FORMES FRUSTES IN MYELOPROLIFERATIVE DISORDERS

14

other hand, was more specific, binding only to the glioma cells. No significant binding of these supernatants was detected on microtitre plates treated as for a standard assay but without the addition of any cells. None of these supernatants was found to bind to a non-transformed human fibroblast line (MRC5).

FORMES FRUSTES IN MYELOPROLIFERATIVE DISORDERS Identification by the Growth of an Endogenous Erythroid Clone In Vitro in Patients with Arterial Vascular Disease C. D. REID

Discussion

Malignant gliomas

are

often infiltrated

by lymphocytes

which may have a role in host defence against the tumour. By using the hybridoma technique we have demonstrated that B lymphocytes from within gliomas can be immortalised and cloned for further study. The frequency of successful fusion is low but the resulting hybrids are stable. The new immunoglobulins produced by the fused cells represent those released in the vicinity of the tumour in vivo and are thus of considerable clinical interest. Patients with gliomas may produce circulating antibodies to their own tumours, i4 and in this study we have shown that antibodies which bind to glioma cells are produced by the lymphocytes within the tumour. The antigenic determinants recognised are as yet unknown but may well be cell-surface molecules expressed in greater quantities in rapidly growing tumour cells.

Large amounts of supernatant can be obtained and concentrated from growing hybridomas. Such concentrated supernatants are suitable for the detailed immunochemical characterisation of the glioma cell surface. Furthermore such antibodies may be isotopically labelled and used for the localisation of intracerebral disease by brain scanning as well as being potentially useful for targeting toxic agents to the tumour cell surface. We thank Mr John Ellis for his expert technical assistance and Dr P. Edwards, Dr M. O’Hare, and Prof. M. Neville for providing LICR-LONHMy2 and their helpful discussion. Mr P. Carey and Mr J. Lanigan kindly contributed patients to this study.

Correspondence

should be addressed

to

K. S

REFERENCES

Bergevin PR. Brain In. Bergevin PR, Blom J eds Guide to therapeutic oncology Baltimore: Williams and Wilkins 1979 492-96 2 Apuzzo ML, Mitchell MS Immunological aspects of intrinsic glial tumors J 1.

Neurosurg 1981; 55: 1-18 Makaley MS, Brooks WH, Roszman TL, Bigner DD, Dudka L, Richardson S. Immunobiology of primary intracranial tumors. J Neurosurg 1977; 46: 467-76. 4 Garson GA, Quindlen EA, Kornblith PL Complement fixation by IgM and IgG autoantibodies on cultured human glial cells.J Neurosurg 1981; 55: 19-26 5. Pfreundschuh M, Shiku H, Takahashi T, Ueda R. Ransohoff J, Oettgen HF, Old LJ. Serological analysis of cell surface antigens of malignant human brain tumors. Proc 3

Natl Acad Sci 1978, 75: 5122-26 6. Phillips JP, Eremin O, Anderson J. The presence of lymphoreticular cells in human brain tumours and in normal brain Br J Cancer (in press) 7 Schnegg JF, Diserens AC, Carrel S, Accolla RS, de Tribolet N. Human glioma associated antibodies detected by monoclonal antibodies Cancer Res 1981, 41: 1209-13 8. Wikstrand CJ, Pegram CN, Bourdon MA Expression of human fetal brain antigens by human glioblastoma cells as defined by monoclonal antibodies. Proc Am Ass Can Res

1981; 22: 1206 (Abstr.) 9.

