Terminal deoxynucleotidyl transferase in acute leukemias by direct immunofluorescence

TdT (terminal deoxynucleotidyl transferase) can be detected by radio enzymatic assay, biochemical assay in cell extracts, serum or plasma, and intracellularly in the smear by indirect immunofluorescent methods. The IgG fraction of anti-TdT serum is conjugated with fluoresceinisothiocyanate and used directly on the cytospin smears of methanol fixed bone marrow/blood smears. The mice thymocytes and peripheral mononuclear cells of healthy donors were used as positive and negative controls, respectively, for TdT. 64% of our cases of ALL were found to be TdT+. The lymphoblasts of L1 morphology (FAB classification) were more frequently positive for TdT as compared to blasts with L2 morphology. 71% of our cALLa positive blasts in acute lymphoblastic leukemias were TdT+ve as compared to 58% of T-ALL blasts. 75% of PAS positive ALL cases were positive for TdT as well. Only 57% of the cases when acid phosphatase showed unipolar positivity (T type) were positive for TdT. 12% of cases with acute myeloid leukemia (6/47) were TdT+ve and 33% of CML in blastic crisis had TdT+ve blasts. Biochemical assay and IF assay for TdT were in good correlation in our study.     

Spurious detection of terminal deoxynucleotidyl transferase in phytohemagglutinin-stimulated lymphocytes

Expression of terminal transferase (TdT) is believed to be restricted to primitive lymphoid cells; recently, however, indirect immunofluorescent (IF) assays have been used to demonstrate the apparent presence of TdT in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes and in various nonlymphoid malignancies. Using an IF assay, we found that a heteroantiserum to TdT reacted with cultured and PHA-stimulated human peripheral blood mononuclear cells, but we were unable to confirm the presence of TdT in these cells using immunoblotting and biochemical assays. We conclude that the IF results are spurious and most likely represent recognition by the heteroantiserum of inducible protein(s) other than TdT.     

Association of terminal deoxynucleotidyl transferase with Ku

Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein-protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.     

A micromethod for determination of terminal deoxynucleotidyl transferase (TdT) in the diagnostic evaluation of acute leukemias

Summary A micromethod for the determination of TdT in peripheral leukocytes and bone marrow cells has been developed that allows unequivocal identification and quantitation of TdT in less than 1 x 10⁶ leukocytes from ALL patients, i.e., in 1 ml of peripheral blood and/or 0.5 ml of bone marrow obtained during routine clinical sampling. The method involves disruption of cell pellet with high salt and detergent followed by centrifugation of extracts at 12,000 x g and partial purification on phosphocellulose matrix by a batch elution technique using a standard laboratory microcentrifuge. Using this microassay, TdT activities have been determined in 500 samples of peripheral blood and bone marrow of 240 adult patients with acute leukemias (86 ALL, 108 ANLL, 44 blastic CML, two acute leukemias following P. vera). From an analysis of our data based on TdT activity, cell surface markers and growth patterns in soft agar and observations published in the literature, it can be concluded that the frequencies of TdT + phenotypes in the various clinicalmorphological diagnostic groups are approximately 95% in ALL, 10% in ANLL, 50% in AUL, and 35% in blastic CML. Since the presence of high TdT activity is clearly associated with clinical response to specific forms of chemotherapy in blastic CML and most probably, also in ANLL, the determination of TdT should be considered in all cases of acute leukemias to objectively define prognostically important subgroups which can not be diagnosed by conventional means.Abbreviations ALL acute lymphoblastic leukemia - ANLL acute non-lymphoblastic leukemia - ATP adenosine triphosphate - AUL acute undifferentiated leukemia - B cell bone marrow related lymphocyte - CFU-c Colomy-forming unit in soft agar culture (myeloid committed stem cell) - CM chronic myelogenous leukeima - E-rosette sheep erythrocyte rosette - (³H) dGTP tritium-labeled deoxyguanosine triphosphate, incorporated into nucleic acid as monophosphate: (³H) dGMP - HTLA human T-lymphoeyte antigen - MDS myelodysplastic syndrome - MPS myeloproliferative syndrome - MPS-AP in acute or blastic phase - Oligo d(pA)_12-18 polymer of deoxyadenylic acid, chain length 12–18 residues - PB-T peripheral blood T cell - slg surface immunoglobulins - T cell thymus-related lymphocyte - TdT terminal deoxynucleotidyl transferase - TdT specific TdT activity in bone marrow mononuclear cells <0.10 units/10⁸ cells - TdT+ TdT activity >0.10 units/10⁸ cells - thy-T thymocyte This research was supported in part by the following grants: ACS PDT-95, NCI-CA-17404, NCI-CA-19267, NCI Program Project Grant 3 PO1 CA-20194, NCI Grant CA-08748, the Gar Reichman Foundation and the Zelda R. Weintraub Cancer Fund, and a research career development award no. 1 KO4-CA-00545 from the National Cancer Institute to MJM     

Histochemical demonstration of terminal deoxynucleotidyl transferase in leukemia

White blood cells from 22 patients with leukemia and lymphoma were studied for the presence of terminal deoxynucleotidyl transferase with a peroxidase-antiperoxidase method. The enzyme was detected in leukemic cells of 5 patients with acute lymphoblastic leukemia and 1 patient with chronic myelogenous leukemia, whereas 16 patients with different forms of leukemia or lymphoma were negative for this enzyme. Comparative studies using a biochemical and an indirect immunofluorescence assay revealed complete concordance between these three methods.     

