Use of Terminal Deoxynucleotidyl Transferase in the Diagnosis of Leukemia

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 13, No. 2 Copyright 1983, Institute for Clinical Science, Inc. Use of Terminal Deoxynucleotidyl Transferase in the Diagnosis of Leukemia EDWARD E. MORSE, M.D., J. GAFFNEY, B.A., E. DONSKOY, M.D., PATRICIA PISCIOTTO, M.D., A. ALTMAN, M.D., J. QUINN, M.D., and I. G O LD SCHNEID ER, M.D. Division of Haematology, Department of Laboratory Medicine, University of Connecticut Health Center, Farmington, CT ABSTRACT Terminal deoxynucleotidyl transferase (TdT) was determ ined by immunofluorescence in 30 p atien ts w ith leukem ia. In acute lym phocytic leukem ia the proportion of cells positive for TdT was 19 to 77 percent during relapse (12 cases) and less than one percent during rem ission (three cases). In seven cases of m yeloproliferative disease and two cases of lymphoma, the TdT was less than one percent. In one case of generalized lymphoblastic lymphoma and five cases of chronic myelocytic leukem ia with lymphoblastic crisis, the cells positive for TdT w ere moderately increased. The presence of TdT in blast cells appears to have diagnostic, therapeutic, and prognostic significance. Introduction Terminal deoxynucleotidyl transferase (TdT) activity was d iscovered in calf thym us gland during isolation studies of deoxyribonucleic acid replication enzymes.2 It is now known that the purified enzyme has a molecular weight of approximately 58,000 daltons and consists of a single polypeptide chain.4 The enzym e, TdT, catalyzes polym erization of deoxynucleoside triphosphates and does not require a tem plate, unlike the replicative deoxynucleotidyl transferases. However, TdT does require an initiator molecule containing a free 3' hydroxyl group to w hich the 5' deoxynucleotides are added. W hile the enzyme has no proven role, it is found only in im m ature lym phocytes and is thought to be involved w ith the im m une process, possibly by producing diversification of antigen receptors in T and B cells.3 Its specific activity can be m easured using tritiated guanosine triphosphate and polydeoxyadenosine w ith at least th ree resid u es as initiato r. T he nano moles of tritium are measured after incorporation into tricholoracetic acid precipitable material. Functional enzyme activity, found in different locations such as thymus, bone marrow and spleen, correlated well with enzym e localized cells by fluorescent antibody studies.4 Fluorescent antibody, specific for TdT, has demonstrated localization of the enzyme in the majority of cortical thymo /83/ $00.90 Institute for Clinical Science, Inc.

2 cytes and in som e prothym ocytes in spleen and bone marrow. There is also evidence that TdT is present in some pre B cells. In rodents during late embryonic d ev elo p m en t and shortly after b irth, postthym ic T dt positive cells can be fo und tra n sie n tly in th e p e rip h e ra l blood.8 A num ber of authors have reported that TdT is a useful marker in the classification of leukem ic cell lines.7 10,13,15 Our experience has been review ed in 30 patients seen over a one year period in w hich TdT assay by immunofluorescent antibody was carried out. Materials and Methods Patients w ere included in this report if they had TdT m easured as part of their diagnostic workup for lym phoproliferative or myeloproliferative disease. Term inal deoxynucleotidyl transferase was determ ined by im m unofluorescent staining te c h n iq u e,17 and differen tial counts w ere perform ed. Bone marrow and peripheral blood w ere treated with 0.1 M N H 4C1 to lyse erythrocytes, and the m ononuclear fraction was spread on a glass slide by cytocentrifuge. In a few cases, smears of cerebrospinal fluid cells were also prepared (red blood cell lysis was not required). All smears were fixed and m aintained at room tem perature in a d essicato r for 48 to 72 hours b efore staining. Rabbit antibody* to bovine TdT (15 /xl) was la y e re d o v er th e cy to centrifu g e smear and incubated for 30 m inutes. The slides were washed three times in phosphate buffered saline (PBS) and 15 /u.1 fluorescein tagged goat antirabbit IgG was added for 30 m inutes. The slides were again w ashed three tim es in PBS and m ounted w ith FA m ountf at ph 9.5 * A kind gift of Dr. F. Bollum, Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, MD. f Difco Laboratories, Detroit, MI. USE OF TdT IN DIAGNOSIS