Terminal deoxynucleotidyl transferase activity in childhood leukemia

The activity of terminal deoxynucleotidyl transferase (Tdt) was measured in mononuclear cells of 70 children with acute leukemia, in whom raised levels of Tdt were noted at diagnosis, relapse, or during remission. Serial measurements of Tdt activity were related to the percentage of blasts in the bone marrow and the clinical course of children both during therapy and after its completion. The Tdt values did not predict relapse in children with acute lymphoblastic leukemia nor did the quantitative determination correlate with the percentage of blasts in the bone marrow.     

Terminal deoxynucleotidyl transferase positive lymphoblastic lymphoma: a study of 15 cases.

The investigation was undertaken to define the features of lymphoblastic lymphoma. Fifteen lymph node biopsies from a group of 82 specimens studied for the enzyme terminal deoxynucleotidyl transferase (TdT) fulfilled morphological criteria for this diagnosis. These criteria required a diffuse infiltrate of relatively uniform, immature lymphoid cells with basophilic cytoplasm; round, oval or lobulated nuclei with evenly dispersed chromatin; rare or inconspicuous nucleoli; and numerous mitotic figures. Examination of 1-micron thick, plastic-embedded, Giemsa-stained tissue sections revealed convoluted nuclei in more than 50% of neoplastic cells in four cases: in six specimens there was an admixture of cells with grooved, hyperlobulated, and round nuclei, and in five the round or oval nuclei were non-convoluted. Specimens from all 15 patients were positive for TdT by fluorescent antibody and biochemical assays. The percentage of cells from involved nodes reacting by indirect immunofluorescence with an antiserum against bovine TdT ranged from 4 to 90% (mean of 52%), and the mean level of biochemically measured enzyme activity was 8.7 units/g of tissue (range of 1.9 to 27.5). Cytochemical stains for acid phosphatase were positive in 13 of the 15 cases. In eight samples more than 50% of cells formed rosettes with sheep erythrocytes, while the E rosettes varied from 14 to 38% in the other seven. The percentage of cells with complement receptors varied widely (range of 6 to 80), but cells bearing surface immunoglobulin or IgGfc receptors were not increased. All patients presented with supradiaphragmatic lymphaedenopathy, eight with an anterior mediastinal mass. Two-thirds of the patients were male, and the mean age was 20 years (range 4 to 46 years). None were leukemic at the time of diagnosis, but eight patients subsequently developed acute lymphoblastic leukemia. Involvement of the central nervous system was observed in four of the 15, and of the testes in two. Ten patients have died of their disease with a median survival of 8 months (range 4 to 20), and five are alive 3--8 months after diagnosis. We observed no differences in clinical findings at presentation, incidence of mediastinal involvement or leukemic dissemination, content of TdT, acid phosphatase staining, or immunologic cell surface characteristics between the convoluted and non-convoluted types of lymphoblastic lymphoma. Distinctive morphologic, cell surface, biochemical, and clinical features of lymphoblastic lymphoma can be identified irrespective of the presence or absence of convoluted nuclei.     

Terminal deoxynucleotidyl transferase in acute myeloid leukaemia

Between January 1980 and May 1981, 1966 marrow or blood samples from leukaemia patients were tested for terminal deoxynucleotidyl transferase (TdT) using nuclear immunofluorescence. The cells were also tested with a panel of immunological markers including monoclonal antibodies. Of 869 TdT positive cases detected, 555 were diagnosed as ALL and 32 as blast crisis of CGL; 226 were provisionally diagnosed as ‘acute leukaemia’ and finally diagnosed as ALL partly on the basis of immunological data; 56 TdT⁺ cases were provisionally diagnosed as acute non-lymphocytic or myeloid leukaemia; 266 cases of AML and 177 cases of CGL in blast crisis were TdT negative.Eleven of the above ‘AML’ cases were anti-cALL⁺ as well as TdT⁺ and were re-diagnosed and treated successfully as cALL. The remaining 45 were anti-cALL negative and finally diagnosed and treated, at least initially, as AML. Eleven of these cases had only 5–10% TdT⁺ cells which could have been normal, non-myeloid cells. Twenty cases had 11–50% TdT⁺ cells and 14 cases had 50–100% TdT⁺ cells. Of these latter two groups, details on 28 patients were available for evaluation. Three cases on review had no definitive myeloid cytochemistry and were haematologically AUL with a null-ALL phenotype (TdT⁺ DR⁺ cALL^?). In 14 cases there was a large overlap (>75%) of the proportion of cells with myeloid cytochemistry (Sudan black, peroxidase or esterases) and TdT; individual blast cells were therefore expressing these markers concurrently. In the remaining cases, mixtures of TdT negative myeloid and TdT⁺ (lymphoid?) cells may have co-existed although this was not proven unequivocally. Twenty-two cases of newly diagnose TdT⁺ ‘AML’ received induction chemotherapy for AML (DAT regime) and only six (37%) obtained a complete remission.It is concluded that TdT positive ‘myeloid’ leukaemias do occur, albeit infrequently (approx. 5%) and may have a relatively poor prognosis.     

