Fluorometric determination of the activity of the biomarker terminal deoxynucleotidyl transferase via the enhancement of the fluorescence of silver nanoclusters by in-situ grown DNA tails.

The activity of terminal deoxynucleotidyl transferase (TdTase) is a biomarker for routine diagnosis of acute leukemia. A method has been developed for the determination of TdTase activity. It is based on the use of silver nanoclusters (AgNCs) whose yellow fluorescence is enhanced by an in-situ grown DNA tail of TdTase-polymerized and guanine-rich DNA at the 3' end of a hairpin DNA. The fluorescence, best measured at excitation/emission peaks of 530/585 nm, increases linearly in the 1 to 35 mU mL-1 TdTase activity range. The detection limit is 0.8 mU mL-1. The method is cost-efficient, selective and convenient. It integrates enhancement of the fluorescence of AgNCs and target recognition into a single process. Graphical abstract Schematic presentation of a method for determination of TdTase activity. It is based on AgNCs fluorescence enhanced by in-situ grown TdTase-polymerized G-rich DNA tail. The method integrates AgNCs fluorescence enhancement and the target recognition into a single process.

A seven-color panel including CD34 and TdT could be applied in >97% patients with T cell lymphoblastic leukemia for minimal residual disease detection independent of the initial phenotype.

A seven-color panel was used to detect minimal residual disease (MRD) in T cell acute lymphoblastic leukemia (T-ALL) via flow cytometry (FCM). Its availability and clinical significance were studied in T-ALL patients with newly diagnosed (n = 64), relapsed (n = 48) and morphologically complete remission (n = 103). The following four features were used to identify immature cCD3+ T cells: CD34+, TdT+, but mCD3-/dim+, and CD45dim+. Among these features, either TdT or CD34 expression was the most useful and were found in 93.8% of patients at diagnosis and 86.7% of patients who relapsed. Although some of the immature markers had disappeared in 23 of 59 cases after therapy, only one case presented with a false negative MRD. Of the 74 consecutive patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT), MRD-positive patients showed a higher relapse rate, a higher cumulative incidence of relapse at 4 years and a shorter median relapse-free survival than MRD-negative patients at post-HSCT(72.7% vs 17.3%, P = 0.000; 100% vs 19.9%, P < 0.0001; and 16 months vs undefined, P < 0.0001). We demonstrated that this panel could be applied to>97% of T-ALL patients to detect MRD and predict relapse after allo-HSCT even in the absence of the initial immunophenotype.

Enzyme markers in acute leukemias: advances during the last decade.

Besides the recent advances in immunology that provided important information for the characterization of leukemia cells, enzyme marker analysis is another area of progress in leukemia research. Assays of enzyme activities, both quantitative enzyme levels and qualitative isozyme changes, are of value in the classification of leukemias. The interest in the study of enzyme markers is due not only to technical improvements and new biochemical possibilities, but also to the involvement of enzyme marker analysis in the so-called "multiple marker analysis," which combines traditional and recently developed techniques in leukemia research. The aims of this review are to describe well-known and potential enzyme markers as well as to stress the relevance of enzyme marker analysis combined with multiple marker analysis for leukemia subclassification and the understanding of normal hematopoietic cell differentiation.

Philadelphia chromosome and terminal transferase-positive acute leukemia: similarity of terminal phase of chronic myelogenous leukemia and de novo acute presentation.

Twenty-eight patients with Philadelphia chromosome (Ph1)--positive and terminal transferase (TdT)--positive acute leukemia (AL) were treated with intensive chemotherapy used for adult acute lymphoblastic leukemia (L-10 and L-10M protocols). Fifteen patients had a documented chronic phase of Ph1-positive chronic myelogenous leukemia preceding the acute transformation (TdT + BLCML) while the remaining 13 patients did not (TdT + Ph1 + AL). An overall complete remission (CR) rate of 71% was obtained with a median survival of 13 months in the responders. Clinical presentation, laboratory data, cytogenetics, response to treatment, and survivals of the two groups of patients are compared. These results appear to be similar, suggesting a common or closely related origin. Since the overall survival of those receiving chemotherapy maintenance is poor, three patients underwent allogeneic bone marrow transplantation (BMT) from histocompatibility leukocyte antigen--matched siblings after they achieved CR. One of them is a long-term survivor (35 + months) with a Ph1-negative bone marrow. New techniques such as BMT should be considered in young patients with a histocompatibility leukocyte antigen--compatible sibling once a CR has been achieved.