OF LEUKEMIA 1 29 for counting. The TdT positive cells w ere expressed as a percent of m ononuclear cells in the preparation. Results Twelve patients with acute lymphocytic leukemia (ALL) by morphologic criteria showed TdT positive staining w ith 18 to 77 percent of the cells being fluorescent. Three patients studied during rem ission showed less than one percent TdT positive cells. Eight patients with chronic myelocytic leukemia (CML) in blast crisis w ere observed. In th ree, m yeloblasts predom inated and the TdT activity was not observed. The other five had blasts w ith lym phoid characteristics and dem onstrated eight p ercen t to 80 p ercen t T dt positive cells. O nly one p a tie n t show ed less than 10 p ercen t positive cells. Four patients w ith myeloproliferative disorders and two patients w ith lym phom a show ed less than one p ercent TdT positive cells. O ne p a tie n t w ith g e n e ra liz e d lym phom a sh o w ed 10 percent TdT positive cells in the bone marrow. One patient with CML illustrated the value of th e T dt test in determ in in g therapy. A 15-year-old white male, JP, TABLE I Terminal Deoxyneucleotidyl Transferase in Leukemia D ia g n o sis N Age (Range) M/F TdT P e rc e n t P o s it iv e C e lls Comment ALL* / T, 10 Null Relapse (2-46) ALL* /1 < 1 3 Null Remission (4-16) CMLf /4 < Myeloblasts Blast Crisis (6-65) Predominant Lymphoma 3 2/1 <1-10 Mediastinum (19-26) Jejeunum Generalized ANLL$ or myelo- 4 2/2 < 1 proliferative (36-65) *Acute lymphocytic leukemia fchronic myelocytic leukemia jacute nonlymphotic leukemia

3 1 30 MORSE, GAFFNEY, DONSKOY, PISCIOTTO, ALTMAN, QUINN, AND GOLDSCHNEIDER d ev elo p ed P h 1 chrom osom e positiv e chronic myelocytic leukem ia in late 1979 and re sp o n d e d to b u su lfa n and h y droxyurea. In m id 1981, he experienced an episode of blast crisis with white blood cells (WBC) 95,900 (22 percent blasts, and a p re d o m in an ce o f m y elo b lasts, myelocytes, and prom yelocytes) w hile TdT was negative. The patient showed a gradual response to cytosine arabinoside (ARAC). In January 1982, he again developed a blast crisis with WBC 109,000 (30 percent blasts of which 65 percent were TdT positive. He show ed a partial response to v in cristin e and p rednisone with WBC decreasing to 2,400 w ith eight percent TdT positive cells in February. In March, his white count increased to 232,000 w ith 84 p ercent T dt positive cells in the peripheral blood. Again, he showed a partial response to vincristine and prednisone. A second patient, SG, a 16-year-old w hite male, presented w ith bone pain and a WBC of 38,000 w ith 14 percent blasts. Bone marrow showed many blasts of the L,! type. He responded initially to the usual therapy for lym phoid leukemia. Three years later he showed relapse in marrow and cerebrospinal fluid (CSF) with larger blasts suggesting L2 lymphoblasts or myeloblasts. In addition, TdT was present in 30 percent of the blasts. He was treated with vincristine, prednisone, cyclophosphamide and daunomycin as w ell as intrathecal methotrexate. The CSF did not clear completely nor did the bone marrow show rem ission until he was given in trath ecal ARAC and hydrocortisone, as w ell as systemic ARAC, thioguanine and L asparaginase. W hile the bone marrow rem ained in remission, the patient showed two more CSF relapses, one year and one-and-ahalf years later. Thirty to 50 percent CSF cells were TdT positive. At each remission, the patient responded to intrathecal ARAC and hydrocortisone. A third patient SS, showed evidences of a mixed variety of leukem ia and was a 17-year-old w hite male who presented with fever and pneum onia. The marrow show ed 64 percent blasts w hich appeared to be myeloblasts and were TdT negative. He failed to achieve remission on daunomycin, ARAC, prednisone and vincristine. He was sent home for terminal care on low dose (0.7 mg per kg) of ARAC. He developed m arked m egaloblastic changes after two months and, supported with transfusions, attained complete remission. Three months later he showed a relapse marrow with 20 percent blasts being TdT positive, six percent positive for common acute lymphocyte leukemic antigen (CALLA), and chromosomal abnorm alities in 37 p ercen t of blasts including 5 p translocation to chromosome 15 and deletions of 6q and 12p. Discussion It has now been amply dem onstrated that TdT arises in the early cells of the lym phoid series. W ith few