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THE MUTATION OF N‐RAS ONCOGENE DOES NOT INVOLVE MYELOID AND ERYTHROID LINEAGES IN A CASE OF MULTIPLE MYELOMA

Multiple myeloma is a B-cell malignancy characterized by the expansion of mature plasma cells producing monoclonal immunoglobulins. However, it has been regarded as a tumour arising at the B, pre-B lymphocyte, or even stem cell level (Caligaris-Cappio et al, 1985; Corradini et d, 1991). The occurrence of the primary neoplastic lesion in an early haematopoietic progenitor, capable of differentiation into the various lineages, has been postulated by Epstein et al(1990). This hypothesis was suggested by the observation that myeloid and erythroid antigens are expressed on malignant plasma cells. To evaluate the possible involvement of these lineages in multiple myeloma, we used the presence of a N-ras oncogene mutation as a tumour cell marker. The patient was a 62-year-old male with light-chain multiple myeloma diagnosed in 1988 in relapse phase. Bence-Jones protein was 3.2 g/24 h. Bone marrow aspirate revealed a 60% plasma cell infiltration. Patient’s bone marrow cells were screened for Kand N-ras mutations in exons I and I1 by direct sequencing. The N-ras codon 61 was found to be mutated and a CAA+AAA transversion was detected. This change led to the replacement of the glycine aminoacid with lysine. In order to obtain myeloid and erythroid cells totally devoided of contaminating myeloma cells, clones were obtained from culturing single CFU-GM/BFU-E in liquid medium at limiting dilution. Cells were separated according to their expression of CD34 antigen by magnetic separation as previously described (Piacibello et al, 1991). CD34 positive cells were cultured at Limiting dilution in Iscove’s Modified Dulbecco’s Medium containing 25% fetal bovine serum, 30 ng/ml human recombinant ‘stem cell factor’, 1.5 U/ml human recombinant erythropoietin and 3% vol/vol supernatant of 5637 cell Line, as source of colony stimulating factors. Clones were scored after 14 d of culture. Five pools of 10 colonies each (six myeloid and four erythroid) were screened to detect the presence of the N-ras codon 61 mutation. Molecular analysis was performed also on CD34 negative cells containing the whole myeloma cell population (as a positive control). Genomic DNA was extracted by cell lysis, heating at 95°C for 10 min. The N-ras exon I1 has been amplified for 3 5 cycles in a thermal cycler. Amplified DNA has been directly sequenced using a primer that was end-labelled with [y-32P]adenosine triphosphate according to a procedure previously described (Neri et al, 199 1). Sequencing analysis showed the absence of the N-ras codon 61 mutation in myeloid and erythroid cells derived from limiting dilution culture, while it was present in the CD34 negative cells (Fig 1). The absence of the N-ras mutation in CD34 positive cells suggests that myeloid and erythroid lineages are not involved in multiple myeloma tumourigenesis. However, it should be