>2%) was seen in all but three cases (92%). Mean percentage of mutations in all cases, IGVH3 family, IGVH3-23 and IGVH3-74 genes was 7,5%, 8%, 9,4% and 7,5%, respectively. Sixty-two percent of the patients were positive for the ΜΥD88 L265P mutation and notably all three unmutated WM cases were negative for this MYD88 point mutation. No correlation between VH or DH gene usage, CDR3 length, and mutational status with clinical parameters, time to treatment initiation, or survival was found.

WM IgH genes repertoire, as expected, differs from that observed in normal B-cells and other B-cell diseases such as MZL, MCL and B-CLL/SLL. In addition to the known hyper-representation of the IGVH3-23 gene, another member of the VH3 family, the IGVH3-74 gene is also over-represented in WM. The high IGVH mutation rate supports a derivation of WM cells from postgerminal center memory B cells in the majority (92%) of WM patients. However, the identification of a minority of WM patients (3 of 36) with unmutated IGHV gene segments, negative for the ΜΥD88 L265P mutation, supports the hypothesis that they represent a subgroup of WM not arising from post-germinal B cells with a different disease pathogenesis.

No relevant conflicts of interest to declare.