exceptions, leukemic cells w hich are TdT positive have other lymphocyte markers. In some patients, particularly children, w ith undifferentiated acute leukemia, the finding of TdT activity in the blasts may allow reclassification as ALL and appropriate treatment.3 Vogler described patients in whom the blast cells contained TdT in the nucleus and intracytoplasmic IgM, suggesting they were pre B cells.18 O ther studies have dem onstrated that typical B Cell ALL is negative for TdT. Lymphocytes from patients with infectious mononucleosis and mitogen stimulated lym phocytes are also n eg ativ e.3 Reactive lymph nodes and non lymphoblastic lymphomas are usually TdT negative.6 Some confusion exists about the cell of origin of rare cases of acute undifferentiated leukem ia w here morphology and special stains (peroxidase) indicate acute myelocytic leukemia (AML),

4 yet TdT activity is found in greater than 10 percent of the blasts. Tw o such cases w ere found in one series of 40 patients with AML. In one case, auer rods w ere present.3 Similarly, G rogan9 rep o rted th ree p atien ts w ith morphologic features of AML with azurophilic granules and punctate nonspecific esterase activity who had positive blasts for TdT. Only one of the three attained a com plete remission. The patient was a 14-year-old girl who show ed poor response to daunorubicin and ARAC, but developed complete remission after vincristine, prednisone, and adriamycin. One of the other two patients was a 38-year-old female who died 40 days after the start of vincristine, prednisone, and L asparaginase. She had a hypoplastic marrow and died of a fungal septicemia. The last patient was a 33-year-old male who had Phj chromosome positive AML and showed a partial response to adriamycin, vincristine, ARAC, prednisone and later hydroxyurea and 6 m ercaptopurine. He did not attain rem ission and d ied 11 months after diagnosis. These w ere the only three cases observed with myeloid features out of 45 patients w ith ALL observed at two major clinics. Therapeutic implications exist because TdT not only id entifies b last cells as probably lym phoid in nature, but also because TdT positive cells have been shown to be sensitive to steroids while TdT negative stem cells appear to be resistant.3,4 T he TdT positive cells have been observed to regenerate rapidly from pluripotential stem cells in rats. Thus, it seems likely that TdT positive leukemias may respond to steroid treatm ent even when the morphology appears myeloid. Janossy11 reported that even blast crisis in CML responded to steroids and vincristine if the blasts showed a large proportion w ith ALL markers. Thus, TdT was an im portant aid in estab lish in g whether or not the blasts were lymphoid. USE OF TdT IN DIAGNOSIS OF LEUKEMIA 131 There was a positive response by 14/15 patients, w hile 21/25 patients who were CALLA n eg ativ e, and T dt n eg ativ e failed to respond to prednisone and vincristine. In his series, two patients had m yeloblasts m orphologically, b u t had ALL and TdT markers; how ever, four patients had lymphoblasts but w ere ALL and TdT negative. Response to therapy seem ed to correlate with markers rather than with morphology, but the num bers w ere small. These results are consonant with those of Marks and McCaffrey12 but disparate with Srivastava et al.16 Ross et al14 reported that loss of TdT activity in one patient appeared to herald the emergence of resistance to chemotherapy and suggested TdT m ight be a m arker for ch em o th erap y sensitiv ity. W hile these observations may reflect the difference betw een the lym phoid blast cells and m yeloid blast cells, Bertazzoni et a l1 recently reported that a substantial series of patients w ith P h1 positive for CM L morphologically and TdT positive blasts showed a better prognosis (on the average, six months longer life) than similar patients w ith TdT negative blasts. The TdT positive patients were treated w ith vincristine and prednisone w hile the TdT negative patients were treated w ith 6 th io g u a n in e and ARAC. Bertazonni and coworkers suggest, as did Bradstock et al5 that TdT could also be expressed in non lym phoid cells. This may indicate a common precursor cell for CML cells and some types of lymphocytes. Indeed, TdT has become an im portant m arker in the leukem ias. It is helpful w hen the proportion of positive cells is high, because this most often signifies lymphocytic origin of the cells and a sensitivity to vincristine and prednisone. W hile further work is required to clarify the importance of TdT positive cells in m yelogenous leukem ia, it seem s clear that the presence of such cells, particularly in chronic myelogenous leukem ia

