Terminal deoxynucleotidyl transferase-containing cells in peripheral blood: implications for the surveillance of patients with lymphoblastic leukemia or lymphoma in remission

An indirect immunofluorescence assay was used to quantitate TdT- containing (TdT+) cells in the mononuclear leukocyte fraction of peripheral blood from normal subjects and patients with acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL). In normal children (10) and adults (10), 0.036% +/- 0.014% (mean +/- SD) and 0.030% +/- 0.015% TdT+ cells were found. In peripheral bloods from 10 children receiving chemotherapy for tumors other than ALL or LL, 0.040% +/- 0.039% TdT+ cells were found. Serial determinations were performed on 15 patients with ALL or LL who were in clinical remission. Eight of these patients remained in continuous remission and always had fewer than 0.11% TdT+ cells in their peripheral blood. Three patients who developed systemic relapse were found to have progressively rising numbers of TdT+ cells in their peripheral blood prior to clinical evidence of relapse. All 3 of these patients had greater than 0.1% TdT+ cells in their peripheral blood from 3 to 8 wk prior to clinical relapse. In 3 other patients, localized extramedullary relapse developed, but no trend was found on serial TdT determinations. Thus, the indirect immunofluorescence assay for TdT detects a small population of cells in normal peripheral blood. In patients with ALL, progressive increases above this normal level were associated with subsequent bone marrow relapse.     

Terminal deoxynucleotidyl transferase as a hematopoietic cell marker.

Terminal deoxynucleotidyl transferase (TdT) is an intracellular protein characteristic of certain primitive lymphocytes in normal thymus and bone marrow. It can be detected by enzyme assay or immunocytochemical staining. High levels of enzyme and increased numbers of TdT+ cells are found in some lymphoblastic leukemias and lymphomas. Analysis for TdT thus provides a useful adjunct in the differential diagnosis of leukemia.     

Phenotypic heterogeneity of TDT+ cells in the blood and bone marrow: implications for surveillance of residual leukemia [see comments]

Terminal deoxynucleotidyl transferase (TdT) is a useful marker for normal lymphocyte precursors and acute lymphoblastic leukemia (ALL). Our previous studies, however, have shown that for monitoring minimal residual disease in the circulation, assay for TdT alone is not sufficiently specific to distinguish leukemia cells from the background of rare normal blood TdT+ cells. In an attempt to increase specificity for leukemic cells, we have used double and triple immunophenotypic analysis to characterize normal circulating and bone marrow TdT+ cells. Overall, normal TdT+ cells were about 1000-fold more frequent in the marrow than in the blood. More than 75% of TdT+ cells in both the blood and marrow expressed the CD34, CD22, and HLA-DR antigens. However, circulating TdT+ cells infrequently expressed CD19 (4.5%) and CD9 (2.3%), compared with their marrow counterparts (74% and 47%, respectively). The brightly staining CD10+ phenotype, frequently associated with ALL blasts, was significantly less common among normal blood (5.7%) than marrow (31%) TdT+ cells. Although T-lineage markers were rarely expressed on TdT+ cells in either site, CD7+ cells were far more prevalent within the circulating TdT+ subset (4%) than among the marrow population (less than 0.2%). The results suggest a selective release of lineage-uncommitted and/or thymus-destined TdT+ cells from the marrow into the circulation. Moreover, since CD19, CD9, and high- density CD10 are frequently found on ALL blasts, staining for these markers on TdT+ cells in the circulation should improve the specificity of assay for residual common ALL cells. Likewise, assay for CD5+ and possibly CD7+ TdT+ cells in either marrow or blood should provide a very sensitive method of detection of T-ALL blasts.     

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.     

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.     

Long-term in vitro correction of alpha-L-iduronidase deficiency (Hurler syndrome) in human bone marrow.

Allogeneic bone marrow transplantation is the most effective treatment for Hurler syndrome but, since this therapy is not available to all patients, we have considered an alternative approach based on transfer and expression of the normal gene in autologous bone marrow. A retroviral vector carrying the full-length cDNA for alpha-L-iduronidase has been constructed and used to transduce bone marrow from patients with this disorder. Various gene-transfer protocols have been assessed including the effect of intensive schedules of exposure of bone marrow to viral supernatant and the influence of growth factors. With these protocols, we have demonstrated successful gene transfer into primitive CD34+ cells and subsequent enzyme expression in their maturing progeny. Also, by using long-term bone marrow cultures, we have demonstrated high levels of enzyme expression sustained for several months. The efficiency of gene transfer has been assessed by PCR analysis of hemopoietic colonies as 25-56%. No advantage has been demonstrated for the addition of growth factors or intensive viral exposure schedules. The enzyme is secreted into the medium and functional localization has been demonstrated by reversal of the phenotypic effects of lysosomal storage in macrophages. This work suggests that retroviral gene transfer into human bone marrow may offer the prospect for gene therapy of Hurler syndrome in young patients without a matched sibling donor.     

