British Journal 4 Haematology, 1981, 49, 479-481.

Short Communication THYMIDINE KINASE ISOENZYMES IN

CHRONIC LYMPHOCYTIC LEUKAEMIA

SUMMARY. The profile of thymidine kinase isoenzymes was determined in peripheral blood lymphocytes from 14 patients with chronic lymphocytic leukaemia (CLL) and 31 controls. Twelve patients with indolent disease showed TK2 isoenzyme activity, while two patients in whom the disease evolved and two patients who presented with aggressive disease exhibited TK1 isoenzyme activity. The demonstration of TK1 activity in the peripheral blood lymphocytes of clinically aggressive CLL suggests that this isoenzyme may be a useful biochemical marker of such behaviour.

Thymidine kinase (TK) catalyses the phosphorylation of thymidine to thymidine monophosphate, an essential precursor of DNA-thymine. In human cells, the enzyme occurs as two isoenzymes designated TKl and TK2 (Ellims et al, 1981). TK1 activity closely parallels changes in cell DNA synthesis, with a marked increase occurring with transition of the cell from the dormant to the dividing state (Adler & McAuslan, 1974). In contrast, TK2 activity remains relatively constant throughout the cell cycle (Adler 8i McAuslan, 1974). We have recently demonstrated for non-Hodgkin’s lymphoma (NHL) a correlation between T K isoenzyme type and cell morphology, in that cell immaturity is associated with the appearance of the TKl isoenzyme and a stepwise increase in this activity with progressive degrees of cell dedifferentiation (Ellims et al, 1981). In this study TK isoenzyme activities were examined in peripheral blood lymphocytes of patients with chronic lymphocytic leukaemia (CLL) in an attempt to determine the possible value of the isoenzymes as markers of clinical behaviour.

MATERIALS AND METHODS

Peripheral blood lymphocytes were isolated by centrifugation on Ficoll-Hypaque and thymidine kinase assayed as previously described (Ellims et al, 1981). TKl and TK2 isozyme activities were identified on the basis of biochemical properties-known to distinguish between the two (Ellims et al, 1981). The TKl isozyme has a specificity for adenosine triphosphate (ATP) as the phosphate donor; while TK2 also uses cytidine triphosphate (CTP), with activity reaching 7 0 4 0 % of that obtained with ATP. Deoxy-CTP produces only 15-20% inhibition of TK1 ATP-mediated activity; but TK2 activity decreases by 7&80%. Thymidine triphosphate (TTP) markedly inhibits (80-90%) the activity ofboth isozymes. At pH 5.0, TK1 activity decreases by 60-70% of that measured at pH 7.4, while TK2 activity falls by only 15-20%. The ratio of enzyme activity with CTP as the phosphate donor

Correspondence: Dr Peter H. Ellims, Monash University Department of Medicine, Alfred Hospital, Commercial Road, Prahran 31 81, Victoria, Australia.

0007-1048/81/1100-0479802.00 0 1981 Blackwell Scientific Publications

479

480 P. H . Ellims, T. Eng G u n and M . B. Van der Weyden

compared to that obtained with ATP was used to discriminate between predominance of TK1 or TK2 isoenzyme activity with a ratio of less than 0.4 considered to indicate predominance of TKl activity (Ellims et a!, 1981).

RESULTS AND DISCUSSION

The control group included 14 healthy donors and 17 patients with non-neoplastic diseases. Control lymphocytes were found to exhibit TK2 activity (mean CTP/ATP 0-71, range 0.63-0.85), with the mean value 0.18 nmol/h/mg protein, range 0.09-0.25. The clinico- pathological findings of the 14 patients with CLL, grouped according to peripheral blood lymphocyte TK status, are shown in Table I. Twelve patients had peripheral blood lymphocyte TK2 isoenzyme activity which is consistent with the predominant small mature peripheral blood lymphocyte characteristic of the disease. In these patients the majority of peripheral blood lymphocytes showed faint immunofluorescence for monoclonal IgM or IgM/IgD. Four patients were found to have TKl isoenzyme status. Two patients initially showed TK2 activity but TKl activity was detected after observation for 2 years and 3 years respectively. Two patients exhibited TKl activity at presentation. In all four patients, peripheral blood lymphocyte TK1 isoenzyme activity was associated with the presence in the peripheral blood of a pleomorphic sub-population of immature lymphocytes resembling prolymphocytes and lymphoblasts. The peripheral blood lymphocytes from three of these patients showed faint immunofluorescence for monoclonal IgM or IgM/IgD but in one patient, change from TK2 to TK1 isoenzyme status was associated with loss of this pattern. Patients with TKl isoenzyme status had Rai clinical stage IV disease (Rai et al, 1975) which in three patients was resistant to intermittent chlorambucil and prednisolone therapy (Sawitsky et al, 1977). Partial control of the disease was achieved by combination chemotherapy with cyclophosphamide, daunorubicin, vincristine and prednisolone together with intermittent

