British lournal of Haematology. 1993, 8 3 , 672-677

Case Reports

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 K- and 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 super- natant 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

Fig 1. Direct sequencing analysis of the N-ras codon 61 in CD34 positive bone marrow cells. PCR-amplified fragment corresponding to codon 61 was obtained from genomic DNA, sequenced and run on a 6% acrylamide gel. The sequence of N-ras codon 61 shows a wild type configuration in CD34 positive cells, whereas this is mutated in CD34 negative. Non-coding strands are shown. The arrow points to the band corresponding to the mutated base pair in CD34 negative cells.

6 72

British Journal of Haematology, 1993, 83 Case Reports 673 pointed out that two alternative explanations may exist. First, it may be supposed that only a minority of the stem cell pool belongs to the tumour population. However, in true stem cell disorders, as for example chronic myelogenous leukaemia, the majority of haematopoiesis is neoplastic, as expected in a multiple myeloma patient with 60% plasma cell infiltration. Second, ras mutations can be a late genetic lesion during myeloma tumourigenesis, even if they are usually considered an early lesion in human tumourigenesis, as it has been shown for myelodisplasias and colon adenomas (Fearon & Vogelstein, 1990). Our results, therefore, do not support the hypothesis of a stem cell origin for multiple myeloma. However, to definitively exclude such origin, further analyses on a large panel of cases are required.

ACKNOWLEDGMENTS

This work was supported by the Associazione Italiana Ricerca sul Cancro (AIRC, Milano, Italy) and by Consiglio Nazionale Ricerche (Progetto Finalizzato ACRO, No. 9202242.PF39). C.V. is a fellow of Comitato Piemontese Gigi Ghirotti.

Department of Medicine and Experimental Oncology, DARIO FFXRERO Division of Haematology, CLAUDIA VOENA University of Torino, MARCO LADETTO 20226 Torino, MARIO BOCCADORO ltaly ALESSANDRO PILERI

PAOLO CORRADINI

REFERENCES

Caligaris-Cappio. F., Bergui, L.. Tesio. L., Pizzolo, G.. Malavasi. F., Chilosi, M., Campana. D., Van Camp, B. & Janossy. G. (1985) Identification of malignant plasma cell precursors in the bone marrow of multiple myeloma. Journal of Clinical Investigation. 76, 1243-1251.

Corradini. P.. Boccadoro, M. & Pileri, A. (1991) Molecular evidence for a B lymphocyte involvement in multiple myeloma. (Abstract). Blood. 78, 505a.

Epstein, J., Xiao, H. & He, X.Y. (1990) Markers of multiple hematopoietic-cell lineages in multiple myeloma. New England journal of Medicine, 332, 664-668.

Fearon, E.R. & Vogelstein, B. (1991) A genetic model for colorectal tumorigenesis. Cell, 61, 759-776.

Neri, A., Chang. C.C., Lombardi, L., Salina, M., Corradini, P., Maiolo, A.T., Chaganti, R.S.K. & Dalla Favera. R. (1991) B cell lymphoma associated chromosomal translocation involves candidate onco- gene lyt-10 homologous to NF-kBp50. Cell, 67, 1075-1087.

Piacibello, W., Ferrero, D., Sanavio, F.. Badoni. R.. Stacchini. A,. Severino, A. & Aglietta, M. (1991) Responsiveness of highly enriched CFU-GM subpopulations from bone marrow, peripheral blood, and cord blood to hemopoietic growth inhibitors. Experi- mental Hematology, 19, 1084-1089.

SEVERE ANAPHYLACTIC TRANSFUSION REACTION ASSOCIATED WITH HLA-INCOMPATIBLE PLATELETS

Life-threatening anaphylactic transfusion reaction is a rare side-effect in platelet transfusion. It has been reported in patients who lacked some kind of plasma protein such as IgA (Fundenberg et al, 1968: Vyas et al, 1968) and the fourth complement component (C4) (Lambin et al, 1984). It has not been described, however, in association with HLA-incompa- tibility .

A 14-year-old Japanese boy was admitted on 30 July 199 1 because of fever, sore throat and pancytopenia. He was diagnosed as having acute myeloid leukaemia (MSa, in FAB criteria) and received remission induction chemotherapy of daunorubicin. Ara-C, 6-MP and prednisolone (DCMP). Six platelet donors, which were not HLA-matched, were chosen from healthy young males, and platelet concentrate (PC) was obtained from each one of the six donors. The PC was transfused through a leucocyte removal filter (PLSOH, Pall Biomedical Product Co., U.S.A.) and the same type of filter was also used in red blood cell transfusion (RCSOTM, Pall Biomedical Product Co., U.S.A.). Despite the attempts to avoid alloimmunization, almost no post-transfusion platelet incre- ment was gained by several PC transfusions. On 5 September, just after the twelfth PC infusion was started, he complained of throat discomfort and itching with urticaria, immediately followed by dyspnoea and marked hypotension. Therefore we washed PC with 1500 ml of saline by the apheresis system to

decrease the plasma concentration: however, anaphylactic reactions, such as urticaria, hot flush and throat discomfort, recurred (a total of seven times) after infusion of 30 ml or less of washed PC, and finally occurred with PC of all six donors. On the other hand, red blood cell transfusions caused nothing more than fevers.

The patient had had no allergic history. The leveb of C3 and C4 in his serum were normal. Immunoglobulin (Ig) G was 1804 mg/dl (normal 802-1556 mg/dl), IgA 119 mg/dl (130-367mg/dl). IgM 237 mg/ml(58-189 mg/dl), IgD 3.3 mg/dl (O-15 mg/dl). and IgE 580 IU/ml(0-500 IU/ml). Both direct and indirect Coombs tests were negative. Possible pyrogenic and chemical contaminations in PC were excluded because there were no reactions to the saline passed through the apheresis system and the leucocyte removal filter. Intracutaneous reactions using the PCs from three respon- sible donors were negative. We could detect anti-HLA A 3 1 antibody alone in his serum, though we checked repeatedly on antibodies against class-I HLA antigens by anti-human globulin-lymphocytotoxicity test, and against human plate- let specific antigens by mixed passive haemagglutination test. The patient’s serum was also examined by agarose gel immunodiffusion against the sera obtained from the three donors responsible for anaphylactic reactions, but no precipi- tating antibodies were detected. With the patient’s informed