Clinical utility of terminal deoxynucleotidyl transferase and adenosine deaminase determinations in adult leukemia with a lymphoid phenotype.

A prospective study was done to assess the clinical utility of terminal deoxynucleotidyl transferase (TdT) and adenosine deaminase (ADA) assays in adult leukemia with a lymphoid phenotype. The study population consisted of 58 patients with adult lymphoblastic leukemia (ALL) at onset, 12 with lymphoblastic lymphoma, 15 with acute unclassifiable leukemia (AUL), and 30 with lymphoid or mixed acute phase of Philadelphia chromosome-positive (Ph' +) chronic myelogenous leukemia (CML). TdT was present in all cases of T-ALL, in 90% of non-T, non-B ALL, and absent in B-ALL; the ADA activity was significantly higher (P less than .01) in T-ALL. TdT was found in 75% of lymphoblastic lymphomas, in 78% of lymphoid, and in 50% of mixed CML transformations; higher ADA activity correlated with TdT positivity in AUL and CML blastic transformations (P less than .001). TdT-positive ALL had a better chance of response to therapy than TdT-negative ALL (P less than 0.01), but survival was not statistically different. TdT was undetectable in the peripheral blood of patients with ALL in complete remission and within the normal range in bone marrow (0.1%-8% of nucleated cells); median ADA activity was as in control subjects. Relapsing ALL patients had TdT and ADA enzymatic activities as before therapy; TdT immunofluorescence test (IF) was positive in 69% of bone marrow and in 100% of CNS relapses. Twenty percent of TdT-positive ALL at onset became TdT-negative in bone marrow at relapse. TdT IF test was instrumental in detecting meningeal leukemia but neither TdT nor ADA could be used as indicators of complete remission or impending relapse because TdT-positive cells were present in normal marrows and wide fluctuations of TdT IF values and of ADA activity were observed in remission.

Terminal deoxynucleotidyl transferase (TdT) expression in acute myeloid leukemia.

Terminal deoxynucleotidyl transferase (TdT) was initially considered as a marker of immature lymphoid cells, but many studies have since provided conclusive evidence for the existence of TdT+ cases of acute myeloid leukemia (AML). The reported incidence of TdT+ AML cases varies largely (from 0% to 55%, average of combined data of the literature 18%, children 19%, and adults 21%) suggesting interlaboratory differences in the types of AML examined, the sensitivity of the method used, and the percentage of positive blasts taken as cut-off value. Significantly higher frequencies of TdT+ AML were reported in studies employing immunocytochemical staining (alkaline phosphatase anti-alkaline phosphatase or immunoperoxidase) than in series using immunofluorescence microscopy or biochemical assays. Statistical analysis of various cut-off levels demonstrates an inverse correlation between cut-off point and incidence. The combined data show that TdT-positivity is more common in the immature cell types (M0, M1), with no correlation with age or sex. Except for contested suggestions of an association with t(6;9) and t(8;21), no clear relationship between karyotype and TdT status has been documented. Although an association between T-cell receptor or immunoglobulin gene rearrangements and expression of TdT in AML was postulated, subsequent studies could not demonstrate this correlation. There was no significant relationship with other immunophenotypic markers except for CD34 positivity suggesting that the TdT+ cells represent an immature population. The percentage of positive cells was usually lower in AML than in ALL; in most cases only a subpopulation of the AML cells was TdT+. Thus, TdT could be viewed as a marker of hematopoietic immaturity. In about one-half of the studies on adults, TdT expression was reported to indicate a poor prognosis; others did not find any prognostic difference between TdT+ and TdT- AML cases. No correlation between TdT-positivity and prognosis was found in childhood AML.

Terminal deoxynucleotidyl transferase positive subpopulations occur in the majority of ANLL: implications for the detection of minimal disease.