Immunoassay of circulating terminal transferase in patients with acute lymphoblastic leukemia: A new technique for diagnosis

The measurement of circulating terminal deoxynucleotidyl transferase (TdT) antigen can be accomplished by the use of an ELISA immunoassay system. TdT was present in either peripheral or bone marrow plasma of five patients with acute lymphoblastic leukemia (ALL) and absent from normal controls using ELISA. The assay was sensitive to a level of 20 ng/ml. The ALL patient values ranged from 160 ng/ml to 420 ng/ml. Use of ELISA for quantitation of TdT may have important diagnostic implications in patients with ALL.     

Prognostic significance of terminal transferase activity in childhood acute lymphoblastic leukemia: a prospective analysis of 164 patients

Whether the level of terminal deoxynucleotidyl transferase (TdT) activity in mononuclear cells from bone marrow and peripheral blood has prognostic significance has been analyzed prospectively in 164 children with T and non-T, non-B marked acute lymphoblastic leukemia (ALL). TdT was measured at diagnosis to assess its value as a predictor of duration of remission and length of survival. It was measured repeatedly during remission to assess whether it could predict relapse. Ninety-seven percent of the children achieved a complete remission of their disease, and 40% relapsed during the study. The level of TdT activity in blasts at diagnosis varied 1000-fold from patient to patient. There was no statistically significant relationship between TdT activity in cells at diagnosis and the achievement of complete remission, the duration of remission, or length of survival. TdT activity was significantly increased in the bone marrow of 65% of patients at the time of marrow morphological relapse, but was rarely increased in marrow from patients with isolated testicular or central nervous system relapse. Wide fluctuations in TdT activity were characteristically seen in mononuclear cells from the marrow and peripheral blood of patients with ALL at all stages of their disease. An isolated high value of TdT activity in the bone marrow or peripheral blood cannot be taken as evidence of impending relapse. Quantitative measurements of TdT activity alone on mononuclear cells from bone marrow and peripheral blood are helpful in differential diagnosis, but cannot guide therapy of children with ALL.     

Terminal Deoxynucleotidyl Transferase Expression in Acute Non-lymphoid Leukaemia: an Analysis by Immunofluorescence

S ummary . Indirect immunofluorescence for terminal transferase enzyme (TdT) was used to study the blasts of 64 patients with acute non-lymphoid leukaemia (ANLL). In 32 patients no TdT positive cells were seen. In 19 cases a small subpopulation of cells expressing TdT was detected; these constituted up to 5% of total nucleated cells, and it was not clear whether these TdT positive cells were part of the leukaemic process or represented residual normal bone marrow lymphoid cells.
The remaining 13 patients had TdT positive cells accounting for 7–90% of the total. In two of these cases TdT was expressed on blasts with myeloid features, representing an aberrant expression of TdT by myeloid cells; in contrast, in three cases mixed populations of TdT positive lymphoid blasts and TdT negative myeloid blasts were observed. In the remaining cases it was not possible to determine whether the TdT positive cells had definite lymphoid or myeloid features. Cytogenetic analysis showed no evidence of the Philadelphia chromosome. Response to treatment was assessed in 11 of the 13 patients. Only one patient remitted with the initial choice of therapy (DAT); four failed to respond to initial regimes of vineristine and prednisone (V & P) while the other five patients did not respond to myelotoxic combinations (DAT). Only one patient subsequently entered complete remission on second line therapy (V & P). This group of patients with TdT⁺ ANLL had a particularly bad prognosis, and appeared to differ from cases of TdT positive acute undifferentiated leukaemia, which often respond to V & P therapy.     