Simultaneous evaluation for terminal deoxynucleotidyl transferase and myeloperoxidase in leukemia

A technique for dual staining of cells using terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO) is described. The technique has been applied to cells of two patients. One patient had chronic myelomonocytic leukemia evolving into acute myelomonocytic leukemia. The other patient had chronic myelogenous leukemia in blast crisis. Our findings indicate that TdT and MPO are exclusive markers except for a rare precursor cell with dual staining in one patient. This study supports the concept of acute mixed leukemia.     

Demonstration of terminal deoxynucleotidyl transferase in thymocytes by immunofluorescence.

The cellular and subcellular distribution of terminal deoxynucleotidyl transferase (DNA nucleotidylexotransferase; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase, EC 2.7.7.31) in thymocytes and peripheral lymphocytes from rat, mouse, and calf was studied by immunofluorescence using rabbit antiserum to homogeneous transferase from calf. Terminal transferase was readily detected in approximately 75% of cortical thymocytes, but not in medullary thymocytes or lymph node lymphocytes. The enzyme appeared to be present predominantly in the cytoplasm of positive thymocytes in ethanol-fixed cell smears and frozen sections. The reactivity of anti-terminal-transferase for thymocytes could be neutralized with purified calf enzyme. Results of experiments in which thymocytes were separated on 7-step discontinuous Ficoll density gradients suggested that cortical thymocytes are heterogeneous with respect to terminal deoxynucleotidyl transferase content.     

Intracellular distribution of terminal deoxynucleotidyl transferase in rat bone marrow and thymus

A subset of bone marrow cells that contains terminal deoxynucleotidyl transferase (DNA nucleotidylexotransferase; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase, EC 2.7.7.31) can be identified in adult rats by immunofluorescence using affinity-column-purified antibody to homogeneous calf transferase. The transferase-positive cells comprise approximately 1.8% of bone marrow cells. Correcting the specific activity of terminal transferase in total bone marrow cells (0.21 units per 10(8) cells) for the percentage of transferase-positive bone marrow cells (1.8%) gives 11.7 units per 10(8) cells, a value approximately half that found for transferase-positive thymocytes.Fluorescence appears to be restricted to the nucleus of transferase-positive bone marrow cells, in contrast to the predominantly cytoplasmic fluorescence of small thymocytes from adult rats. Some large thymocytes contain intranuclear transferase fluorescence patterns similar to those seen in bone marrow. These thymocytes are especially numerous in neonatal rat thymus, where they are localized in the subcapsular region of the cortex. Thymocytes with combined patterns of nuclear and cytoplasmic transferase are also present. In addition, Thy-1.1 antigen, which is present on thymic and prethymic cells but not on the majority of post-thymic cells in the rat, is also present on transferase-positive bone marrow cells.The results suggest that the transferase-positive subset of bone marrow cells may contain the immediate progenitors of cortical thymocytes in the rat. The nuclear location of fluorescence may indicate the site of physiological activity of terminal transferase in thymocytes and their precursors.     

Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT; EC 2.7.7.31) adds nucleotides to DNA ends generated during V(D)J recombination that are subsequently processed by proteins involved in general double-strand break (DSB) repair pathways. We report an association between TdT and a 55-kDa protein in lymphoid cells. This protein, identified as hPso4, is a homolog of the protein encoded by the PS04/PRP19 gene in Saccharomyces cerevisiae that has pleiotropic functions in DNA recombination and error-prone repair. Purified hPso4 binds double-stranded DNA in a sequence-nonspecific manner but does not bind single-stranded DNA. hPso4 protein is induced 15- to 30-fold in cells by gamma radiation and chemical mutagens but not by UV treatment. Loss of hPso4 expression induced by siRNA results in accumulation of DSBs, apoptosis, and decreased cell survival after DNA damage. We conclude that hPso4 plays a major and previously undefined role in mammalian DNA DSB repair.     