A series of 60 acute nonlymphocytic leukemias (ANLL) was analyzed for the expression of terminal deoxynucleotidyl transferase (TdT). The detected TdT+ cells were studied in detail by use of double marker analyses for TdT and differentiation markers, such as myeloid markers (CD13 and CD33), B cell markers, T cell markers, and the precursor antigen CD34. In 15 (25%) of these leukemic cell samples, we found no TdT+ cells or low percentages of CD10+TdT+ cells; the latter probably represent precursor B cells. In the other 45 (75%) ANLL myeloid marker+TdT+ CD10- cells were detected, ranging from 0.1-10% (n = 24) or over 10% (n = 21) of mononuclear cells. Interestingly, a higher frequency of CD34 positivity was found on the TdT+ cells as compared to the TdT- cells, suggesting that the TdT+ cells represent an immature leukemic subpopulation. Therefore, it may be speculated that the TdT+ subpopulation contains the clonogenic ANLL cells. In two patients, in whom immunologic marker analysis was performed at initial diagnosis as well as at relapse, an expansion of the TdT+ subpopulation was documented at relapse, which may reflect a reduced differentiation capacity of the leukemic cells. Previous studies on a series of nonleukemic bone marrow and blood samples revealed that normal counterparts of myeloid marker+TdT+ cells are rare in bone marrow (less than 0.03%, if they occur at all) and that such cells are not detectable in blood. Therefore myeloid marker TdT double stainings may be useful to monitor the TdT+ leukemic subpopulation in patients with a TdT+ ANLL during and after chemotherapy. Our preliminary results on the follow-up of two such patients support this hypothesis.

Regeneration of TdT+, pre-B, and B cells in bone marrow after allogeneic bone marrow transplantation.

In 15 children transplanted with allogeneic bone marrow for acute leukemia and in complete remission, regeneration of the early stages of the B cell system was studied. Bone marrow aspirates taken before and longitudinally after BMT were investigated for pre-B and B cells by immunofluorescence techniques; in some cases, TdT+ cells were also determined. Normal values were derived from bone marrow samples taken from 23 healthy individuals who served as bone marrow donors. In normal bone marrow, B cells outnumber pre-B cells and the latter are more numerous than TdT+ cells. Before BMT, the numbers of BM pre-B were outside the normal range in all cases; B cell numbers were abnormal in most of the 11 patients studied, probably due to the antileukemic remission induction/consolidation therapy. After BMT, two distinct patterns of regeneration of the B cell system were observed. In 9 patients, TdT+ cells were considerably increased early after BMT. This was followed by a rise in pre-B cells, with values well above the normal range, and resulting in ratios of TdT+:pre-B cells and of pre-B cells:B cells that were transiently greater than 1. In the other 6 patients, the regeneration of TdT+ cells varied and the reconstitution of the pre-B cells was more gradual than in the first group, with pre-B-to-B cell ratios less than 1 during the whole observation period. The only consistent difference between the patients of the two groups, possibly relevant to the regeneration of the B cell lineage, was the duration of corticosteroid therapy, which was much longer in the 6 patients with slow-pace reconstitution. The pace of regeneration of the B cell system in the bone marrow was correlated with the recovery of the humoral immunity, as indicated by a significant increase in specific antibody titers after the second vaccination with diphtheria-tetanus-poliomyelitis vaccine in 7 of 9 patients in the rapid-pace group, versus 2 of 6 patients in the slow-pace group.

Terminal deoxynucleotidyl transferase (TdT)-containing peripheral blood mononuclear cells during remission of acute lymphoblastic leukemia: low sensitivity and specificity prevent accurate prediction of relapse.

Serial samples of peripheral blood were obtained from 75 children with acute lymphoblastic leukemia in remission. An immunofluorescence assay was used to quantitate TdT-containing (terminal deoxynucleotidyl transferase-containing) cells in the mononuclear leukocyte fraction of these specimens. Nine relapses in 8 patients were preceded by elevations (0.12-0.70%) in peripheral blood terminal deoxynucleotidyl transferase-containing cells noted 1-33 weeks prior to relapse. No such elevations were observed prior to 6 relapses in 4 patients. Peripheral blood terminal transferase deoxynucleotidyl-containing cells were elevated (0.12-0.61%) in 22 children who did not relapse over a 5-82 week period of observation. The sensitivity (67%) and specificity (68%) of this assay are inadequate to establish which patients have minimal residual leukemia.

Early relapse of acute lymphoblastic leukemia is not predictable by serial biochemical assays of terminal transferase activity in cells from peripheral blood.

Serial samples of peripheral blood were collected from 37 children with acute lymphoblastic leukemia (ALL) in remission. Activity of terminal transferase (TdT) was assayed by a biochemical technique. False positive results were obtained infrequently (approx. 1%), in contrast to experience with bone marrow analyses. However, early relapse of disease was not predictable in ALL by repeated measurement of TdT in circulating mononuclear cells during remission.

Detection of circulating 'terminal transferase-positive' cells does not predict relapse in acute lymphoblastic leukemia.