Immunofluorescent and Biochemical Studies of Terminal Deoxynucleotidyl Transferase in Treated Acute Leukaemia

S ummary . Indirect Immunofluorescence (IF) for terminal deoxynucleotidyl trans-ferase (TdT) was used in conjunction with the biochemical assay of TdT enzymatic activity to study human leukaemias before and during therapy. In addition, non-leukaemic marrows were analysed to compare the enzyme expression on normal cells. An excellent correlation was observed between the IF and biochemical methods when specimens contained greater than 5% TdT⁺ cells (by IF); below this level the biochemical assay was less reliable, while the sensitive IF test could detect isolated TdT⁺ cells among greater than 10 000 TdT negative cells. The IF method also had the advantage of allowing further immunological characterization of TdT⁺ cells, by simultaneous labelling of membrane antigens with appropriate antiscra. TdT⁺ cells expressing la-like antigens (but lacking other antigens associated with B- and T- lymphoid differentiation) were frequently found in low numbers in remission marrows from acute lymphoblastic leukaemia (ALL) patients. However, similar cells were also observed in remission acute myeloid leukaemia, as well as in non-leukaemic regenerating marrows, and marrow from normal donors. The presence of these normal TdT⁺ precursor cells therefore precluded the use of either IF or biochemical TdT tests for estimating the degree of residual disease or predicting early relapse in patients with non-T, non-B ALL. In contrast, the detection of TdT⁺ cells with T lymphoid antigens (HuTLA⁺) but lacking Ia antigens, in thymic (T-cell) -ALL, but not in normal marrow, allowed the use of this combination of markers to detect minimal residual disease in T-ALL.     

Terminal Deoxynucleotidyl Transferase (TdT) in Human Foetuses

The liver, bone marrow and cord blood of 30 human foetuses between the 15th and 21st weeks of gestational age were examined. Liver suspensions were investigated both by immunofluorescent and biochemical assay; cord blood suspensions and bone marrow touches with immunofluorescent assay. The results indicate the existence of a few TdT+ cells in the liver, cord blood and bone marrow of human foetuses in the ages studied. The peaks for each organ were: 15 + 1.60 (mean + SD) TdT+ cells in the liver, 12 + 2 in the bone marrow and 18 + 2.83 in the cord blood. In all the organs TdT+ cells decrease in the later weeks. In the liver, the results of the immunofluorescent assay were confirmed by the biochemical activity.     

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.     

Regeneration of CALLA (CD10+), TdT+ and double-positive cells in the bone marrow and blood after autologous bone marrow transplantation.

In this prospective study we investigated the frequency of CD10+, TdT+ and CD10+TdT+ mononuclear cells in the bone marrow (BM) and peripheral blood (PB) before and after autologous bone marrow transplantation (ABMT). 49 patients treated for acute lymphoblastic or myeloblastic leukaemia, malignant lymphoma or multiple myeloma were included. A significant increase in CD10+ cells occurred in BM in both children and adults after ABMT. In children, we also found a significant increase in CD10+ cells in PB. In individual patients remaining in remission, up to 34% CD10+ cells having a normal Ig kappa/lambda light chain ratio were recorded after ABMT. In children, the percentage of TdT+ and CD10+ TdT+ cells increased significantly in BM. In most cases the CD10/TdT-ratio was greater than 1.0, but during early regeneration after ABMT this ratio was less than 1.0 in several patients remaining in complete remission. In patients remaining in remission, CD10+TdT+ cells were detected in the blood in only 2 out of 140 samples tested, and the proportion of these cells never exceeded 0.03%. We conclude that quantitation of CD10+TdT+ cells in peripheral blood is helpful in the evaluation of complete remission in patients treated for pre-B-ALL.     

Terminal deoxynucleotidyl transferase (TdT) in human foetuses. An immunofluorescent and biochemical study

The liver, bone marrow and cord blood of 30 human foetuses between the 15th and 21st weeks of gestational age were examined. Liver suspensions were investigated both by immunofluorescent and biochemical assay; cord blood suspensions and bone marrow touches with immunofluorescent assay. The results indicate the existence of a few TdT+ cells in the liver, cord blood and bone marrow of human foetuses in the ages studied. The peaks for each organ were: 15 +/- 1.60 (mean +/- SD) TdT+ cells in the liver, 12 +/- 2 in the bone marrow and 18 +/- 2.83 in the cord blood. In all the organs TdT+ cells decrease in the later weeks. In the liver, the results of the immunofluorescent assay were confirmed by the biochemical activity.     