Sikora K, Phillips J Human monoclonal antibodies to glioma cells. Br J Cancer 1981; 43: 105-07

10 Edwards PAW

Smith CM, Munro Neville A, O’Hare MJ A human-human system based on a fast-growing mutant of the ARH-77 plasma cell leukaemia-derived line. Unpublished 11 Littlefield JW Selection of hybrids from matings of fibroblasts in vitro and their preserved recombinants Science 1964, 145: 709 12. Kennett RH Fusion by centrifugation of cells suspended in polyethylene glycol. In Kennett RH, McKearn TJ, Bechtol KB, eds. Monoclonal antibodies. New York: Plenum Press. 1980: 365-67 1 3. Watson JV. Dual laser beam focussing for flow cytometry through a single crossed cylindrical lens pair Cytometry 1981; i: 14-19 14 Phillips JP, Sujatanond M. Martuza RL, Quindlen EA, Wood WC. Kornblith PL, Dohan Jr FC Cytotoxic antibodies in pre-operative glioma patients. A diagnostic assay Acta Neurochir 1976; 35: 43-52

hybridoma

I. CHANARIN

J. LEWIS Clinical Research Centre and Northwick Park Middlesex

Hospital, Harrow,

The myeloproliferative disorders, including polycythaemia rubra vera, arise as a result of a single-cell mutation. A characteristic of the abnormal haemopoietic clone is that it can form erythroid colonies in vitro in the absence of added erythropoietin. Such endogenous erythroid clones were consistently found in two of seven patients with peripheral vascular disease. These two patients had mean platelet counts of 600 x 109/l and 630 x 109/l Culture of blood and bone-marrow cells from patients with raised platelet counts secondary to a variety of other disorders failed to yield such colonies. The presence of endogenous erythroid clones provides early evidence of a myeloproliferative disorder.

Summary

Introduction POLYCYTHAEMIA rubra vera, chronic myelofibrosis, essential thrombocythaemia, and chronic myelocytic leukaemia, are often grouped together as myeloproliferative disorders. They result from a single-cell mutation, as has been demonstrated in women heterozygous for the X-chromosome-linked isoenzymes of glucose-6-phosphate dehydrogenase (G-6-PD). Normal cells have either the A or the B G-6-PD variant. In the mutant there is segregation of either the A or the B G-6-PD isoenzyme into the clone of abnormal cells, which implies that it originates from a single

cell.I In-vitro culture of haemopoietic progenitor cells reveals a further characteristic of these disorders. In culture of cells from normal subjects erythroid colonies develop only when substantial amounts of erythropoietin, a hormone which stimulates erythropoiesis, are added.2 However, progenitor cells from patients with myeloproliferative disorders can grow in vitro in the absence of added erythropoietin2-S and give rise to so-called endogenous erythroid colonies (EEC). These abnormal clones have been demonstrated in all the

myeloproliferative disorders.6,7 The mutation that will lead eventually to one of the myeloproliferative disorders must occur months and even years before the clinical and haematological features, but the early and also the atypical presentation of these disorders should be recognisable by the demonstration of EEC in, culture. We describe the presence of such EEC in cultures of cellsI from two patients with peripheral vascular disease whose only haematological abnormality was a slight elevation of the, platelet count and in cultures of cells from a young woman with a very large spleen due to splenic-vein thrombosis as the only abnormality in an otherwise normal pregnancy.

Patients and Methods Patient 1 A 62-year-old man presented with long-standing ischaemic changes in the toes of both feet. Ischaemia of his right toes was noted

15 first in 1972; and in August, 1979, because of recurrent infections of both feet, a bilateral lumbar sympathectomy was performed. The left third toe was amputated in February, 1980. On admission in January, 1981, the left first and second toes were blue and tender, but all peripheral pulses were readily detectable. There were no other physical findings. An arteriogram showed sluggish flow in the metatarsal arteries, and the digital arteries were not visualised. A peripheral-blood count showed:

haemoglobin (Hb),

15.1g/dl

;

10.3 xl 09 II; platelets,

haematocrit, 0-45; white-cell count,

(range, 545-630 x 109/1); and erythrocyte sedimentation

630 x 109/1 rate, 15 mm/h. thrombin, and

Cryoglobulins

were not

detected. Prothrombin,

partial thromboplastin times were all normal. was 236 g/dl, and antithrombin III 95%; platelets Fibrinogen showed no spontaneous aggregation and aggregated normally in response to adrenaline, ADP, and collagen. Red-cell mass was 32’ 1 ml/kg and plasma volume was 41 .7. ml/kg; both are normal values. The serum uric acid concentration was raised (542 mol/1). The bone-marrow trephine showed a patchy increase in megakaryocytes and a slight increase in reticulin. Iron was present. Patient 2 A

60-year-old woman presented in August, 1980, with a

5-week

history of a painful left calf and thigh. No pulses were detected in the left leg below the femoral artery, and a pulsed ultrasound scan showed a 4

cm

block in the femoral artery.