Terminal deoxynucleotidyl transferase activity and blast cell characteristics in adult acute leukemias

Terminal deoxynucleotidyl transferase (TdT) measurements in patients with leukemias and other hematopoietic disorders were correlated with cell surface phenotypes and hematological and cytochemical characteristics.High TdT activity was found in thymus and in all T-and non-T/non-BALL patients in active phase. Low TdT activity (< 2 U/mg DNA) was found in blood or bone marrow from normal persons. ALL patients in remission, immunologically characterized T-cell disorders (T-cell CLL, Sezary syndrome, mycosis fungoides and infectious mononucleosis). B-cell ALL, most AML and other hematopoietic disorders. DNA polymerase activity was high in immature cells when compared to mature leukocytes. About $\text{1}\text{3}$ of AML patients which had high TdT activity were indistinguishable from other AML patients on the basis of blast cell characteristics. Two patients at initial diagnosis had the morphological and cytochemical characteristics of lymphoblasts and high TdT activity, but on relapse, the blasts had the typical characteristics of myeloblasts and low TdT activity.Among 10 patients with Ph¹ chromosome positive acute leukemia $\text{4}\text{5}$ patients with lymphoid-appearing blasts and $\text{2}\text{5}$ patients with myeloid-appearing blasts had high-TdT activity.The results of this study show that although about $\text{1}\text{3}$ of AML patients may have high TdT activity, it is still a useful marker for the diagnosis of ALL. In addition, Ph¹ chromosome positive acute leukemia may represent a distinct entity although related in its origin to both non-T/non-B ALL and blastic phase of CML.     

Terminal deoxynucleotidyl transferase activities and glucocorticoid receptors in leukaemia.

The relation between terminal deoxynucleotidyl transferase (TdT) activity, glucocorticoid (GC) receptors and the effect of vincristine-prednisolone (VP) therapy on fresh leukaemia cases was examined. Five of 6 TdT+ leukaemias showed high levels of GC receptors and a favourable response to VP therapy, whereas 1 acute lymphoblastic leukaemia (ALL) and 3 of chronic myelogenous leukaemia (CML) cases in blast crisis with no TdT activity showed low level of GC receptors and poor response to VP therapy. Significant correlation (r = 0.821, P less than 0.01) was observed between TdT activity and the number of GC receptor sites in these cases. X2 test showed significant difference (P less than 0.01) between TdT+ and TdT- leukaemias in the effect of VP. A significant difference (P less than 0.01) was also observed between VP-effective and ineffective leukaemias in the number of GC-receptor sites by unpaired t test. Therefore GC receptors may be responsible for the effect of VP on TdT+ leukaemias.     

High terminal deoxynucleotidyl transferase activity in acute myelogenous leukemia.

