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Rapid detection of MYD88-L265P mutation by PCR-RFLP in B-cell lymphoproliferative disorders

Accumulating evidence suggests that chronic lymphocytic leukemia (CLL), multiple myeloma (MM) and Waldenstrom’s macroglobulinemia (WM) have been associated with precursor conditions, termed CD5+ monoclonal B-cell lymphocytosis (MBL) for CLL,1 and monoclonal gammopathy of undetermined significance (MGUS) for MM2 and WM.3 However, the outcome for MGUS seems to be different, depending on the class of immunoglobulin expression. Immunoglobulin type G (IgG) and immunoglobulin type A (IgA) MGUS progress to MM, while immunoglobulin type M (IgM) MGUS is considered to be a distinct biological and clinical entity, and has been reported to progress most often to WM.3 However, the point at which MGUS progresses to the above malignancies remains still vague.

Long-term follow-up of patients with IgM-MGUS reveals that they are at increased risk for WM,3 a B-cell lymphoproliferative disorder (BLD) characterized by lymphoplasmacytic cell infiltration in bone marrow (BM) and the presence of IgM monoclonal gammopathy in the serum.4 Although the pathogenesis of WM has not been defined, familial clustering suggests that genetic factors may have a role in the development of the disease.5 Recently, Treon et al.6 revealed a novel recurring mutation (L265P) in the myeloid differentiation primary response 88 (MYD88) gene in patients with WM. Whole-genome sequencing, confirmed by Sanger sequencing, revealed that approximately 90% of patients with WM and 100% with non–IgM-secreting lymphoplasmacytic lymphoma (LPL) carried the mutation.6 On the contrary, MYD88-L265P was absent in the paired normal tissue of the above patients, as well as in 10 patients with MM (including two with IgM-MM), but it was expressed in 7% of patients with marginal-zone lymphoma. Interestingly, it was found that only 2 of 21 patients with IgM-MGUS (10%) were positive for MYD88-L265P using Sanger sequencing, while one of them developed WM.6 Subsequently, the same group, using an allele-specific PCR (AS-PCR) protocol applied in BM samples, detected the mutation in 93% of patients with WM and in 54% of patients with IgM-MGUS,7 while Landgren et al.8 detected the MYD88-L265P mutation in CD19+ BM cells of 5 out of 9 IgM-MGUS patients (56%), by Sanger sequencing. Additional studies confirmed that MYD88-L265P is a marker highly characteristic of WM, but it is also rarely present in other BLD, as splenic lymphoma with villous lymphocytes (SLVL).9, 10

To expand on this topic, a quick and reliable PCR-RFLP experimental protocol was generated to assess the status of MYD88-L265P expression in patients with WM and other LPDs. Twelve (12) patients with WM, 12 patients with B-cell chronic lymphocytic leukemia (CLL) and 8 with SLVL were enrolled in the study. Furthermore, consecutive samples of peripheral blood (PB) and BM isolated CD19+ cells, derived from a patient with monoclonal IgM-MGUS, were also analyzed for the presence of MYD88-L265P mutation. The diagnosis of the enrolled patients was based on standard diagnostic criteria,11 and all patients consented for their participation in the study according to Helsinki declaration.

Genomic DNA was extracted from BM and/or PB using the QIamp DNA blood mini kit (Qiagen, Valencia, CA, USA) and the detection of the L265P mutation was performed by PCR amplification of exon 5 of MYD88 gene, followed by restriction fragment length polymorphism (RFLP) analysis, as the presence of the mutation results in the generation of BsiEI restriction enzyme site. In particular, the forward and reverse primers, designed with the aid of the Oligo-6 software (NBI, Plymouth, MN, USA), were 5′-IndexTermCTGGCAAGAGAATGAGGGAATGT-3′, and 5′-IndexTermAGGAGGCAGGGCAGAAGTA-3′, respectively. For each PCR, a total of 100–200 ng of genomic DNA was amplified in a 30-μl reaction using 62.5 μM of each deoxynucleotide triphosphate, 20 pmol of each primer, 1.5 mM MgCl2 and 0.8 U Taq Polymerase (Bioron, Ludwigshafen, Germany) in a buffer supplied by the manufacturer. The PCR conditions were 2 min at 94 °C followed by 32 cycles of 94 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s and 5 min at 72 °C after the last cycle. A 489 bp fragment was amplified by PCR and subjected to BsiEI digestion (New England Biolabs, Beverly, MA, USA) for 4 h at 37 °C. The mutated allele contains a BsiEI site resulting in 289 bp and 200 bp fragments, whereas the wild-type allele does not (Figure 1). All PCR and digestion procedures were carried out in the PCR engine apparatus PTC-200 (MJ Research, Watertown, MA, USA), and the PCR and digestion products were analyzed in 2% TBE (Tris-borate-EDTA) agarose gels. Additionally, PCR-RFLP results were confirmed by direct sequencing of purified CD19+ cells. The B-cell isolation was performed by magnetic sorting using the Easy Sep Kit on a Robosep instrument (STEMCELL Technologies Inc., Vancouver, Canada), resulting in a >90% B-cell purity (CD19+ cells). The CD19- fraction was used as the normal paired sample.