A

peripheral-blood count showed: Hb, 14.44 g/dl ; haematocrit, 0-44; white-cell count, 9 -4 x 109/1 ; and platelets 635 x 109/1 (range, 520-779 x 109/1). Prothrombin, thrombin, and partial thromboplastin times were normal, and fibrinogen was 224 g/dl. Platelets showed

no

spontaneous

aggregation

and

responded

normally ADP, adrenaline, and collagen. Uric acid was 265 pmol/l. The red-cell mass was 28 -5ml/kg and plasma volume 35’ 6 ml/kg; both these values are normal. The leucocyte alkaline phosphatase (LAP) score was within the normal range (47). Colloid scan of liver and spleen was normal. Bone-marrow trephine showed to

increase in megakaryocyte numbers and paratrabecular fibrosis. Iron was present. an

Patient 3 At an antenatal clinic in May, 1979, this 26-year-old woman was noted to have a massive splenomegaly (20 cm below the costal margin), which was firm and non-tender. For at least 10 years she had dried herself after a bath by evaporation, since towelling caused very unpleasant skin irritation. She was otherwise well. The blood count showed: Hb, 11.3 3 g/dl ; normal red-cell indices; white-cell count, 4-4x10/1; and platelet count, 105 x 10911. Bone-marrow aspirate and trephine were normal and, as she was well, no

investigations were undertaken during her pregnancy, which was uncomplicated, with normal labour and a healthy infant. In August, 1980, splenectomy was carried out. Histology of the spleen showed congestion and splenic-vein thrombosis. There were no megakaryocytes. Over the next year her platelet count increased to over 2000 x 109/1, and her red-cell mass rose to 34 ml/kg; her arterial oxygen level and oxygen affinity of haemoglobin were normal. There were pronounced increases in cellularity and megakaryocyte numbers of the bone marrow. The LAP score was 130 (normal range, 35-100). Polycythaemia rubra vera was diagnosed.

Erythroid Colony Cultures Cultures were carried out in methylcellulose from the mononuclear cells of peripheral blood and bone marrow. The details have been described elsewhere.8 Bone-marrow colonies (CFU-E) were scored after 7 days of culture, and both peripheralblood and bone-marrow erythroid bursts (BFU-E) at 14 days. For patients 1 and 2 there were EEC in all cultures carried out, and these were most marked in peripheral blood (see table). In contrast, there was no colony growth in the absence of added in cultures of peripheral-blood mononuclear cells from four normal volunteers or in cultures of bone-marrow cells from seven other subjects (four normal volunteers and three patients without haematological disorder). Cultures from six patients with secondary thrombocytosis (platelet counts, 500-1100x109/1) associated with malignancy or chronic inflammatory disease (three bone-marrow and six peripheral-blood cultures) also failed to grow colonies in the absence of erythropoietin, and spontaneous colony growth was not seen in peripheral-blood cells from five randomly selected patients with peripheral vascular disease but without haematological

erythropoietin

abnormality. Patient 3 was first studied before the techniques for erythroid culture were established in this laboratory, and when her peripheral-blood count was normal. At that time we could not establish erythroid colony growth from either normal subjects or patients with the exception of patient 3, whose cells grew readily under conditions that were clearly far from optimum. Subsequently (after splenectomy and the appearance of thrombocytosis) EEC were cultured from both her peripheral blood and bone marrow.

