Correspondence 663 RESPIRATORY BURST ACTIVITY IN MDS AND MPS PATIENTS

Patients with myelodysplastic syndromes (MDS) and certain myeloproliferative syndromes ( M P S ) have an increased risk for serious bacterial infection. One reason for this may have been an impaired granulocytic function seen in MDS and some MPS patients (Elias et al, 1987). This decreased bursting activity of granulocytes from patients with MDS has been found in vitro with activators of G-protein- dependent pathways of granulocytic activation (e.g. the bacterial polypeptide f-MLP), but not with activators of protein kinase C (e.g. the phorbol ester TPA) (Elias et al, 1987). In keeping with these studies Lowe et al(1994) have reported that a fraction of MDS patients (those with < 7 mV chemiluminescence response), roughly representing SO% of the patients investigated, revealed significantly lower response to f-MLP compared to either normal controls or the other MDS patients. Furthermore, following priming with GM-CSF, these patients showed altered chemilumines- cence patterns. The authors could show that the receptor for fMLp and the NADPH oxidase system were normal in these patients, suggesting that a defect in tMLP induced signalling was responsible for the observed phenomenon.

Using a different assay to determine respiratory burst activity, namely a sensitive single-cell flow cytometric assay (Rothe et al, 1988), we have observed a similar phenomenon in a group of MDS and MPS patients. In order to address the question of whether the G-protein dependent impairment could be due to activated ras-protooncogenes, a moIecular lesion frequently observed in MDS and M P S patients (Liu et al, 1987; Neubauer et al, 1994; reviewed in Bos, 1989), we

analysed our patients using a slot-blot and Southern procedure as described (Neubauer et al, 1991) for the presence of ras-mutation in codons 12. 13 and 61 of the N- and K-ras protooncogene. Ras genes encode for small G- proteins and play an important role in signal transduction of certain receptor tyrosine kinases (reviewed in McCormick, 1993).

Fig 1 shows a typical example of a normal control (A-C) and a patient with CMML (D-F), where A and D represent the flow data of unstimulated granulocytes, B and E of !MLP stimulation ( 1 0 - 6 ~ ) in the presence of cytochalasin B ( 5 mg/ml), and C and F TPA at 0.5 m. Out of 13 patients analysed, seven showed impaired burst activity following fMLP stimulation. Two of 13 patients (both CMMoL) had a mutated ras-protooncogene (one patient at N-ras codon 13 ASP, the other at N-ras codon 12 VAL), one of whom also had an impaired respiratory burst after iMLP. and the other patient showed a normal response following fMLP stimu- lation. The remaining 11 patients revealed wild-type ras genes.

These data corroborate the data by Lowe and coworkers and extend the findings to the molecular analysis of the ras-genes. However, mutated ras-genes cannot be the only reason for the observed impairment in the fMLP- induced signalling pathway, because most of the patients in our study (6/7) with decreased response to tMLp harboured wild-type, not mutated, ras-protooncogenes. Other G-proteins may be responsible for the observed phenomenon.

100

a

A 1

I \ I I I 1 I 1

I00 2 0 0

i"i C I

Fig 1. A normal control (A-C) and a patient with CMML (D-F): (A) and (D) flow data of unstiiulated granulocytes: (B) and (E) fMLp stimulation M) in the presence of cytochalasin B (5 mg/ml); (C) and (F) "PA at 0 .5 m.

664 Correspondence Universitatsklinikum Rudolf Virchow, ANDREAS NEUBAUER Abteilung Innere Medizin mS Hiimatologie, Spandauer Damm 1 3 0 , 14050 Berlin, Germany

Lineberger Comprehensive Cancer Center, EDISON LIU 16. University of North Carolina at Chapel Hill, NC 27599-7295, U.S .A .

Lowe. G.M.. Dang. Y., Watson. F., Edwards, S.W. & Galvani, D.W. (1994) Identication of a subgroup of myelodysplastic patients with a neutrophd stimulation-signalling defect. British Journal of Haernatology, 86, 761-766.

McCormick. F. (1993) How receptors turn ras on. Nature, 363, 15-

Neubauer, A., Dodge, R., George, S.L., Davey, F.R., Silver, R.T., Schfler, C.A., Mayer, R., Ball, E.D.. Wurster-Hill, D.. Bloomtield, C.D. & Liu. E.T. (1994) Prognostic importance of mutations in the

REFERENCES

Bos. J.L. (1989) Ras oncogenes in human cancer: a review. Cancer Research. 49, 4682-4689.

Elias, L., Van Epps, D.E., Smith, K.J. & Savage, B. (1987) A trial of recombinant a 2 interferon in the myelodysplastic syndromes. II. Characterization and response of granulocyte and platelet dysfunction. Leukemia, 1, 111-115.

