J Clin Pathol: first published as 10.1136/jcp.20.5.699 on 1 September 1967. Downloaded from J. clin. Path. (1967), 20, 699 Incidence and pathogenesis of megaloblastic erythropoiesis in multiple myeloma A. V. HOFFBRAND,1 J. R. HOBBS, S. KREMENCHUZKY, AND D. L. MOLLIN From the Departments of Haematology and Chemical Pathology, Royal Postgradute Medical School, Ducane Road, London, and the Medical Research Council Group for Investigation into Megaloblastic and Sideroblastic Anaemias SYNOPSIS Intermediate megaloblastic changes occurred in six (19 %) of 32 patients with multiple myeloma and trivial megaloblastic changes in a further ten (31 %). Folate deficiency was the pre- dominant cause of these changes and in at least two patients was sufficiently severe to contribute to anaemia. Folate deficiency appeared to be due to excess folate utilization by the tumour and was related to the amount of paraprotein produced daily. Five of the 32 patients had subnormal serum B12 levels. Reduction in the serum B12 level was related to the reduction in the normal circulating immunoglobulins and occurred despite normal B12 absorption. Possible explanations for this finding are discussed. copyright. Subnormal serum vitamin B12 concentrations have Larsson, 1962; van Dommelen, Olie, and Slagboom, been reported in patients with multiple myeloma by a 1964). number of authors (Mollin and Ross, 1952; The present study was undertaken to determine the Mandema, 1956; Mandema, Faber, de Vries, and incidence, severity, and cause of B12 deficiency in Nieweg, 1956; Killander and Larsson, 1962; Larsson, patients with multiple myeloma. Since megaloblastic 1962; Forshaw, 1963; Hansen, 1964). In some ofthese anaemia in other conditions in which thereis prolifera- patients megaloblastic anaemia was also observed tion of primitive tissue is almost invariably due to http://jcp.bmj.com/ and in the patients of Mandema (1956), in two folate deficiency (Swendseid, Swanson, Meyers, and patients of Larsson (1962), and in the patient of Bethell, 1952; Girdwood, 1953), the incidence and Forshaw (1963) the anaemia responded to large pathogenesis of folate deficiency was investigated in doses of vitamin B12. The anaemia in another patient the same group of patients and this paper reports the of Larsson (1962) and the patient of Bichel (1964) results of both studies. responded to liver therapy. Nonetheless, true Addisonian pernicious anaemia PATIENTS STUDIED on September 26, 2021 by guest. Protected appears to be rare in myeloma and it has only been established by the demonstration of histamine-fast Thirty-two randomly selected patients with myeloma achlorhydria associated with malabsorption of were studied. Their ages ranged from 42 to 81 (mean 67 vitamin B12 corrected by intrinsic factor in three years) and they included 17 males and 15 females. All the cause patients were investigated while in hospital for inves- patients (cases 2,3, and 5 ofLarsson, 1962). The tigation or treatment. of subnormal serum B12 concentrations in patients In each patient, the diagnosis was made because the with myeloma without pernicious anaemia is un- patient showed clinical and/or radiological evidence of certain. It has been suggested that they are due to bone lesions ofthe myeloma type, abnormal and increased malabsorption of vitamin B12 associated with numbers of plasma cells in the bone marrow, a charac- bacterial contamination of the small intestine teristic narrow 'monoclonal' band on electrophoresis of (Larsson, 1962) or to excess B12 utilization by the the serum proteins and/or Bence-Jones proteinuria. Ten tumour (Mandema et al., 1956; Killander and patients were studied at the time myeloma was first diagnosed, and in the remaining 22 patients myeloma was 'Present address: Department of Haematology, St. Bartholomew's known to have been present from two months to nine Hospital, London E.C.1. years previously. Fifteen patients were receiving no specific Received for publication 29 March 1967. therapy, seven patients were receiving melphelan (from 699 J Clin Pathol: first published as 10.1136/jcp.20.5.699 on 1 September 1967. Downloaded from 700 A. V. Hoffbrand, J. R. Hobbs, S. Kremenchuzky, and D. L. Mollin 0-25 to 4 mg. daily), one patient urethane (3 mg. daily), myeloma globulins (mean 11-6 days) (Drivsholm, 1964). one patient prednisone (5 mg. b.d.), and eight were having For the same serum concentration, therefore, YA protein deep x-ray therapy for bone lesions. In the patients on has a faster turnover (estimated as 10-8 % ofthe total pool) cytotoxic drugs, serum for microbiological assay was than YG protein (estimated as 6% of the total pool). In taken at least 24 hours after the last dose of the drug. order to compare our patients, we have arbitrarily con- sidered them all to be 71 kg. in weight, with a plasma METHODS volume of 50 7 ml. per kg., with the plasma containing 71% of the total pool of myeloma globulin (averages of B12 CONCENTRATIONS These were