Discussion Neither the first two patients nor patient 3 at presentation fulfilled the criteria for the diganosis of a myeloproliferative disorder. For the diagnosis of essential thrombocythaemia the minimum requirement stipulated by the Polycythaemia Vera Study Group9 is a platelet count in excess of 1000 x 109/1

NUMBERS OF ERYTHROID COLONIES GROWN IN METHYLCELLULOSE CULTURE FROM BONE-MARROW AND PERIPHERAL-BLOOD CELLS WITH AND WITHOUT ADDED ERYTHROPOIETIN

Epo +=

in parentheses

with no added erythropoietin. Figures with added erythropoietin (2 IU/ml); Epoare percentage EEC of total colonies of each type. *Results of cultures of 10 marrow and 15 penpheral-blood samples from normal volunteers, patients with peripheral vascular disease, and patients with secondary

thrombocytosis.

=

16

(in the absence of any underlying cause). Additional features are splenomegaly (in 50% of patients), neutrophil leucocytosis, and a history of bleeding. Hypochromic anaemia is not uncommon.’" Our three patients showed none of these features, other than splenomegaly in patient 3. The only pointers to a diagnosis of a myeloproliferative disorder were hyperuricaemia in one patient, a possible increase in megakaryocyte numbers in the bone marrow, and a slight increase in bone-marrow fibrosis. In the absence of other confirming data these findings are totally inadequate to support a diagnosis of myeloproliferative disorder. Plateletfunction tests were unhelpful. The normal red-cell mass (with iron-replete bone marrow) in patients 1 and 2 ruled out polycythaemia. The observation of EEC in cultures of both bone-marrow and peripheral-blood progenitor cells makes the existence of myeloproliferative disease virtually certain. Although there has been one report of spontaneous colony growth from normal bone marrow,12 other studies have not found such growth,3-6,13 and we have not observed it. The abnormal progenitors in the myeloproliferative disorders may be. extremely sensitive to the very low levels of erythropoietin in the culture medium, rather than being truly independent of

erythropoietin.4 Spontaneous bleeding and vascular thrombosis are features of the myeloproliferative disorders. Although the tendency to bleed may relate to defective aggregation and adhesion of platelets, the nature of the thrombotic tendency is less clear; both seem to correlate with platelet numbers. 14 The demonstration that a subgroup of patients with peripheral vascular disease show characteristics of a myeloproliferative disorder has considerable implications for their management. The platelet’ count can be restored to normal by administration of radioactive phosphorus, and this may be beneficial in the long term. Correspondence should be addressed to I. C., Northwick Park Hospital and Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ. REFERENCES 1. Adamson

JW, Fialkow PJ, Murphy S, Prchal JF, Steinmann L. Polycythaemia vera: cell and probable clonal origin of the disease. N Engl J Med 1976; 295: 913-16. 2. Eaves CJ, Eaves AC. Erythropoietin (Ep) dose-response curves for three classes of erythroid progenitors in normal human marrow and in patients with polycythaemia vera. Blood 1978; 52: 1196-1210. 3. Prchal JF, Axelrad AA. Bone marrow responses in polycythaemia rubra vera. N Engl J stem

4. 5.

Med 1974; 290: 1382-85. Zanjani ED, Lutton JD, Hoffman R, Wasserman LR. Erythroid colony formation by polycythaemia vera bone marrow in-vitro. J Clin Invest 1977; 59: 841-48. Prchal JF, Adamson JW, Murphy S, Steinmann L, Fialkow PJ. Polycythaemia vera, the in-vitro response of normal and abnormal stem cell lines to erythropoietin. J Clin

Invest 1978; 61: 1044-47 JD, Levere RD. Endogenous erythroid colony formation by peripheral blood mononuclear cells from patients with myelofibrosts and polycythaemia vera. Acta Haematol 1979; 62: 94-99. 7. Eaves AC, Eaves CJ. Abnormalities m the erythroid progenitor compartments in patients with chronic myelogenous leukaemia. Exp Haematol 1979; 7 (suppl 5): 65-75. 8. Reid CDL, Baptista LC, Deacon R, Chanarin I. Megaloblastic change is a feature of colonies derived from an early erythroid progenitor (BFU-e) stimulated by monocytes in culture. Br J Haematol (in press). 9. Laszlo J. Myeloproliferative disorders (MPD): myelofibrosis, myelosclerosis, extramedullary haematopoiesis, undifferentiated MPD and haemorrhagic thrombocythaemia. Semin Haematol 1975; 12: 409-32 10. Gunz FW. Haemorrhagic thrombocythaemia: a critical review. Blood 1960, 15: 6. Lutton