Serial samples of peripheral blood were obtained from 35 children with ALL over a period of 18 months. The mononuclear cells were examined for TdT by indirect immunofluorescence using an unpurified anti-calf thymus TdT as the primary antibody. This analysis failed to distinguish those children who were destined to relapse (n = 9) from those who remained in continuous complete remission. Rather, the exhibition of fluorescence was linked to the co-existence of infection, with a negative predictive value of 0.91. Putative 'TdT-positive' cells were concentrated in the T-lymphocyte fraction and the very process of E-rosette formation seemed to contribute to this phenomenon. It appears as if the anti-TdT reagent recognizes not only TdT but also a variety of antigens which are expressed on or in immature and activated lymphocytes.

Effects of antileukemic agents on the activity of terminal deoxynucleotidyl transferase from human normal thymic and leukemic cells "in vitro".

Several widely used and experimental antileukemic drugs have been tested on the activity of Terminal deoxynucleotidyl Transferase (TdT) purified from normal human thymocytes and T-derived acute lymphoblastic leukemia peripheral blood lymphoblasts. The majority of these inhibitors were equally potent inhibitors of the enzyme from thymocytes or leukemic lymphocytes. Adriamycine and etoposide were more potent inhibitors of the enzyme purified from thymocytes. Vincristine was a more potent inhibitor of TdT extracted from leukemic lymphocytes than from thymocytes. These results are discussed in terms of possible functions for TdT in the two types of cells and on the value of TdT as an indicator for clinical treatment.

Therapeutic and prognostic implications of glucocorticoid receptors and terminal deoxynucleotidyl transferase in acute leukemia.

Glucocorticoid receptors (GR) have been reported to predict clinical responsiveness to glucocorticoid therapy and to possess prognostic significance in leukemia. A raised level in terminal deoxynucleotidyl transferase (TdT) also seems to suggest clinical responsiveness to prednisone and vincristine therapy. We have investigated these two biochemical markers in the leukemic blasts in 23 patients with acute myeloid leukemia (AML) and in 19 patients with acute lymphoblastic leukemia (ALL) and have compared the binding data with clinical glucocorticoid sensitivity, with response to chemotherapy, with survival and with TdT. In 25 of these patients we have also investigated the in vitro glucocorticoid sensitivity by measuring dexamethasone inhibition of radiolabelled uridine and thymidine incorporation into the leukemic blasts. In patients with AML, GR levels were mostly high but did not correlate with the in vitro sensitivity, response to chemotherapy or survival. On the other hand, in the 19 patients with ALL there seemed to be a correlation between GR and in vitro sensitivity and between GR and clinical responsiveness to glucocorticoid therapy. Furthermore, patients with low levels of either TdT or GR seemed to be associated with poor prognosis. TdT activity and GR, however, did not associate with one another. TdT is a useful marker for the identification of lymphoblasts but, by itself, is not connected with glucocorticoid sensitivity. Gr, when applied to ALL, could supplement TdT determination by predicting response to therapy.

Laboratory assessment of nutritional status.

Malnutrition is one of the major causes of increased morbidity and mortality among hospitalized patients. The availability of nutritional therapy for these patients has made clinicians aware of the need for reliable methods of nutritional assessment. A variety of anthropometric, biochemical, and immunologic parameters has been used as indicators of protein-calorie malnutrition. Recently, the concentration of several rapid-turnover visceral proteins (transferrin, thyroxine-binding prealbumin and retinol-binding protein) has been shown to be a very sensitive parameter for indicating both the efficiency of nutritional therapy and conditions of borderline protein intake in apparently healthy children. Likewise, several immunologic parameters (including T cells, delayed hypersensitivity response, and complement components) have been shown to correlate with morbidity, mortality risk, sepsis, and death.

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

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 10(6) 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 clinical-morphological 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.

Avidin-biotin amplification procedures for the detection of human terminal deoxynucleotidyl transferase in cell smears.

The authors have developed fluorescent avidin and avidin-peroxidase assays to detect human terminal deoxynucleotidyl transferase (TdT) in cell smears. These assays used specifically purified rabbit anti-calf TdT antibody, biotinylated goat anti-rabbit IgG antibody, and either fluorescein isothiocyanate-avidin or avidin-biotinylated horseradish peroxidase complex. The use of phosphate-buffered saline rather than TRIS-buffered saline or borate-buffered saline was essential to obtain optimal results in the fluorescent avidin assay. Fixation of cells with either 0.5% paraformaldehyde or 10% neutral buffered formalin was found to be superior to either no fixation or fixation with cold methanol or cold 0.1% glutaraldehyde in ethanol. The sensitivities of the fluorescent avidin and avidin-peroxidase assays were shown to be identical, based on staining intensities and percentages of positive cells when both assays were performed on cells from 14 patients with acute lymphoblastic leukemia (ALL), and 5 patients with lymphoblastic lymphoma (LL).