deter- availabledatainmyeloma patients, Gabuzda, 1962). Thus, SERUM VITAMIN M g. mined by microbiological assay using the z strains of fora given serum concentration of myeloma globulin, Euglenagracilis as test organism as described by Anderson per 100 ml., the total pool would be M x 71 x 50-7 x (1964) with a normal range from 160 to 925 ,u.g. per ml. 100° = 50 7 M and for in the VITAMIN B12 ABSORPTION This was measured by the g., patients equilibrium, urinary excretion method of Schilling (1953) using an oral average daily productions of YA would be 10-8% of dose of 1 ,ug. radioactive "8Co-B12 and an intramuscular 50-7M = 5 5 Mg., andofyG6% of 50-7M = 30 Mg. flushing dose of 1,000 ,ug. non-radioactive B12. Normal In order to obtain the total daily M protein production, subjects excrete more than 10% ofthe oral dose in 24 hours the daily Bence-Jones protein loss in the urine was meas- and patients with Addisonian pernicious anaemia excrete ured, corrected to 71 kg. from the patient's body weight, less than 5% and usually less than 2-5 %in 24 hours. and added to the production of M protein, calculated above. SERUM FOLATE CONCENTRATIONS These were estimated by It is recognized that this calculation only gives an microbiological assay using Lactobacillus casei as test approximate value since plasma and total body pools and organism as described by Waters and Mollins (1961). The the half life of the plasma proteins all show individual normal range is from 6-0 to 21-0 mug. per ml. variation. Nevertheless, this variation is mostly less than ± 25% of the taken means. SERUM MYELOMA PARAPROTEINS These were recognized by their narrow 'monoclonal' electrophoretic mobility, or so- LOSS OF NORMAL HUMORAL IMMUNITY This was estimated copyright. called 'M' band. They were identified by immunoelectro- bymeasuringtheconcentrationof the serum immunoglob- phoresis as YG, yA, or Bence-Jones protein. Their light ulins (yG, YA, and YM) and taking their average, each chains were shown to be of one type only and identified expressed as a percentage of the normal mean, e.g., case as K (Kappa) or L (Lambda) (Nomenclature of Human FB had serum concentrations, YG 75%, YA 70%, and Immunoglobulins, Bull. Wld Hlth Org., 1964). YM 70% of normal, giving an average 72% of the normal immunoglobulin concentration. TOTAL SERUM PROTEIN CONCENTRATIONS These were esti- mated by a Biuret method calibrated against Armour HAEMATOLOGICAL FINDINGS standard bovine albumin. Using this method, YG globulin http://jcp.bmj.com/ yields 3% less colour, and YA globulin 8% less colour than INCIDENCE OF MEGALOBLASTIC HAEMOPOIESIS In the same weight of albumin but no correction has been Table I, patients are divided according to whether or made, the error being slight, since, in all but one patient, not they show megaloblastic change in the bone the concentration of myeloma (M) protein in the serum marrow. Six(19 %) ofthe 32 patients (group I) showed was less than half the total serum protein concentration. intermediatemegaloblasticchanges (DacieandWhite, The proportion of M protein in the serum proteins was 1949) and a further 10 (31 %) patients (group II) estimated after electrophoresis on cellulose acetate using showed trivial megaloblastic changes. The remaining the dye amidoschwarz lOB as colouring agent. This dye 16patients(groupIII) had entirely normoblastic bone on September 26, 2021 by guest. Protected yields near uniform uptakes for the same weights of the florid albumin, YG, and YA globulins and recoveries ofpure added marrows. None of the patients showed fractions are reliable to within 0-2 g. per 100 ml. (Hobbs, megaloblastic changes seen in severely anaemic 1965). The concentration of M protein was then calculated patients with uncomplicated megaloblastic anaemia. from the known total serum protein concentration. The appearances of the stained peripheral blood films were largely, but not invariably, consistent with URINARY PROTEIN was estimated by a Biuret method after these bone marrow findings. Macrocytes and hyper- ovemight precipitation in 10% trichloracetic acid and segmented polymorphs (polymorphs with more than redissolving the precipitate in N sodium hydroxide. The five nuclear lobes) were more frequent in the stained content of M protein in urine was estimated after electro- peripheral blood films ofgroup I, less frequent in those phoresis of concentrated urine by the method used to III. estimate serum M protein. of group II, and rarely present in those of group RELATION OF MEGALOBLASTIC HAEMOPOIESIS TO OTHER PRODUCTION OF M PROTEIN An estimate of the DAILY all of the 32 daily output of M protein was made using the following HAEMATOLOGICAL FINDINGS Nearly (30) assumptions. It has been shown that YA myeloma glob- patients were anaemic at the time of the present ulins have a shorter half life (mean 6-4 days) than yo studies (Table I). There was no direct correlation J Clin Pathol: first published as 10.1136/jcp.20.5.699 on 1 September 1967.