British]oumul ofHuematofogy, 1976,32,487.

The Patterns of Fetal Haemoglobin Production in Leukaemia B. L. SHERIDAN,* D. J. WEATHERALL,t J. B. CLEGG,~ J. PRITCHARD,* W. G. WOOD,? S. T. CALLENDER,~ 1. J. DURRANT,? w. R. MCWHIRTER,~ M. ALI,$J. W. PARTRIDGET AND E. N. THOMPSON** *Department of Haematology, University of Liverpool; t Nufield Department of Clinical Medicine, University of Oxford; $Departments of Child Health, University of Dundee, * * Welsh National School of Medicine; 7 Warwick Hospital; and S Stloseph’s Hospital, Hamilton, Ontario (Received 18 September 1975 ; acceptedfor publication 9 October 1975) SUMMARY. Elevated levels of haemoglobin F (Hb F) have been found in a wide range of haematological malignancies, but very high levels were found only injuvenile chronic myeloid leukaemia (JCML), and erythroleukaemia occurring in infancy. In both these disorders a reversion to a fetal form of erythropoiesis may occur, as judged by both the structure of the Hb F and by the disappearance of Hb A, and the carbonic-anhydrase isozymes during the course of the illness. The clinical picture of JCML is not always associated with a reversion to fetal erythropoiesis; there appears to be a heterogeneity of conditions with this clinical label. Thus the reversion to a completely fetal pattern of erythropoiesis seems to occur in a variety of leukaemias which start in early life. This change is associated with a uniformly bad prognosis. Of a group of 17 patients with acute myeloid leukaemia I 5 developed an increase in the level of Hb F about 60 days after the commencement of treatment; significantly greater increases were observed in those achieving a clinical remission. The level of Hb F usually declined during remission but high levels persisted in a few cases. Increased levels of Hb F were found also in patients with other haematological malignancies who had undergone periods of marrow aplasia during treatment. In all cases the Hb F was heterogeneously distributed throughout the red cells. Analysis of 715 or yCB3 peptides of Hb F from a variety of leukaemias gave glycine compositions ranging from 0.20 to 0.85 residues with many values in the fetal range; all cases with a reversion to fetal erythropoiesis had values in the fetal range. Attempts to confirm the ‘fetal’ origin of the cells containing Hb F by means of other markers was possible only in the cases ofJCML and in one child with erythroleukaemia. These studies indicate that in some forms of leukaemia there may be a genuine reversion to fctal erythropoiesis while in others the emergence of cells containing Hb F appears to be part of a rapid regeneration process occurring after a period of marrow aplasia. The diagnostic and prognostic value of these observations is discussed. There have been many reports of elevated levels of fetal haemoglobin (Hb F) in the peripheral Correspondence: Professor D. J. Weatherall, Nuffield Department of Clinical Medicine, The Radcliffe Infirmary, Oxford OX2 6HE.

487

488

B. L. Sheridan et a1

blood of patients with different forms of leukaemia (Weatherall et al, 1974). However, the only type of leukaemia in which high levels of Hb F are found consistently is juvenile chronic myeloid leukaemia (JCML) (Hardisty et al, 1964, and it has been suggested that the raised level ofHb F in this condition results from the proliferation of a cell line with predominantly fetal properties (Weatherall et al, 1968). In other forms of leukaemia Hb F levels are not consistently raised and the relationship of reactivation of Hb F production to the leukaemic process is not clear. The object of the present study was to examine further the patterns of haemoglobin, redcell enzyme and red-cell antigen production in JCML and to compare and contrast them with those observed in other forms of leukaemia. It was hoped that in this way we might be able to obtain a clearer idea about the relationship of the reactivation ofHb F synthesisto the underlying leukaemic process, both in JCML and other forms of leukaemia. Some of these findings have appeared in a preliminary form (Weatherall et d, 197sa). METHODS Haematological methods, including leucocyte alkaline phosphatase (LAP) estimation and serological studies, followed standard techniques (Dacie & Lewis, 1970). Haemoglobin was analysed by starch-gel electrophoresis using a tris-EDTA-borate buffer system, pH 8.5, as described by Weatherall & Clegg (1972). The intracellular distribution of Hb F was examined by the acid-elution method of Kleihauer et al(1957) or the fluorescent anti-Hb-F antibody technique (Wood et al, 1975), and the level of Hb F was determined by alkali denaturation using a modification of the method of Betke et a1 (1959) described by Pembrey et a1 (1972). Absolute amounts of Hb F (as mg/dl) were derived from the % alkaline resistant Hb and the Hb value in g/dl. Haemoglobin A, levels were estimated by elution after cellulose acetate electrophoresis (Weatherall & Clegg, 1972) and the red cell carbonic anhydrase isozymes were estimated visually after starch-gel electrophoresis (Weatherall & MacIntyre, 1967). The relative rates of a-, 8- and y-chain synthesis in peripheral blood samples were studied by ["Hlleucine incorporation using previously described methods (Weatherall et a\, 1969, 197Sb). For structural analysis large quantities of Hb F were separated on Amberlite IRC-50 columns using developer 2 (Allen et al, 1958). The u and y chains were separated on CM-cellulose columns using an 8 M urea/mercaptoethanol/phosphate buffer system, pH 6.7 (Clegg et al, 1966).The y-chain fractions werepooled, dialysed free of urea against 0.5% v/v HCOOH, freeze dried and fingerprinted (Clegg et al, 1966). The glycinelalanine composition of peptide y15 was determined as previously described (Weatherall et al, 197~b). In some cases the glycine /alanine composition of peptide yCB3, prepared by cyanogen bromide cleavage of y chains prepared from purified Hb F, was determined (Schroeder et al, 1968). PATIENTS STUDIED Haemoglobin F estimations were carried out on all patients with haematological malignancies attending the Haematology Unit at the Liverpool Royal Infirmary over an I 8 month period. Consecutive studies of Hb F levels were performed on all patients admitted from Liverpool