TABLE I. Clinicopathologic findings in patients with CLL according to peripheral blood lymphocyte thymidine kinase findings

Thymidine kinase status

TK2 TK1

No. of patients Age: years (mean) Sex (M/F) Peripheral lymphocytes x 109/1 (mean) Rai clinical stage

0-111 IV

(nmol/h/mg protein) Peripheral blood lymphocyte thymidine kinase

CTP/ATPt

12 4 70 (60-81)* 72 (55-73)

39 (11-72) 76 (30-93)

9 0 3 4

2/ 1 3/'

0.16 (0.10-0.25) 0.62 (0.20-1.5) 0.8 (0.7-0.9) 0.3 (0 .21435)

* Mean value; figures in parentheses indicate the ran e. t Ratio of enzyme activity measured with CTP to t a at obtained with ATP.

Thymidine Kinase Isoenzymes in CLL 48 1

leukapheresis, but over a period ranging from 6 months to 13 months all three patients died, one from disseminated Herpes zoster and two from septicaemia. The remaining patient with TKl isoenzyme status has had a splenectomy and the disease has been partially controlled for 25 months with intermittent chlorambucil and prednisolone therapy.

A change from a clinically indolent to aggressive disease is a well-documented occurrence for chronic lymphocytic leukaemia and has been associated with the appearance of immature lymphocytes in the peripheral blood (Enno et al, 1979) and enhanced in uitro leucocyte thymidine uptake (Moayeri & Sokal, 1979). The detection of peripheral blood lymphocyte TK1 isoenzyme activity in this study is consistent with this reported cytological immaturity and enhanced spontaneous thymidine uptake. In addition the appearance of the TK1 isoenzyme in CLL appears to be a potential biochemical marker for clinically aggressive disease, a concept which is supported by the finding of TKl activity in peripheral blood leucocytes of patients with lymphosarcoma cell leukaemia (Ellims et al, 1981).

ACKNOWLEDGMENTS

Lymphocyte immunofluorescence studies were performed by members of the Clinical Immunology Unit, Peter M c C a h m Clinic, Melbourne. This study was supported by the Anti-Cancer Council of Victoria and the National Health and Medical Research Council of Australia.

Monash University Department of Medicine, A p e d Hospital, Prahran, Victoria, Australia

PETER H. ELLIMS T. ENG GAN MARTIN B. VAN DER WEYDEN

REFERENCES

ADLER, R. & MCAUSLAN, B.R. (1974) Expression of thymidine kinase variants as a function o f the replicarive state of cells. Cell, 2, 113-117.

ELLIMS, P.H., VAN DER WEYDEN, M.B. & MEDLEY, G. (1981) Thymidine kinase isoenzymes in human malignant lymphoma. Cancer Research, 41, 691495.

ENNO, A., CATOVSKY, D., O’BRIEN, M., CHERCHI, M., KUMARAN, T.O. & GALTON, D.A.G. (1979) ‘Prolyrnphocytoid’ transformation of chronic lymphocytic leukaemia. British Journal .f Haema- tology, 41, 9-18.

MOAYERI, H. & SOKAL, J. (1979) In uitro leukocyte

thymidine u take and prognosis in chronic lym-

66, 773-778. RAI, K.R., SAWITSKY, A., CRONKITE, E.P.,

CHANANA, A., LEVY, R.N. & PASTERNAK, B.S. (1975) Clinical staging of chronic lymphocytic leukemia. Blood, 46, 21 9-234.

SAWITSKY, A., RAI, K.R., GLIDEWELL, O., SILVER.

(1977) Com arison of daily versus intermittent

treatment of patients with chronic lymphocytic leukemia. Blood, 50, 1049-1059.

phocytic leu E emia. American journal of Medicine,

R.T. & PARTICIPATING MEMBERS OF CALGB

chlorambuci P and prednisolone therapy in the