Early T-cell Precursor Research Articles

96 Childhood ALL: from the randomized clinical trials to an evidence-based approach

Early T-cell precursor acute lymphoblastic leukaemia was recently recognised as a distinct leukaemia and reported as associated with poor outcomes. We aimed to assess the outcome of early T-cell precursor acute lymphoblastic leukaemia in patients from the Italian Association of Pediatric Hematology Oncology (AIEOP) centres treated with AIEOP-Berlin-Frankfurt-Münster (AIEOP-BFM) protocols.In this retrospective analysis, we included all children aged from 1 to less than 18 years with early T-cell precursor acute lymphoblastic leukaemia immunophenotype diagnosed between Jan 1, 2008, and Oct 31, 2014, from AIEOP centres. Early T-cell precursors were defined as being CD1a and CD8 negative, CD5 weak positive or negative, and positive for at least one of the following antigens: CD34, CD117, HLADR, CD13, CD33, CD11b, or CD65. Treatment was based on AIEOP-BFM acute lymphoblastic leukaemia 2000 (NCT00613457) or AIEOP-BFM acute lymphoblastic leukaemia 2009 protocols (European Clinical Trials Database 2007-004270-43). The main differences in treatment and stratification of T-cell acute lymphoblastic leukaemia between the two protocols were that in the 2009 protocol only, pegylated L-asparaginase was substituted for Escherichia coli L-asparaginase, patients with prednisone poor response received an additional dose of cyclophosphamide at day 10 of phase IA, and high minimal residual disease at day 15 assessed by flow cytometry was used as a high-risk criterion. Outcomes were assessed in terms of event-free survival, disease-free survival, and overall survival.Early T-cell precursor acute lymphoblastic leukaemia was diagnosed in 49 patients. Compared with overall T-cell acute lymphoblastic leukaemia, it was associated with absence of molecular markers for PCR detection of minimal residual disease in 25 (56%) of 45 patients; prednisone poor response in 27 (55%) of 49 patients; high minimal residual disease at day 15 after starting therapy in 25 (64%) of 39 patients (bone marrow blasts ≥10%, by flow cytometry); no complete remission after phase IA in 7 (15%) of 46 patients (bone marrow blasts ≥5%, morphologically); and high PCR minimal residual disease (≥5 × 10–4) at day 33 after starting therapy in 17 (85%) of 20 patients with markers available. Overall, 38 (78%) of 49 patients are in continuous complete remission, including 13 of 18 after haemopoietic stem cell transplantation, with three deaths in induction, five deaths after haemopoietic stem cell transplantation, and three relapses. Severe adverse events in the 2009 study were reported in 10 (30%) of 33 patients with early T-cell precursor acute lymphoblastic leukaemia versus 24 (15%) of 164 patients without early T-cell precursor acute lymphoblastic leukaemia and life-threatening events in induction phase IA occurred in 4 (12%) of 33 patients with early T-cell precursor acute lymphoblastic leukaemia versus 7 (4%) of 164 patients without early T-cell precursor acute lymphoblastic leukaemia. No difference was seen in the subsequent consolidation phase IB of protocol I.Early T-cell precursor acute lymphoblastic leukaemia is characterised by poor early response to conventional induction treatment. Consolidation phase IB, based on cyclophosphamide, 6-mercaptopurine, and ara-C at conventional (non-high) doses is effective in reducing minimal residual disease. Although the number of patients and observational time are limited, patients with early T-cell precursor acute lymphoblastic leukaemia treated with current BFM stratification and treatment strategy have a favourable outcome compared with earlier reports. The role of innovative therapies and haemopoietic stem cell therapy in early T-cell precursor acute lymphoblastic leukaemia needs to be assessed.None.