5 132 MORSE, GAFFNEY, DONSKOY, PISCIOTTO, ALTMAN, QUINN, AND GOLDSCHNEIDER blastic phase indicates a better prognosis than in TdT negative cases. References 1. B e r t a z o n n i, U., B r u s a m o l in o, E., I s e r n ia, P., S c o v a s s i, A. I., T o r s e l l o, S., L a z z a r in o, M., and B e r n a s c o n i, C.: Prognostic significance of terminal transferase and adenosine deaminase in acute and chronic myeloid leukemia. Blood 60: , B o llu m, F. J.: Oligodeoxyribonucleotide primers for calf thymus polymerase. J. Biol. Chem. 235: PC 18-20, B o l l u m, F. J.: T erm inal deoxynucleotidyl transferase as a hem atopoietic cell marker. Blood 54: , B o l l u m, F. J. and G o l d s c h n e id e r, I.: TdT and lymphocyte differentiation. Membranes, Receptors and the Immune Response. New York, Alan R. Liss Inc., 1980, pp B r a d s t o c k, K. F., H o f f b r a n d, R. V., G a n e - s h a g u r u, K., L l e w e l l i n, P., P a t t e r s o n, K., W o n k e, B., P i z z o l o, G., P r e n t i c e, A. G., B e n n e t t, M., B o l l u m, F. J., a n d J a n o s s y, G.: T e r m in a l d e o x y n u c le o t i d y l tr a n s f e r a s e e x p r e s s i o n in a c u te n o n l y m p h o i d le u k e m i a. An a n a l y s i s b y i m m u n o f l u o r e s c e n c e. B rit. J. H a e m a t. 47: , C a s t e l l a, A., D a v e y, F. R., K u r e c, A. S., and T h o m p s o n, N. A.: Terminal deoxynucleotidyl transferase activity in non hematologic and hematologic neoplasms. Ann. Clin. Lab. Sci. i2: , C o l e m a n, M. S., G r e e n w o o d, M. F., H u t t o n, J. J., B o l l u m, F. J., L a m p k in, B., and H o l l a n d, P. H.: Serial observations on term inal deoxynucleotidyl transferase activity and lymphoblast surface markers in acute lymphoblastic leukemia. Cancer Res. 36: , G o l d s c h n e i d e r, I.: O ntogeny of term inal deoxynucleotidyl transferase containing lymphocytes in rats and mice. Terminal Transferase in Immunobiology and Leukemia. Bertazzoni, U., ed. New York, Plenum Press, 1982, pp G r o g a n, T. M., I n s a l a c o, S. J., S a v a g e, R. A., and V a i l, M. L.: ALL w ith prom inent azurophilic granulation and punctate acidic nonspecific esterase and phosphatase activity. Amer. J. Clin. Path. 75: , H o f f b r a n d, A. V., G a n e s h a g u r u, K., J a n o s s y, G., G r e a v e s, M. F., C a t o v s k y, D., and W o o d r u ff, R. K.: Terminal deoxynucleotidyl transferase. Levels and membrane phenotypes in diagnosis of acute leukemia. Lancet 2: , J a n o s s y, G., W o o d r u f f, R. K., P ip p a r d, M. J., P r e n t ic e, G., H o f f b r a n d, A. V., P a r t o n, A., L i s t e r, A., B u n c h, C., and G r e a v e s, M. F.: Relation of lymphoid phenotype and response to chemotherapy. Cancer 43: , M a r k s, S. M., B a l t im o r e, D., and M c C a f f r e y, R.: Terminal transferase as a predictor of initial responsiveness to Vincristine and Prednisone therapy in the blast crisis of chronic myelocytic leukem ia. New Eng. J. Med. 298: , M c C a f f r e y, R., S m o l e r, D., and B a l t im o r e, D.: Terminal deoxynucleotidyl transferase in a case of childhood lym phoblastic leukemia. Proc. Nat. Acad. Sci. 70: , Ross, D. D., W ie r n ik, P. N., S a r in, P. S., and W h a n g - P e n g, J.: Loss of TdT activity as a predictor of emergence of resistance to chemotherapy in a case of CML in blast crisis. Cancer 44: , S a r i n, P. S. and G a l l o, R. C.: T erm inal deoxynucleotidyl transferase in chronic m yelogenous leukem ia. J. Biol. Chem. 249: , S r iv a s t a v a, B. J. S., K h a n, S. H., M in o w a d o, J., G o m e z, G. A., and R a k o w s k i, I.: TdT activity in blastic phase of chronic myelocytic leukemia. Cancer Research 37: , S t a s s, S. A., S h u m a c h e r, A. R., K e n e k l is, F. P., and B o l l u m, F. J.: Terminal deoxynucleotidyl transferase immunofluorescence on bone marrow smears: Experience in 156 cases. Amer. J. Clin. Path. 72: , V o g l e r, L. B., C r i s t, W. M., B o c k m a n, D. E., P e a r l, E. R., L a w t o n, A. R., and C o o p e r, M. D.: Pre B cell leukemia. A new phenotype of childhood lym phoblastic leukem ia. New Eng. J. Med. 298: , 1978.