Evaluation of methods of detecting terminal deoxynucleotidyl transferase in human hematologic malignancies. Comparison of immunofluorescence and enzymatic assays.

Terminal transferase (TdT) activity and antigen have been measured in 267 specimens of human bone marrow and peripheral blood by using a biochemical assay for enzymatic activity and an immunofluorescence test for antigen. Oligo p(dA)50 and dGTP were used as reagents in the biochemical assay and either rabbit anti-calf TdT or rabbit anti-human TdT was used as the primary antibody for immunofluorescence. Because both false-positive and false-negative detection of TdT antigen occurs, the biochemical assay of TdT activity is considered the standard against which immunofluorescence assays must be measured. If specimens of cells contained TdT activity, then the immunofluorescence detected antigen in 91% of cases (rabbit anti-calf TdT) and 95% of cases (rabbit anti-human TdT). When no TdT activity was detected, the immunofluorescence test was positive in 7.8% of cases (rabbit anti-calf TdT) and 5.2% of cases (rabbit anti-human TdT). When air-dried slices were shipped by air mail to a distant location before being stained for immunofluorescence, TdT antigen was detected in only 33% of matched pair cases which contained TdT activity. From this study, the authors conclude that with current methodology, immunofluorescence tests for TdT antigen must be carried out on slides prepared in the testing laboratory and that such tests are reliable in more than 90% of cases. However, because a small percentage of results are false positives and false negatives, the authors suggest that if a patient's clinical response is not consistent with the immunofluorescence TdT result, an enzymatic assay for TdT activity be carried out.

Terminal deoxynucleotidyl transferase activity in neoplastic and nonneoplastic hematopoietic cells.

Terminal deoxynucleotidyl transferase activity was investigated by enzyme assay and (or) an indirect immunofluorescence method with specimens from 151 patients who had a variety of neoplastic and nonneoplastic conditions: leukemia, 62; non-Hodgkin's lymphoma, 36; Hodgkin's disease, seven; lymph nodes without neoplasms, 21; normal peripheral blood, 15; normal bone marrow, ten. Immunologic studies were done on samples from 82 of these patients. Increased terminal deoxynucleotidyl transferase activity was found by both methods in patients who had acute lymphoblastic leukemia and the lymphoid blast crisis of chronic myelocytic leukemia and by the immunofluorescence method in patients who had lymphoblastic lymphoma. A single patient with acute monoblastic leukemia was found by both technics to have increased enzyme activity. Three B-cell proliferations were positive by the enzyme assay; none was positive with the immunofluorescence method. In the remaining 42 B-cell proliferations, the levels of terminal deoxynucleotidyl transferase activity were found to be normal by both the enzyme assay and the immunofluorescence method. Cytoplasmic positivity was observed in as much as 10% of the cells in 13 specimens that were otherwise negative and in eight samples in association with nuclear positivity. A comparison of terminal deoxynucleotidyl transferase activity in microunits/mg protein (enzyme assay) with the percentage of positive cells (immunofluorescence method) yielded a correlation coefficient of 0.62 (P less than 0.01).

Biochemical enzyme analysis in acute leukaemia.

This report summarises the current knowledge regarding the clinical utility of biochemical enzyme markers for both diagnostic and therapeutic purposes in acute leukaemia. The enzymes studied most extensively in this field are terminal deoxynucleotidyl transferase, adenosine deaminase, 5'-nucleotidase, purine nucleoside phosphorylase, and acid phosphatase, esterase, hexosaminidase isoenzymes. For each enzyme, the quantitative and qualitative characteristics in various immunologically defined subclasses of acute leukaemia are described. The quantitative evaluation of enzyme activities represents an adjunctive classification technique which should be incorporated into the multivariate analysis, the "multiple marker analysis." By qualitative characterisation pronounced heterogeneity of leukaemia subsets is uncovered. The application of 2'-deoxycoformycin, a specific inhibitor of adenosine deaminase, and the potential usefulness of two other enzymes as targets for treatment with selective agents is discussed. The concept that gene products expressed at certain developmental stages of normal cells can similarly be detected in leukaemic cells (which therefore seem to be "frozen" or "arrested" at this particular maturation/differentiation stage) is supported by the results obtained in enzyme studies. Besides their practical clinical importance for classification and treatment of acute leukaemias, biochemical enzyme markers constitute a valuable research tool to disclose biological properties of leukaemic cells.