Expression of the c-kit (CD117) Molecule in Normal and Malignant Hematopoiesis

The c-kit proto-oncogen (CD117) has been shown to be present in several cell types including normal and neoplastic hemopoietic cells. Among normal BM cells, CD117 expression has been found in about half of the CD34+ precursors including progenitors committed to the ery-throid, granulo-monocytic, and megakaryocytic cell lineages. In addition, strong CD117 expression is detected in bone marrow mast cells as well as in a small subset of NK cells displaying strong reactivity for CD56, and in a relatively important proportion of CD3−/CD4−/CD8− prothymocytes. These results suggest that CD 117 expression can be detected in both myeloid and lymphoid lineages although for the lymphoid lineage it would be restricted to a small NK-cell subset and early T-cell precursors. In acute leukemias CD117 expression was initially associated with AML. Nevertheless, at present it is well established that CD117 expression may also be found in a relatively important proportion of T-ALL while it is usually absent in B-lineage ALL. Moreover, recent studies have shown that in about one-third of multiple myeloma cases and patients with monoclonal gammopathy of undetermined significance plasma cells display reactivity for CD117. The prognostic influence of CD117 expression has not yet been clearly established. The analysis of this marker may also be of value for the investigation of minimal residual disease (MRD). It has been suggested that CD117 in combination with other antigens may be of great help for the identification of leukemia-associated phenotypes that could be used to monitor MRD in both acute myeloid leukemias and multiple myeloma patients achieving morphological complete remission.

Day 11 mouse fetal thymus: phenotype and search for the point of commitment

This phenotype study of uncommitted hematopoietic cells (UHC) and early T cell precursors (ETCP) in the thymus provides an understanding of the commitment process from UHC to ETCP. The study of genes that are involved in this process depends on the proper identification of these early cells. Nonetheless, most current phenotype studies of these cells are based on the observations from the late stages of fetal or adult thymocytes. Though conclusions drawn from these studies are insightful, cell maturation occurs so fast during thymus development that some important phenotypic nuances go unnoticed if one only looks at late-stage cells. Furthermore, even though early-stage thymocytes are phenotypically similar to those of late stage, they may have different properties. In order to study those thymus populations at the very beginning of their differentiation and commitment, 11-day mouse fetal thymuses were tested. This shows, using day-12 CFU-S as a measure, that at this stage of development there are UHC present. Thymocytes at this very initial stage of development are isolated for the first time, and their phenotypes as well as their differentiation potentials are analyzed. The result shows that UHC are detected only in the Mac-1 − C-kit + subset, which comprises 2% of the total thymus population. Surprisingly, 93% of the C-kit + population is Mac-1 +, which generates T cells with a frequency of 1/72. This indicates that the Mac-1 molecule is a differentiation marker rather than a lineage-specific marker. In addition, C-kit + thymocytes at this stage do not express any T cell markers, such as Thy1.2, IL-2Rα, CD2 and Mel-14. These thymocytes are very akin to UHC, since they express Sca-1, Wga, and CD34, and are likewise similar to ETCP, as they express Pgp high, Mhc-I high, Hsa low, and FcR low. Moreover, they express a high level of adhesion molecules such as Lfa-1, Icam-1 and Lpam-1. As expected, these C-kit + all contain the hematopoietic cell marker CD45. Low expression of CD4 (the typical marker associated to the earliest T cell precursor in adult thymuses) is also found in 10% of the C-kit + population. While the C-kit + population at this stage is more homogeneous than at any other stage of fetal thymus development, there are still markers (B220, CD5, Tsa, Mac-1, CD4, and Sca-1) that can split this population into other subsets. However, the majority of these markers are present in the Mac-1 + C-kit + population, indicating that the C-kit + population is essentially made of two populations (Mac-1 + and Mac-1 − subsets). Interestingly, two major single-positive populations appear to emerge from this C-kit + population one day later, namely Thy1.2 + IL-2Rα − and Thy1.2 − IL-2Rα +. These two major single-positive populations seem to be derived directly from the Mac-1 + rather than from the Mac-1 − subset of the C-kit + population. Thus, these data suggest that important phenotypes are present during the early differentiation process. These phenotypes have never been shown before. Hopefully, this study will open up a new avenue for the study of very early stage T cell sublineages and their relationship to uncommitted thymic hematopoietic cells.