Although leukocytes from all 13 acute lymphoblastic leukemia patients examined had high terminal deoxynucleotidyl transferase (terminal transferase) activity (20 to 100 units/mg of cellular DNA, where 1 unit equals 1 nmole of nucleotide polymerized in 1 hr) and those from 21 acute myelocytic leukemia patients had low terminal transferase activity (0.2 to 2 units/mg of cellular DNA), the bone marrow and peripheral blood leukocytes from 2 patients with acute myelocytic leukemia, diagnosed on the basis of clinical features and the morphology, cytochemistry, and cytogenetics of the leukemic cells, had terminal transferase activity (39 to 52 units/mg of cellular DNA) equivalent to that found in leukemic lymphoblasts. These results bring under question the specificity of high terminal transferase activity outside of the thymus as a marker for leukemic lymphoblasts and, secondarily, the derivation of acute lymphoblastic leukemia cells in all cases from thymocytes. Perhaps malignant transformation in a pleuripotent stem cell with derepression of the genome for terminal transferase could account for high terminal transferase activity observed in certain leukemic cells.     

Terminal deoxynucleotidyl transferase (TdT) in RadLV-induced C57BL thymomas

Newborn C57BL mice exposed to RadLV at birth were followed and periodically sampled for thymus and bone marrow TdT activity. Tumors detected at or before 100 days had mostly low TdT levels. Large thymic lymphocytic tumors detected after 100 days had elevated total TdT content per gland. Serial samples showed a changing TdT pattern with time. The mid period shifted from low levels to normal-to-high levels for both TdT content and total TdT per gland. No unusual bone marrow TdT activity change preceded the appearance of thymomas. TdT marks a unique precursor cell highly sensitive to RadLV associated with lymphomagenesis. RadLV induced both TdT⁺ and TdT⁻ thymic lymphomas and both types were found to occur after lymphoma initiation by injection of RadLV into newborns.     

Terminal deoxynucleotidyl transferase in acute lymphoblastic leukemias and chronic T cell proliferations

Cells from patients with acute lymphoblastic leukemias (ALL) (28 cases), T-derived chronic lymphocytic leukemias (CLL) (6 cases), Sezary syndrome (3 cases) and thymomas (3 cases) were studied for both immunological membrane phenotype and terminal deoxynucleotidyl transferase (TdT) content (using chromatography of cell extracts on phosphocellulose). Cells from 25 cases of ALL contained TdT. T-derived ALL had relatively homogenous TdT values whereas a wider range of TdT activity was found in non-T non-B ALL. Cells from 3 ALL cases had no detectable enzyme; two were B-derived ALL, one T-derived ALL. Cells from the three thymomas and from one case each of Sezary syndrome and T-CLL had TdT activity. The significance of TdT in neoplastic disorders is discussed in the light of the known distribution of TdT in various subpopulations of normal mature T-cells and T-cell precursors.     

Prognostic significance of terminal deoxynucleotidyl transferase activity in acute nonlymphoblastic leukemia

Bone marrow and/or peripheral blood samples from 133 (75%) of a total of 177 consecutive previously untreated protocol patients with acute nonlymphoblastic leukemia (ANLL) were analyzed for terminal deoxynucleotidyl transferase (TdT) activity at the time of presentation. Twenty-nine (22%) were found to exhibit TdT activity (greater than or equal to 0.10 U/10(8) cells, TdT+) as measured in a biochemical microassay. There were no differences between TdT+ as compared with TdT-negative (TdT-) patients with respect to age, sex, French-American-British (FAB) classification, or the presence of Auer's rods. Remission induction rates were higher for the TdT- patients, with 68% v 48% for the TdT+ patients (P = .05). TdT- patients also experienced longer remissions (P = .003) than TdT+ patients, especially in the Auer's rod-positive subgroup (P = .002). None of five patients with TdT+ ANLL treated with vincristine and prednisone as initial therapy achieved complete remission; all required induction regimens containing daunorubicin or amsacrine in combination with cytosine arabinoside and 6-thioguanine. It is concluded that TdT activity in ANLL indicates biphenotypia or lineage infidelity and is associated with a poor prognosis on chemotherapy protocols currently used for the treatment of ANLL.     