The ontogeny of terminal deoxynucleotidyl transferase positive cells in the human fetus

The ontogeny of cells containing the enzyme terminal deoxynucleotidyl transferase (TdT) in human fetal liver, bone marrow, and thymus has been studied using a highly specific antiserum to TdT together with monoclonal antiprecursor cell antibodies in double and triple marker immunofluorescence. TdT+ cells were first observed in fetal liver at 12 wk of gestation and accounted for 55% of the lymphoid-like cells isolated after Ficoll-Hypaque separation. TdT+ cells were first observed in the bone marrow 16 wk after gestation. Like TdT+ cells in normal infant bone marrow, the majority of TdT+ cells in fetal liver and bone marrow expressed both BA-1 and RFB-1 antigens. This suggests that fetal TdT+ cells include progenitors of the B lineage (BA-1+) and perhaps of thymocytes (RFB-1+). Nevertheless, TdT was not observed in fetal thymocytes until after 20 wk of gestation, although thymic blasts and the majority of thymocytes were strongly RFB-1+ from 12 wk of gestation. These results clearly show that fetal thymus is first populated by TdT, RFB-1+, BA-1 cells, but does not exclude the fact that a second "wave" of TdT+ prothymocytes, possibly bone marrow derived, also exists.     

Induction of terminal deoxynucleotidyl transferase and Lyt antigens with thymosin: identification of multiple subsets of prothymocytes in mouse bone marrow and spleen.

Thymosin (fraction 5 and synthetic alpha 1 peptide) induced prothymocytes in mouse bone marrow and spleen to express terminal deoxynucleotidyl transferase (TdT; DNA nucleotidylexotransferase; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase, EC 2.7.7.31) or Lyt-1+, 2+, 3+ alloantigens (or both) after brief incubation in vitro. Three antigenic phenotypes were generated: (i) TdT+ Lyt+, (ii) TdT- Lyt+, and (iii) TdT+ Lyt-. The TdT+ Lyt+ phenotype was expressed by 80% of prothymocytes in bone marrow and 30% of prothymocytes in spleen from normal mice. The TdT- Lyt+ phenotype was expressed by 81% of prothymocytes in bone marrow from athymic mice. More than 80% of TdT+ bone marrow cells from normal and athymic mice expressed Lyt antigens after thymosin treatment. We interpret these observations as suggesting that (i) most TdT+ hemopoietic cells in normal and athymic mice are thymocyte progenitors; (ii) two independent lineages of prothymocytes exist, one that expresses TdT and another that does not, (iii) commitment of prothymocytes to the TdT+ cell pathway is partially regulated by a thymic feedback mechanism; and (iv) the bone marrow preferentially produces TdT+ prothymocytes, whereas the spleen may serve as a repository for TdT- prothymocytes. A model of T-cell development is presented in which the thymus functions as a compound organ to process TdT+ and TdT- thymocytes progenitors and to generate two lines of T cells.     

Adenosine deaminase, terminal deoxynucleotidyl transferase (TdT), and cell surface markers in childhood acute leukemia.

The purpose of this report is to compare measurements of enzymatic activities and cell surface markers as methods of distinguishing subtypes of lymphoid leukemias of childhood. Twenty-six children ages 2-15 yr were studied. Terminal deoxynucleotidyl transferase (TdT) activity was high in blasts from all 20 children with either null or T cell acute lymphoblastic leukemia. The activity of adenosine deaminase per cell was higher (P less than 0.005) and that of TdT lower (p less than 0.05) in T than in null cell lymphoblasts, although there was some overlap in values. Blasts from 3 children with acute lymphoid leukemia were positive for surface-associated immunoglobulins. The neoplastic lymphoid cells from these children differed from T and null cell leukemic lymphoblasts by having very low levels of TdT and adenosine deaminase activity. Measurements of adenosine deaminase and TdT may complement measurements of cell surface markers and distinguish biochemical subtypes of acute lymphoid leukemia.     

Evaluation of a new enzyme-linked immunoassay for terminal deoxynucleotidyl transferase in haematologic malignancies

We have evaluated a new solid phase enzyme immunoassay (EIA) for detection of terminal deoxynucleotidyl transferase (TDT). The EIA is greater than 100 times more sensitive than previously used tests for enzyme activity. In 284 clinical specimens of human peripheral blood and bone marrow, the EIA detected TdT antigen in 97% of peripheral blood and 100% of bone marrow samples that were positive for enzymatic activity. The excellent sensitivity and specificity of this new test suggests that it can be used in clinical situations where quantitative TdT measurements are desired.     