Suppression of hematopoiesis by activated T-cells in infectious mononucleosis associated with pancytopenia

Two patients with primary Epstein-Barr virus (EBV) infection followed by pancytopenia were studied. They showed increased numbers of DR-positive, activated T-cells and serological evidence of persistent EBV infection over a 12 and 18 week period. Bone marrow granulocyte-macrophage colony formation (CFU-GM) was investigated by limiting dilution assay (LDA) and methylcellulose assay. CFU-GM of bone marrow mononuclear cells (BMMNC) was markedly suppressed in both patients during the pancytopenic phase. Removal of bone marrow T-cells by E-rosetting resulted in a significant increase of CFU-GM to normal levels in BMMNC of both patients, while no significant increase was observed in the BMMNC of normal subjects. CFU-GM in BMMNC from Patient 1 in the recovery phase was normal when DR-positive T-cells were within normal levels, irrespective of the presence or absence of T-cells. These results suggest that pancytopenia due to infectious mononucleosis in these patients was due to bone marrow suppression by activated T-cells. In vitro studies with Con A activated T-cells and their culture medium showed that suppression of CFU-GM by T-cells was mediated by a lymphokine.     

Stromal-mediated down-regulation of CD13 in bone marrow cells originating from acute myeloid leukemia patients

The metallopeptidase CD13 is expressed on normal myeloid cells of monocytic and granulocytic origin and on the surface of leukemic blasts in most acute myeloid leukemias (AML). To study the mechanisms regulating lineage restricted CD13 expression in AML we determined normalised CD13 mRNA levels in bone marrow cells and peripheral blood cells of 27 AML patients. Cells of bone marrow origin had lower levels of normalised CD13 mRNA than cells of peripheral blood origin, even though fluorescence intensity and fraction of cells expressing CD13 on the surface was unchanged. In particular, AML patients with very low levels of normalised CD13 mRNA in bone marrow cells showed an increase in CD13 mRNA expression in peripheral blood. To evaluate the effects of bone marrow microenvironment on CD13 mRNA expression, we cultured leukemic myeloid cells with and without murine stromal cells. Bone marrow cells with high and low CD13 surface expression that entered the stromal layers all down-regulated CD13 mRNA expression as compared to cells in suspension above. For peripheral blood cells within stromal layers, CD13 mRNA expression was diminished in only 3 out of 6 cases. The ambiguous effect of stromal cells on peripheral blood cells may illustrate a differentiation-dependent response towards stroma. We determined the polyadenylation status of CD13 mRNA for 9 bone marrow aspirates and 7 peripheral blood samples. Polyadenylation was diminished in bone marrow cells from AML patients with low levels of normalised CD13 mRNA, raising the possibility of involvement of mRNA instability in regulation of CD13 mRNA expression in this subgroup of patients.     

Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray

A novel antibody microarray has been developed that provides an extensive immunophenotype of leukaemia cells. The assay is a solid phase cell-capture technique in which 82 antigens are studied simultaneously. This paper presents the analysis of 733 patients with a variety of leukaemias and lymphomas from peripheral blood and bone marrow. Discriminant Function Analysis of the expression profiles from these 733 patients and 63 normal subjects were clustered and showed high levels of consistency with diagnoses obtained using conventional clinical and laboratory criteria. The overall levels of consensus for classification using the microarray compared with established criteria were 93.9% (495/527 patients) for peripheral blood and 97.6% (201/206 patients) for bone marrow aspirates, showing that the extensive phenotype alone was frequently able to classify the disease when the leukaemic clone was the dominant cell population present. Immunophenotypes for neoplastic cells were distinguishable from normal cells when the leukaemic cell count was at least 5 x 10(9) cells/l in peripheral blood, or 20% of cells obtained from bone marrow aspirates. This technique may be a useful adjunct to flow cytometry and other methods when an extensive phenotype of the leukaemia cell is desired for clinical trials, research and prognostic factor analysis.     

Terminal-deoxynucleotidyl transferase (TdT): serial observations on patients with leukemia

Terminal-deoxynucleotidyl transferase (TdT) bone marrow determinations were performed on 67 patients with leukemia using the indirect immunofluorescence technique. A total of 103 smears were evaluated on 32 patients with acute lymphoblastic leukemia. With some exceptions, TdT levels were elevated at onset, declined during induction except in resistant cases, decreased during remission on chemotherapy, showed slight elevation during remission off chemotherapy, and rose during relapse in those cases previously positive. The most important finding was that patients in remission may have elevated TdT levels. Those were usually less than 10%. A total of 124 bone marrow smears were evaluated on 29 patients with acute myeloid leukemia. In general, values in all categories were below 1%, with a few elevated between 1% to 10%. Six patients with chronic myelogenous leukemia in blast crisis had 13 bone marrow smears evaluated. Five were in myeloblastic crisis and had values of less than 1%; 1 was lymphoblastic which had 50% positive cells at onset. In our experience, TdT determinations are of value in lymphoblastic leukemia in diagnosis, in predicting response to therapy, and in detecting early relapse.