Fetal Huemoglobin in Leukaemia

489

to the M.R.C. 6th Adult Leukaemia Trial between the months of September 1973 and September 1974. Similarly all patients with leukaemia attending the Nufield Department of Medicine at the Radcliffe Infirmary, Oxford, between 1973 and 1974 were screened for Hb F levels. Haemoglobin analysis and red cell enzyme and antigen studies were carried out between 1968 and 1974 on an additional group of patients referred from centres outside Liverpool and Oxford with the diagnosis of JCML. Normal control samples were obtained from the Liverpool Regional Blood Transfusion Service.

Haematological Malignancies (Table I) Haemoglobin F levels were measured during the course of the disease in nine patients with JCML, 64 patients with acute myeloblastic leukaemia (including myeloid, myelomonocytic, TABLE I. Overall incidence of raised haemoglobin F values in different forms of haeinatological malignancy

Diagnosis

No. of patients

No. with increased Hb F

Range of Hb F

Mean & SE

(%I Acute myeloblastic leukaemia Erythroleukaemia Acute lymphoblastic leukaemia Chronic myeloid leukaemia Chronic lymphatic leukaemia Juvenile chronic myeloid leukaemia* Myeloma Hodgkin’s disease Non-Hodgkin’s lymphoma Polycythaemia Vera My elofibrosis

I2

0.4-10.3 0.7-41.7 0.6-24.7 0.4-7.1 0.2-3.3 4.9-70.0 0.3-9.5 0.2-7.0 0.3-2.9 0.2-2.4

7

0.2-12.0

64 9 I9

43 8

30

I9

44 9 39 93

IS

60

48 16

12

9 22

49 27

2.01f 0.23

8.07k 4.59 2.78f 1.24 1.54+0.25 0.96ko.10 35.0+ 8.90 I.47k0.27 1.36k0.13 0.96&0.07 0.83 k0.06 2.11 k0.76

* The figures for H b F are those found on presentation. The two atypical cases are included. If these are excluded the mean presenting Hb F value is 43.3%. monocytic and promyelocytic), nine patients with erythroleukaemia, 19 patients with acute lymphoblastic leukaemia (age range 14-65 years), 30 patients with chronic myeloid leukaemia, 44 patients with chronic lymphatic leukaemia, 39 patients with myelomatosis, 93 patients with Hodgkin’s disease, 60 patients with non-Hodgkin’s lymphoma, 48 patients with polycythaemia Vera and 16 patients with myelofibrosis. Details of cases of particular interest are given in the following sections.

Juvenile Chronic Myeloid Leukaernia (Cases 1-9, Table 11) Case I , G.B., a male child aged 2 years, presented in May 1968 with malaise, abdominal swelling and bruising on the legs. Examination showed pallor, echymoses and petechiae on the limbs, and marked hepatosplenomegaly. The haematological findings were : Hb I I. 8 g/dl, W C C IOO ooo/pl (neutrophils 34%, metamyelocytes s%, myelocytes 9%, eosinophils 16%, basophils I%, lymphocytes 16%, monocytes 9%, blasts 7%) and platelets 50 ooo/pl. Marrow aspiration showed myeloid hyperplasia; a Philadelphia chromosome was not present.

