Waldenström's macroglobulinemia (WM) is a rare B-cell disorder characterized by the accumulation of monoclonal lymphoplasmocytic cells in bone marrow in association with a serum monoclonal immunoglobulin (Ig)M protein. Patients may present with organomegaly, anemia, hyperviscosity, cryoglobulinemia, amyloidosis and autoimmune disease.
The genetic basis of this disorder is poorly understood. Cytogenetic abnormalities differ from those commonly reported in other B-cell neoplasms. Translocations involving immunoglobulin heavy-chain gene (IgH) are a common genetic change in multiple myeloma (MM) (75%) and a recurrent abnormality in many B-cell lymphomas such as t(11;14)(q123;q32) in mantle cell lymphoma, t(14;18)(q32;q21) in follicular lymphoma and t(9;14)(p13;q32) in lymphoplasmocytic lymphoma. On the other hand, the incidence of IgH rearrangements is low in WM (<3%).1, 2 The translocation t(11;18)(q21;q21), a specific abnormality, observed in about 20% of extranodal marginal zone lymphomas has been reported in few cases of patients with WM.3, 4 However, this translocation was not detected in the tumor cells of 24 WM patients using well-validated probe for its detection.1 Deletions of chromosome 13q14 are present in approximately 50% of patients with MM or B-cell chronic lymphocytic leukemia (B-CLL), but are rare in WM at the time of diagnosis (<5%) and with a low frequency in patients after treatment (15%).5 The deletion of the long arm of chromosome 6 is a recurrent abnormality reported in about 50%, in cases of WM mainly in advanced disease and is frequently associated with clonal evolutions (1Schop et al. Blood, 2005; 106: 290; abstract).
Limited studies are available with regards to the ploidy status in WM. DNA content studies have shown that the clonal cells are likely diploid (Barlogie B, personal communication, 2002). With the exception of one patient with monosomy 9, no aneuploid cell populations were detected in tumor cells from 15 patients with WM by interphase fluorescent in situ hybridization (FISH) using centromeric probes for chromosomes frequently found to be aneuploid in MM and B-CLL (7, 9, 11, 12, 15 and 17).1 Nevertheless, some sporadic karyotype aberrations consistent with aneuploidy have been reported in the literature and the set of probes precedently enumered can miss an aneupolidy, like trisomy 4 reported as the sole abnormality in one case of WM patient.6
We analyzed by conventional cytogenetic (CC) a cohort of 39 patients with a diagnosis of WM, according to the Workshop classification (at diagnosis in 23 patients and after a median evolution of 3.8 years in 13). The observation of three patients with trisomy 4 detected by CC prompted us to search additional cases of this abnormality, by interphase FISH (IP-FISH) using a centromeric probe from chromosome 4. The results of the karyotypes and IP-FISH are detailed in Table 1. In our study, among 37 patients with evaluable metaphases and 13 with abnormalities, none had a deletion or rearrangement of 6q and one harbored a t(11;18)(q21;q21) (No. 19). However, we found three patients with a whole trisomy 4 (8%) (Nos. 4, 24 and 33) and one with a partial trisomy 4 (No. 29). Trisomy 4 was the sole abnormality in two patients (Nos. 4 and 24) and was associated with trisomy 18 and the gain of a marker chromosome in the third. Metaphase cytogenetic studies in WM are hampered by the low proliferative activity of the lymphoplasmocytic cells as in the MM or monoclonal gammapathies. In the larger studies published yet, detection of chromosomal abnormalities was weak with CCs. Louviaux et al. Blood 1998; 92: 184b; abstract reported only 12 patients out of 45 with chromosomal abnormalities (27%), and Schop et al.1 13 patients among 35 (37%). These results are comparable with our finding of 13 patients with abnormalities among 37 (35%).
FISH on interphase cells has greatly improved the detection of chromosomal abnormalities as it is independent of cells proliferative capacity. Thus, we decided to search for additional cases with trisomy 4 by IP-FISH on cytogenetic cryopreserved pellets in our cohort of 39 patients. To our knowledge, chromosomal aneuploidy was never screened with centromeric probe (CEP) probe for chromosome 4 in WM. The three complete trisomies 4, already seen by CC, were confirmed. As expected, the partial trisomy 4 did not include the chromosome 4 centromere. Among the 35 others patients, we further found four other cases with trisomy 4. Two were present in patients in whom CC failed (Nos. 35 and 37), one was present in a patient with a normal karyotype (No. 10) and one in a patient with a clonal 17p deletion (No. 2). The normal metaphases observed in patient 10 may probably derive from the normal myeloid cells in the bone marrow. Two independent clones coexisted in patient 2, one detected by CC and the other by IP-FISH as shown by a simultaneous FISH with p53 and CEP4 probes. As deletion 6q was the most frequent cytogenetic abnormality detected using IP-FISH analysis in WM, we have searched this aberration with a unique probe located at 6q21. Among the 29 patients studied, we found six 6q deletions (21%), a percentage in a smaller range compared to the study of Schop et al. Blood, 2005; 106: 290; abstract (39%) conducted with a panel of 6q probes. Only one patient, among the four with trisomy 4 tested, showed a 6q deletion, suggesting that 6q deletion may be an additionnal abnormality. Trisomy 4 is a new recurrent chromosomal abnormality in WM with a prevalence of at least 18%. If partial trisomy 4 is included, the prevalence of trisomy 4 reached 20%. IP-FISH studies were performed on stored bone marrow samples previously cultured for metaphase analysis. It is tempting to speculate that the proportion of WM patients with trisomy 4 could be higher with IP-FISH studies performed on selected lymphoplasmocytic cells. In fact, in our cohort, the percentage of clonal involvement of the bone marrow was lower than 30% in 10 patients. The mean percentage of bone marrow lymphoplasmocytic cells was higher for patients with trisomy 4 (63%) than in the others (43%). Moreover, in the study of Schop et al. Blood, 2005;106: 290; abstract the incidence of deletion 6q was higher in patients analyzed by cIgM-FISH (54% instead of 39%).