Terminal deoxynucleotidyl transferase expression in acute myelogenous leukemia and myelodysplasia as determined by flow cytometry

The significance of terminal deoxynucleotidyl transferase (TdT) expression in acute myelogenous leukemia (AML) remains controversial. Therefore, we studied TdT expression by flow cytometry in 120 previously untreated patients with AML or myelodysplastic syndrome (MDS) to determine the distribution of TdT-positive blasts and the intensity of TdT expression and to seek clinically significant associations. TdT expression measured by flow cytometry (flow TdT%) was heterogeneous, ranging from 0.1% to 87% (median, 8.5%), and 74 patients (62%) had at least 5% TdT-positive blasts. TdT positivity was associated with the M0 or M1 subtype and with expression of CD34 and CD7. No significant correlation was found between TdT expression and type of cytogenetic abnormality or rearrangement of immunoglobulin or T-cell receptor genes. Remission lasted longer in patients with a flow TdT% < 5 than in patients with a flow TdT% > 5 (median, 95 weeks vs 55 weeks, p = 0.02); however, complete remission rates did not differ when patients were classified by initial flow TdT%. Survival was slightly better for patients with flow TdT% less than 5%. Among patients with a flow TdT% > 5%, those with a higher TdT intensity survived longer than those with a lower intensity. These data suggest that quantitative TdT measurement may contribute to prognostic estimate in AML patients.     

Terminal deoxynucleotidyl transferase in human lymphomas: possible existence of forms with high and low molecular weights.

Optimized methods for extraction and enzyme assay in crude tissue preparations were used to determine the amounts of terminal deoxnucleotidyl transferase (TdT) in malignant lymphomas. The TdT concentration was increased only in lymphoblastic lymphomas (LL) and was as high in these tumours as in the white blood cells from untreated patients with acute lymphoblastic leukaemia (ALL). The enzymes extracted from such lymphomas and from the leukaemic lymphoblasts had the same properties. Moreover, forms of TdT with low and high mol. wt were found in the LL tumours, similar to other reports of TdT-positive leukaemias. The overall study points at some basic biochemical identity of certain lymphoblastic malignancies, irrespective of whether the transformed cells are in solid tumours or are disseminated in the blood.     

Enumeration of terminal deoxynucleotidyl transferase positive cells in leukemia/lymphoma by flow cytometry

The method to fix single cells in suspension and its application for the detection of TdT-positive cells by flow cytometry are described. In comparison to formalin-methanol fixation, formalin-acetone fixation resulted more formation of aggregated cells which caused non-specific staining. In our assay system 80% cells in human thymus were TdT-positive. Levels of TdT in normal human peripheral blood was 0.5 +/- 0.6% (means +/- 1 S.D., n = 50). A total of 104 patients with leukemia/lymphoma was examined for TdT. TdT-positive cells were detected in ALL (87.5% cases), AML (57.1% cases), CML-BC (58.3% cases) and ML (17.6% cases). Mean channel of fluorescence intensity of the TdT-staining in AML was significantly reduced in comparison with that in ALL. When antigen density of TdT was very low, fluorescence microscopy gave false negative results and flow cytometry could detect this dim fluorescence.     

Serial observations on terminal deoxynucleotidyl transferase activity and lymphoblast surface markers in acute lymphoblastic leukemia.

Terminal deoxynucleotidyl transferase activity and cell surface markers were measured in peripheral lymphoid cells from 27 children with acute lymphoblastic leukemia in various phases of their disease. Lymphoblasts from untreated patients had smooth surface ultrastructure but heterogeneous surface receptors. Greater than 60% of lymphoblasts from 4 to 7 untreated patients formed rosettes with sheep red blood cells. Transferase activity was variable, ranging from 8 to 210 units/10(8) blasts, but it was consistently elevated at diagnosis and in relapse. Transferase levels did not correlate with the presence of lymphoblast surface receptors. During induction therapy transferase activity decreased rapidly, but it remained elevated in peripheral lymphoid cells even when blasts were not detectable in peripheral blood smears. Patients in remission had normal surface receptors and undetectable or minimally elevated levels of transferase. Terminal transferase activity may be a sensitive biochemical marker for a primitive cell population and may be important in the evaluation of therapeutic effectiveness in acute lymphoblastic leukemia.     

High terminal deoxynucleotidyl transferase activity in pediatric patients with acute lymphocytic and acute myelocytic leukemias.