Fucose and galactose receptor and liver recognition by lymphoma cells

A syngeneic model system for the study of metastases is described. The system consisted of 2 lymphoma clones (A/63-I and A/63-2) derived from a single thymoma (A/63) induced by a wild-type Abelson-Moloney viral complex. Phenotype and genotype analyses revealed that both clones were derived from transformation of early T-cell precursors. An in vivo study of the colonizing potential following intravenous (i.v.) injection of clones showed that only the A/63-I cell clone colonized the liver. This observation was confirmed by quantitative analysis of organ distribution of both cell clones consecutive to i.v. injection of 125IUdR-labelled cells. In the same way, an in vitro study of the invasive potential of both clones was performed on frozen liver sections and showed that only the A/63-I cell clone had the ability to attach to liver. This specific adhesion was inhibited by L-fucose, D-galactose, N-acetyl-D-galactosamine (D-GalNAc) and with D-galactose- and L-fucose-containing neoglycoproteins. Differences in cell surface carbohydrates of the 2 cell clones were detected using various lectins: peanut agglutinin (PNA), Dolichos biflorus (DBA), Aleuria aurantia (AAA) and Galactia tenuiflora agglutinins (GTA). A/63-I was found to react strongly with PNA, DBA and GTA, and the removal of sialic acid by neuraminidase treatment increased DBA and PNA receptor sites of A/63-2 as compared to A/63-I. The present data suggest that cell-surface GalNAc, galactosyl and fucosyl residues are responsible for the ability of the A/63-I cell clone to recognize liver tissue probably through binding to a Kupffer-cell-associated lectin.

Isolation of an early T-cell precursor (CFU-TL) from human bone marrow

CD2-CD3-CD4-CD8- human bone marrow (BM) cells were previously shown to generate T-cell clones in vitro. This capacity was abolished after treatment of this population with anti-CD7 monoclonal antibody and complement. In this study, using rosetting with sheep erythrocytes, complement-dependent cytotoxicity, and specific immunoadherence method, we isolated a minor BM subset that contained more than 80% CD7+CD2-CD3- CD4-CD8- cells with small lymphoid cell morphology. They comprised most early T-cell precursors (CFU-TL) as they displayed high capacity to generate T-cell clones when cultured in limiting dilutions. CFU-TL nature of these cells was also confirmed by the sequential expression of mature T-cell specific markers on their surface after in vitro induction. This BM subset also contained 2% to 3% CFU-GM precursors. Together, these results pointed to the existence of BM CD7+CD2- precursors with high differentiation potential and showed the commitment of most of them to T-cell lineage.

Isolation of an early T-cell precursor (CFU-TL) from human bone marrow

CD2-CD3-CD4-CD8- human bone marrow (BM) cells were previously shown to generate T-cell clones in vitro. This capacity was abolished after treatment of this population with anti-CD7 monoclonal antibody and complement. In this study, using rosetting with sheep erythrocytes, complement-dependent cytotoxicity, and specific immunoadherence method, we isolated a minor BM subset that contained more than 80% CD7+CD2-CD3-CD4-CD8- cells with small lymphoid cell morphology. They comprised most early T-cell precursors (CFU-TL) as they displayed high capacity to generate T-cell clones when cultured in limiting dilutions. CFU-TL nature of these cells was also confirmed by the sequential expression of mature T-cell specific markers on their surface after in vitro induction. This BM subset also contained 2% to 3% CFU-GM precursors. Together, these results pointed to the existence of BM CD7+CD2- precursors with high differentiation potential and showed the commitment of most of them to T-cell lineage.

Peripheral blood T lymphocytes of patients with chronic B cell lymphatic leukemia express Ia-like antigens

15.7 +/- 3.5% of sheep rosette-forming cells (SRFC) were isolated from the peripheral blood of 11 patients with B-cell chronic lympholeukemia (B-CLL). SRFC did not express surface immunoglobulins, antigens of nondifferentiated blasts and antigens of early T-cell precursors, while NK-cell antigen expression was low. 6 of 11 patients revealed 44.6 +/- 19.6% of Ia-like antigens in SRFC. Ia-like antigen expression was 4.8 +/- 0.6% in SRFC isolated from the peripheral blood of 19 healthy donors. The expression of Ia-like antigens in T cells of patients with B-CLL is suggested to be related to the activation of regulatory T-lymphocyte subpopulation.