Trisomy 4 was the sole abnormality in two of the three karyotypes where it was identified and in one sporadic case reported previously. Thus, trisomy 4 could be a specific primary genetic event in WM. Moreover, the detection of one partial trisomy 4 defined an interesting region with coding genes for oncogenes. One of them, the c-KIT gene, is located on chromosome 4q12 and is expressed in about half of the CD34+ precursors and mast cells. Activating mutations of c-KIT are found in mast cell disease but also in acute myeloid leukemia (AML), preferentially in core binding factor AML. In bone marrow biopsies of WM patients, an excess of mast cells have been found. It was shown that the expression of CD40 ligand on bone marrow mast cells supports the growth of lymphoplamocytic cells in patients with WM. We decided to look for c-KIT gene abnormalities in our patients with trisomy 4. Molecular analysis was performed in three patients. Coding sequence from exons 8 to 13 and 17, the most frequently mutated, were sequenced. A Val for Met substitution at codon 541 was identified in one patient only at the RNA level. This variant has been already reported in other tissues and tumors, but little is known about its significance. On the one hand, some authors considered this variation as a polymorphism identified in 1.5–9% of healthy subjects. On the other hand, there are accumulating elements suggesting an impact in human pathology. First, the Met541Leu variant was significantly more frequent in normal tissues of CML patients than of healthy subjects (8.8 vs 1.5%). In addition, this variant was found as an authentic somatic event that occurred in CML cells from one patient during blastic crisis, whereas it was not detected in chronic phase. Second, it was recently observed in seven out of seven dedifferentiated and three out of seven well-differentiated liposarcoma. Third, P Dubreuil et al. (unpublished results) recently identified this variant at the RNA level in pathological skin biopsies of 31 of 169 mastocytosis patients (18.3%), and in the genomic DNA of only 1/71 (1.5%) healthy subjects (P<0.005). Interestingly, genomic DNA sequencing of normal blood cells in 10 patients with Met541Leu variant identified in pathological tissues did not reveal any allelic variation, suggesting that it may represent a true somatic event in mastocytosis.
We identified trisomy 4, a new recurrent chromosomal abnormality in WM, with a prevalence of 18% using IP-FISH on unselected cells. Further studies are needed to better define the exact prevalence of this aberration on selected cells, to search for partial trisomy 4 and to precise the molecular pathogenesis links of this abnormality in WM. A French prospective multicentric study is in process to precise the clinical and biological features, the response to treatment and the outcome of WM patients with different chromosomal abnormalities.
Schop R, Kuehl W, Van Wier S, Ahmann G, Price-Troska T, Bailey R et al. Waldenström macroglobulinemia neoplastic cells lack immunoglobulin heavy chain locus translocations but have frequent 6q deletions. Blood 2002; 100: 2996–3001.
Avet-Loiseau H, Garand R, Lode L, Robillard N, Bataille R . 14q32 translocations discriminate IgM multiple myeloma from Waldenström macroglobulinemia. Semin Oncol 2003; 30: 153–155.
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Hirase N, Yufu Y, Abe Y, Muta K, Shiokawa S, Nawata H et al. Primary macroglobulinemia with t(11;18)(q21;q21). Cancer Genet Cytogenet 2000; 117: 113–117.
Schop RF, Jalal SM, Van Wier SA, Ahmann GJ, Bailey RJ, Kyle RA et al. Deletions of 17p13.1 and 13q14 are uncommon in Waldenström macroglobulinemia clonal cells and mostly seen at the time of disease progression. Cancer Genet Cytogenet 2002; 132: 55–60.
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Terré, C., Nguyen-Khac, F., Barin, C. et al. Trisomy 4, a new chromosomal abnormality in Waldenström's macroglobulinemia: a study of 39 cases. Leukemia 20, 1634–1636 (2006). https://doi.org/10.1038/sj.leu.2404314
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