Specificity of TdT5 as a marker for ALL was evaluated by determining its activity in cells from normal control subjects and from 35 pediatric patients with ALL, AML, Hodgkin's disease and disseminated Burkitt's lymphoma. We evaluated the DNA polymerase activity, cell surface phenotypes (E rosettes, EAC rosettes, Smlg and la-like, HTLA and cALL antigens), and hematological and cytochemical characteristics in both the normal and patient groups. DNA polymerase alpha + beta and DNA polymerase gamma activity were indiscriminately high in all immature cells as found in ALL, AML, Burkitt's lymphoma and phytohemagglutinin-stimulated normal lymphocytes, when compared to mature leukocytes found in normal individuals or in patients whose cancer was in remission. High TdT activity was found in 24 of 26 T and non-T/non-B ALL patients in active phase as well as in two of three AML patients one of whom had Auer rods. Thus, TdT, although valuable for monitoring ALL patients, may have limitations in separating AML from ALL.     

Nuclear terminal deoxynucleotidyl transferase in leukaemic infiltrates of testicular tissue.

Early detection of testicular leukaemia and the identification of residual leukaemic cells in treated patients are important aims in the management of males with acute lymphoblastic leukaemia (ALL). In most cases of ALL ( greater than 95%) the blast cells express terminal deoxynucleotidyl transferase (TdT), a nuclear enzyme. We have therefore standardized the immuno-fluorescence and -peroxidase techniques (using anti-Tdt antibodies) for identifying TdT cells in the normal thymus, as well as in samples of testis with heavy leukaemic infiltrates (positive controls). TdT cells can be identified in formalin (but not in Bouin''s or Carnoy''s) fixed paraffin-embedded tissues, and the preservation of morphological details is excellent. The method is nevertheless difficult to standardize and also requires the use of deoxyribonuclease (DNase) for the digestion of sections. However, in frozen tissue sections, stronger staining of TdT cells was found, even without DNase treatment. Good morphology was preserved when cut sections were fixed immediately in the cryostat. In the second part of the study 15 samples from treated boys were analysed to see whether the technique is suitable to identify residual minimal leukaemic infiltrates. In 5 patients scanty disseminated TdT cells were detected, and in 2 patients small clumps of TdT cells were seen. The results indicate that the immunohistological identification of TdT ALL blasts may be the method of choice.     

Terminal deoxynucleotidyl transferase activity and cell surface antigens of two unique cell lines (NALM-1 and BALM-2) of human leukemic origin.

Two unique cell lines, NALM-1 and BALM-2 derived from lymphoblast-like cells of chronic myelogenous leukemia and rare B cell acute lymphoblastic leukemia patients, respectively, were compared with fresh parent cells from the patients and with a Philadelphia chromosome positive K-562 cell line previously established from a chronic myelogenous leukemia patient in blastic phase. NALM-1 resembled the parent cells in the presence of Philadelphia chromosome, non-T/non-B acute lymphoblastic leukemia specific antigens and lack of T or B cell markers, whereas BALB-2, like the parent cells, had two chromosome markers and bore kappa, delta and mu immunoglobulins. NALM-1 lacked Epstein-Barr virus genome, whereas BALM-2 showed the presence of Epstein-Barr virus genome. K-562 cells lacked all the antigen markers examined. All cells had high DNA polymerase alpha activity and low DNA polymerase gamma activity. NALM-1, like the parent cells and unlike K-562 cells, had high terminal deoxynucleotidyl transferase activity of about 200 mu/mg DNA, whereas BALM-2, like its parent cells, had terminal deoxynucleotidyl transferase activity of 1-2 mu/mg DNA (1 u = 1 nmole Mn++-dGTP/h on dA12-18 initiator). Terminal deoxynucleotidyl transferase was characterized by its chromatographic and sedimentation behavior, thermal sensitivity and specific inhibition by streptolydigin and terminal deoxynucleotidyl transferase antisera. These results indicate that NALM-1 and K-562 may represent different phenotypes of cells in CML blastic crisis. Moreover, NALM-1 and BALM-2 seem to have retained the characteristics of original leukemic cells from which they may have been derived.