706-23. 11. Ozer FL, Truax

WE, Miesch DC, Levin WC. Primary haemorrhagic thrombocythaemia. Am J Med 1960; 28: 807-23. 12. Golde DW, Bersche H, Cline MJ. Polycythaemia vera: hormonal modulation of erythropoiesis in-vitro. Blood 1977; 49: 399-405 13. Lacombe C, Cassedevall N, Varet B, Polycythaemia vera: in-vitro studies of circulating erythroid progenitors Br JHaematol 1980; 44: 189-99 DA. 14. Triplett Quantitative disorders of platelets thrombocytosis and thrombocythaemia. In: Triplett DA, Harms CS, Newhouse P, Clark C, eds. Platelet function Chicago American Society of Clinical Pathologists. 1978: 179-92.

Preliminary Communication SENSITIVITY OF HUMAN NATURAL KILLER CELLS TO HYPERTHERMIA

J. AZOCAR

E. J. YUNIS M. ESSEX

Sidney Farber Cancer Institute and Department of Microbiology, Harvard University School of Public Health, Boston, Massachusetts, U.S.A.

Division of Immunogenetics,

Summary

Significantly less natural killer (NK) activity is exhibited by human lymphocytes when

temperatures above 37°C. directly on the NK effector cells. Hyperthermia The effect is partially abrogated by treatment of the heated the NK assay is carried

out at

seems to act

cells with human

leucocyte interferon. INTRODUCTION

CELL-MEDIATED immunity is widely thought to play a major part in the control of infections. Natural killer (NK) cells, a subset of lymphocytes, have the ability preferentially to kill virus-infected cells and tumour cells without prior sensitisation.I-6 NK cells, which have been well characterised in the mouse and in man, show no majorhistocompatibility-complex restriction for their cytotoxic effect. 1-7 It has been proposed that NK cells are a first-level mechanism for protection against tumour growth, and a correlation between defective NK activity in vivo and an increased incidence of malignant diseases both in human beings and in mice supports this concept" NK activity can be modified in vitro by interferon, prostaglandins, and metabolic inhibitors. 11-14 Because fever is common in viral infections and often occurs during the course of neoplastic and autoimmune diseases, we studied the effect of hyperthermia on the in-vitro NK activity of human

lymphocytes. METHODS

Effector NK cells were obtained from the peripheral blood of a healthy donor. Lymphoid cells were separated on a ’FicollHypaque’ gradient as described by B6yum, 15 washed three times with RPMI 1640 medium, and finally resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal-calf serum. K-562, a human myeloid leukaemia cell line used as target cells,16 was cultured in disposable polystyrene flasks in RPMI 1640 medium supplemented with 20% fetal calf serum (Grand Island Biological Co.) at 37°C in an atmosphere with 5% CO2 for 4 days before they were used for the NK assay; each flask contained 10 ml of cell suspension with an initial cell concentration of 5 x 105 cells/ml. Cytotoxicity was determined in triplicate by measuring chromium-51 release from labelled K-562 target cells. Effector and target cells were mixed in different proportions in a total volume of 200 µl of RPMI 1640 with 10% fetal calf serum and 5 x 10-5mo1l1 2-mercaptoethanol in standard conical 96-hole microplates (Limbro Scientific Inc.). Identical plates were incubated at 37°C, 38 - 5°C, or 40°C, in an atmosphere with 5% CO2 for 4 h. 100 µl of supernatant was then collected from each well, and cytotoxicity was determined from the following formula: control supernatant—cpm ininmedium cpm in intesttestsupernatant—cpm medium control Specific lysis Specific lysis (%) =cpm maximum release-cpm in medium control

x x100 100

cpm

There was always less than 20% spontaneous release, and cpm in triplicates of the test varied by less than 5%. Effector cells were also