Intrathymic differentiation: Thymocyte heterogeneity and the characterization of early t-cell precursors

Multiparameter FCA of unfractionated or isolated subpopulations of thymocytes reveals at least seven subpopulations in the mouse thymus. One of these subpopulations, designated dLy1 has been isolated and characterized extensively. The data reviewed here indicate that the dLy1 thymocyte subpopulation, whether derived from the adult or fetal thymus, represents an early stage in intrathymic differentiation. The immature status of dLy1 cells was suggested by the extensive similarity to a predominant cell type that occurs early in fetal thymic ontogeny. Its precursor role was demonstrated by its capacity to generate cortical and medullary-type thymocytes in vivo. Its expression of Ly1, Thy-1 and mRNA specific for the beta-chain of the T-cell antigen receptor support its commitment to a T-cell developmental pathway. In summary, dLy1 thymocytes appear to be the earliest committed T cells yet to be described, isolated and characterized. Further investigation should reveal whether this subpopulation of thymocytes contains subsets of cells in earlier states of maturation and/or precursors already committed to more than one T-cell lineage.

Establishment of continuous cultures of Thy1.2 +, Lytl +,2 −T cells with purified interleukin 3

IL-3 is a recently described lymphokine that induces the expression of the enzyme 20-α-hydroxysteroid dehydrogenase (20αSDH) in an early T-cell precursor. We demonstrate that purified IL-3 can be used to establish continuous cultures of a discrete subpopulation of T cells with virtually 100% efficiency from normal, unstimulated splenic lymphocyte populations. Once established over a period of approximately 4–6 weeks, the cultures can be readily cloned either in soft agar or by limiting dilution. All of the established lines are Lyt1 +,2 −, la −, Ig −, Tdt −, 20αSDH +, a phenotype characteristic of helper T cells; they are therefore distinct from continuous cultures of T cells established with IL-2. Although initiation of these cell lines was absolutely dependent on IL-3, once established all of the cell lines were independent of exogenously added lymphokines for their growth in vitro. However, all of the cell lines were found to constitutively produce IL-3 at high levels. None of the cell lines constitutively produced IL-2, but could be readily induced to produce this lymphokine by treatment with phorbol myristic acetate. The ability to produce IL-3 and IL-2 is a further indication that all the cell lines are helper T cells. The possible mechanisms by which IL-3 allows the specific differentiation and/or amplification of T cells of helper phenotype in tissue culture are discussed.

Cultured human leukemic non-T/non-B lymphoblasts and their stimulating capacity in “one-way”mixed lymphocyte reaction. Suggestive evidence for early T-cell or B-cell precursors

It is now well recognized that a large proportion of cases with acute lymphoblastic leukemia are classified as non-T/non-B neoplastic disease. The origin of leukemic non-T/non-B cells is at present not known. It has been shown that fresh or cultured leukemic T lymphoblasts exert no stimulating capacity while leukemic B lymphoblasts exert a strong stimulation in "one-way" mixed lymphocyte reaction. It has also been shown that fresh leukemic cells from some patients with non-T/non-B acute lymphoblastic leukemia possess a strong stimulation while leukemic cells from other patients with this disease possess no stimulation on allogeneic lymphocytes. The present study shows that cultured leukemic lymphoblasts from 3 non-T/non-B cell lines (NALL-1, NALM-6 and NALM-16) consistently exert a strong stimulation on allogeneic lymphocytes. On the other hand, cultured leukemic lymphoblasts from 2 non-T/non-B cell lines (REH and KM-3) consistently fail to stimulate in "one-way" mixed lymphocyte reaction. Our data clearly support the speculation that leukemic non-T/non-B cells which possess the stimulating capacity may represent less differentiated leukemic B lymphoid cells (pre-B cells) and leukemic non-T/non-B cells which possess no stimulating capacity may represent less differentiated leukemic T lymphoid cells (per-T cells).