CD7-positive acute leukemia lacking T cell receptor gene rearrangements and terminal deoxynucleotidyl transferase expression suggesting pre-T cell origin

In this report, we describe a case of acute leukemia possessing a unique immunophenotype. Leukemic cells isolated from peripheral blood were analyzed by automated fluorescence-activated flow cytometry. They demonstrated the following antigen pattern: CD7+, CD38+, transferrin receptor+, HLA-DR+, common ALL antigen (CALLA)-, and terminal deoxynucleotidyl transferase (TdT)-. No expression of B cell or myeloid antigens was observed. The observed antigen pattern suggests a pre-T cell origin. In addition, the tumor cells contained germline T cell receptor beta (T beta) and gamma (T gamma) chain genes, and a germline immunoglobulin heavy chain (JH) gene. These findings support the concept that pre-T cell leukemias may demonstrate germline T beta and T gamma genes as well as lack TdT expression. In addition, our results suggest that CD7 expression may be one of the earliest events in T cell differentiation, occurring prior to both T beta an T gamma gene rearrangements, and TdT expression.     

Coexpression of p165 myeloid surface antigen and terminal deoxynucleotidyl transferase: a comparison of acute myeloid leukaemia and normal bone marrow cells

A double immunofluorescence technique, using antibodies to terminal transferase (TdT) and a 165-kilodalton myeloid differentiation antigen (p165), has been used to investigate the phenomenon of TdT expression in cases of acute myeloid leukaemia (AML). Five cases of AML were shown to have significant (18-90%) numbers of leukaemic cells that concurrently expressed both TdT and p165 myeloid surface antigen. Examination of nonleukaemic bone marrow cells showed that the vast majority of normal TdT+ cells are p165 negative. However, in 5 of the 11 samples analyzed, rare cells staining for both p165 and TdT were found. These results suggest that some cases of TdT+ AML may arise from the clonal expansion of rare "biphenotypic" precursor cells existing in normal bone marrow.     

Terminal deoxynucleotidyl transferase and HLA-DR expression appear unrelated to prognosis of acute myeloid leukaemia

Mononuclear cells from peripheral blood or bone marrow from 314 patients with acute myeloid leukaemia were examined for the presence of nuclear terminal deoxynucleotidyl transferase (304 patients), surface membrane expression of HLA-DR (314 patients) and the common acute lymphoblastic leukaemia antigen (281 patients). All patients were treated with identical remission induction chemotherapy, and morphological diagnosis was carried out in a central laboratory. The overall complete remission rate was 70%. There were no significant correlations between the immunological markers and complete remission rate, duration of remission, or survival.     

Measurement of terminal deoxynucleotidyl transferase mRNA in clinical samples: a new parameter in analysis of leukemia cells

A 1750 base pair cDNA to human terminal deoxynucleotidyl transferase (TdT) has been cloned. This cDNA detects a dominant 2200 base pair messenger RNA species in normal and leukemic cells synthesizing the enzyme. A quantitative dot blot assay was utilized to survey a number of clinical samples from patients with TdT positive and negative leukemias as well as cells from normal volunteers. A linear relationship was detected between the amount of TdT mRNA and the amount of enzyme activity in bone marrow cells. The assay is sensitive enough to detect normal TdT levels in bone marrow, and distinguish these levels from the lack of such mRNA in peripheral blood and bone marrow of patients with myeloid leukemia. Elevated levels of mRNA were found in two cases of patients in clinical remission. The prognostic significance of these observations must await further study.     

Comparison of purine degradative enzymes and terminal deoxynucleotidyl transferase in T cell leukaemias and in normal thymic and post-thymic T cells

Adenosine deaminase (ADA), ecto 5' nucleotidase (5'NT), purine nucleoside phosphorylase (PNP) and terminal deoxynucleotidyl transferase (TdT) were measured in the cells of patients with acute or chronic T cell leukaemia and compared with normal putative prothymocytes (large, blast-like cortical thymocytes), cortical and medullary thymocytes and peripheral blood T lymphocytes. Distinct patterns of enzyme activities were found in the individual types of T cell leukaemia. Mean ADA, TdT and 5'NT activities in thymic acute lymphoblastic leukaemia (Thy-ALL) were 41.9 u/10(8) cells, 31.1 u/10(8) cells and 4.7 u/10(6) cells respectively; in chronic T cell leukaemia they were 7.1 u/10(8) cells, 0.6 u/10(8) cells and 18.1 u/10(6) cells respectively. Mean PNP activity was similar between these two groups of leukaemia (68.6 u/10(6)cells in Thy-ALL and 77.9 u/10(6) cells in chronic T cell leukaemia). The activities of these four enzymes in OKT4+ chronic T cell leukaemia did not differ significantly from those in the OKT8+ chronic T cell leukaemia cases. The activities of TdT, ADA, PNP and 5'NT in Thy-ALL closely resembled those in normal immature thymocytes, and in the chronic T cell leukaemias showed a similar pattern of enzyme activities to that of mature T lymphocytes. These findings are consistent with surface phenotypic studies of T cell malignancies which suggest that different T cell leukaemias represent malignant proliferation of T cell clones arrested at different stages of T cell differentiation. They also demonstrate the value of biochemical markers in defining the different subtypes of acute and chronic leukaemia.