Figure 1

MYD88-L265P mutation established by PCR-digestion by BsiEI. (a) Serial dilutions of CD19+ cells of a patient with MW to identify the sensitivity of PCR-RFLP protocol. M: 100 bp DNA ladder (New England Biolabs). Lane 1: 100% CD19 cells; lane 2: 100% CD19+ cells; lane 3: 50% CD19+ cells; lane 4: 25% CD19+ cells; lane 5: 12.5% CD19+ cells; lane 6: 6.25% CD19+ cells; lane 7: 3.125 CD19+ cells; lane 8: 1.56% CD19+ cells. (b) Sequence chromatogram of MYD88-L265P mutation. The mutated nucleotide (c.794T>C) is indicated by a red arrow.

The MYD88-L265P mutation was detected in 11 out of 12 patients with WM (91.7%), in 1 out of 8 patients with SLVL (12.5%), while it was absent in all CLL patients. The sensitivity of PCR-RFLP was assessed by PCR analysis of samples obtained by serial dilutions of DNA of CD19+ cells of a WM patient in the DNA of CD19 cells, while the results were further verified by direct sequencing. Interestingly, PCR-RFLP protocol exhibited a greater sensitivity than direct sequencing, when applied to total BM cells (12.5 vs 25%). Moreover, the patient with IgM-MGUS carried the MYD88-L265P mutation in isolated CD19+ cells of both BM and PB in all consecutive samples and remained in a stable condition for the last 7 years.

The discovery of MYD88-L265P mutation seems to be a breakthrough in the evaluation of BLD, bearing in mind the difficulty of the diagnosis due to overlapping morphologic, immunophenotypic, cytogenetic and clinical features of these disorders.6, 11 In this study, we reported the presence of the MYD88-L265P mutation in 91.7% of patients with WM analyzed with PCR-RFLP. Similarly with previous studies, only 12.5% of our SLVL patients and none of the CLL samples carried the L265P mutation.9, 10 These findings further support the notion that MYD88-L265P mutation is probably a driver mutation which could give a competitive growth advantage to an early, possibly WM-bound, clone.6, 7 Therefore, the detection of the L265 mutation may be a useful tool in order to differentiate WM from other BLD. The described herein PCR-RFLP protocol is rapid and inexpensive, as it does not require special equipment, and its results can be obtained at the latest within 1–2 days. Moreover, it has a slightly increased sensitivity than direct sequencing when applied to total BM cells, possibly due to inherent problems of the fluorescence-based DNA sequencing.12, 13

It is interesting that we detected the MYD88-L265P mutation in a patient with IgM-MGUS, both in the isolated CD19+ cells of the BM and in the PB. The presence of the mutation, in the absence of disease progression for many years, suggests that this genetic defect alone may not be sufficient to sustain overt neoplastic disease.

In conclusion, this study presents a new method for the rapid and reliable detection of MYD88-L265P mutation in every day clinical practice, thus facilitating the validation of its possible diagnostic and prognostic value in patients with BLD.


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Correspondence to M Speletas.

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Argentou, N., Vassilopoulos, G., Ioannou, M. et al. Rapid detection of MYD88-L265P mutation by PCR-RFLP in B-cell lymphoproliferative disorders. Leukemia 28, 447–449 (2014).

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