490

B. L. Sheridan et al

Treatment with various antileukaemic agents resulted in no improvement and the patient died 6 months later. Case 2, R. T., a 5-year-old boy, presented in early 1973 to Hamilton General Hospital, with a 10 week history of malaise and anorexia. On examination he was febrile, with generalized wasting, widespread purpura and moderate axillary and inguinal lymphadenopathy. His abdomen was distended due to enlargement of the liver and spleen, 2 and 4 cm respectively below the costal margin. Haematological findings were: Hb 8.8 g/dl, WCC 18 8oo/p1 (neutrophils IS%, band forms 26%, metamyelocytes 3%, promyelocytes 2%, myeloblasts 5 % ) platelets 15 ooo/p1 and nucleated red cells 5 per IOO WBCs. Marrow aspiration showed marked myeloid hyperplasia. The LAP score was 15. Treatment with corticosteroids and vincristine produced no improvement and he died about 5 months later. Case 3 , A.M., a 3-month-old male infant, presented to Maryfield Hospital, Dundee, in January 1970 with persistent vomiting and failure to thrive. Examination showed a small pale baby with abdominal distension, enlargement of the liver and spleen, each 3 cm below the costal margin, and moderate axillary and inguinal lymphadenopathy. Haematological findings were: Hb 10 g/dl, W C C 64 5001~1(neutrophils48%, lymphocytes 40%, monocytes 12%) and platelets I I 5 ooo/pl. Marrow aspiration showed marked myeloid hyperplasia. A Philadelphia chromosome was not present but an abnormal small Y chromosome was found in both the patient and his father. The LAP score was reduced. Treatment with 6-mercaptopurine, busulphan and dibromomannitol produced little improvement in either his clinical condition, peripheral blood picture or in the size of the spleen. Several episodes of respiratory tract infection occurred during the next year. Radiotherapy to the spleen in July 1970produced no diminution in size and splenectomy was carried out in October 1970 when it had enlarged to 14 cm below the costal margin. Some improvement in his general condition and platelet count was achieved but only temporarily. A pathological fracture of the lower end of the femur occurred in July 1972, episodes of chest infection necessitated admission in September and November 1972 and he died in March 1973 after a period of persistent vomiting. Post mortem examination showed extensive leukaemic infiltration of the brain, lungs, kidneys, lymph nodes, gastro-intestinal mucosa and bone marrow. Case 4,D.V., was born in October 1968, one of uniovular twins. He was much smaller (3 Ib) than his brother ( 5 lb) and showed evidence of intrauterine malnutrition. At 3 months he was seen again at Warwick Hospital with irritability and inadequate weight gain. Undescended testes and hypospadias were noted at this time. By July 1971 he weighed only 22 Ib and limb wasting had become obvious; at that time the haemoglobin value was 11.2 g/dl and a blood film showed normal white cells and platelets. In November 1971 he had a febrile convulsion with an upper respiratory tract infection. Cervical lymphadenopathy and a petechiael rash on his arms were noted. The white cell count had risen to 37 500 pl (polymorphs 75%, lymphocytes 16%, monocytes 6%). He continued to have severe catarrh and by March 1972 large lymph nodes were noted and the spleen was palpable 5 cm below the costal margin. A papular, purpuric rash was present on the forehead. The white cell count had risen to 40 ooo/pl (polymorphs 5 8 % , lymphocytes 28%, monocytes I%, promyelocytes 6%, myelocytes 4%, metamyelocytes 3%) and the platelet count had fallen to 20 ooo/pl. A marrow aspirate showed marked myeloid hyperplasia. A Philadelphia chromosome was not

syr

3mth

3mth 3 yl 4yr

syr

2. R.T.

3. A.M.

4. D.V.

5. M.deL.

M.H.

2

3mth

8. J.S.

9. D.B.

7.

6. Y.K.

yr

2 yr

G.B.

I.

presentation

Case NO.

Age on

M

F

M

M

M

M

M

M

M

Sex

o

100

I

250 11.0

12.5 9.2 10.3 9.2

11.0

10.6

0 100

11.0

60

8.2

9.6 7.7 9.1

10.2

10.2

11.8 8.6

Hb (gldl)

50

5

20

550

450

0

I0

I00

o

Days from presentation

19600

45000 35000

rosm woo00

68000 7200 I5700 17900 48000

42900

:E

308000 68000

15700

s6ooo

IOOOOO

/pl

WCC

8o000