Liu, E.. Hjelle. B.. Morgan. R.. Hecht, F. & Bishop, J.M. (1987) Mutations of the Kirsten ras protooncogene in myelodysplastic syndrome. Nature, 327, 430-432.

ras protooncogenes in de novo acute myeloid leukemia. Blood. 83,

Neubauer, A.. Shannon, K. & Liu. E. (1991) Mutations in the ras proto-oncogenes in childhood monosomy 7. Blood, 77, 594- 598.

Rothe, G., Oser, A. &Valet. G. (1988) Dihydrorhodamine 123: a new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes. NaturwissenschaJkn, 7 5 , 354-3 55.

1603-1611.

Keywords: myelodysplasia, myeloproliferative syndromes, ras-protooncogenes, respiratory burst activity.

PRIMARY CAUSE OF MEGALOBLASTIC ANAEMIA IN ZIMBABWE

We congratulate Savage et a1 (1994) for their attempt to determine the relative roles of folate and vitamin B12 deficiency in the aetiology of megaloblastic anaemia in Zimbabwe. It is our opinion, however, that the conclusion that vitamin B12 deficiency is the primary cause of megaloblastic anaemia in Zimbabwe cannot be made on the strength of the study they conducted.

For inclusion into their study of 235 medical patients, two or more of the following criteria had to be fulfilled neuro- logical dysfunction consistent with that seen in vitamin B12 deficiency, anaemia, MCV 2 100 fl, pancytopenia, diarrhoea, and glossitis. The inclusion criteria for each of the 235 individual patients were not stated, nor was the frequency with which each of the criteria had been used for patient inclusion into their study. We propose that at least two of the inclusion criteria to some extent select patients who are more likely to have vitamin B12 deficiency than folate deficiency.

‘Neurological dysfunction consistent with that seen in vitamin B,, deficiency’ must be considered to be due to vitamin B12 deficiency, and before serious consideration can be given to folate deficiency, vitamin BI2 deficiency should be excluded (Chanarin, 1990). Thus this criterion clearly favours inclusion of vitamin BI2 deficient patients into their study. The fact that milder forms of vitamin B12 deficiency may have been excluded by the criteria does not alter this fact.

The use of MCV 2 100 fl as an inclusion criterion possibly excluded some patients who had combined megaloblastic and iron deficiency anaemia. Confirmed cases of megalo- blastic anaemia with MCV values < 100fl, anisocytosis and raised RDW occur commonly in the African population in Durban, Natal, South Africa (D. G. Kenoyer, personal communication). Given the fact that nutritional deficiency of folate. with or without increased demand for the same, is more often associated with iron deficiency than is vitamin B12 deficiency (Chanarin, 1990), we think that the use of MCV 2 l O O f l as an inclusion criterion tends to select vitamin B12 deficient patients.

The importance of the need to include pregnant women in a study to determine the relative import of folate and vitamin B12 deficiency in the aetiology of megaloblastic anaemia cannot be over-emphasized. Women of childbearing age represent a significant proportion of the Zimbabwean popu- lation and folate deficiency has been reported in significant proportions of pregnant African women elsewhere (Fleming et al, 1968). Therefore, exclusion of obstetric cases leaves out a large group of subjects whose megaloblastic anaemia is likely to have a folate deficiency aetiology.

The data of Savage et a1 (1994) not only represent the ‘tip of the iceberg’ of vitamin B12 deficiency in Zimbabwe as they stated: more importantly, they represent ‘the nose of the hippopotamus’ of megaloblastic anaemia as a whoie. Clearly, a lot more work needs to be done before firm conclusions can be made.

Department of Haematology, ZIVANAI C. CHAPANDUKA University of Natal Medical School, VINCENT L. NAICKER P.O. Box 1 7 0 3 9 . D. GAYLE KENOYER Congella 401 3, VINOD B. JOGESSAR

Durban, R.S.A.

REFERENCES

Chanarin. I. (1990) The Megaloblustic Anaemias, 3rd edn. Blackwell Scientific Publications, Oxford.

Fleming, A.F., Hendricks, J.D. de V. & Allan, N.C. (1968) The prevention of megaloblastic anaemia in pregnancy in Nigeria. Journal of Obstetrics and Gynaecology of the British Commonwealth,

Savage, D.G., Gangaidzo. I., Lindenbaum. J., Kiire. C.. Mukiiti. J.M.. Moyo, A., Gwanzura, C., Mudenge, B.. Bennie. A.. S i t i a . J., Stabler, S.P. & Allen, R.H. (1994) Vitamin B12 deficiency is the primary cause of megaloblastic anaemia in Zimbabwe. British Journal of Haematology, 86, 844-850.

75,425-432.

Keywords: megaloblastic anaemia, aetiology, vitamin B,, , folate.