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Purpose: Leukemic cells from T-lineage acute lymphoblastic leukemia (ALL) patients are thought to originate from T-lymphocyte precursors corresponding to discrete stages of T-cell ontogeny. Here we sought to determine the influence of leukemic cell apparent maturational stage on treatment outcomes in pediatric T-lineage ALL., Patients and Methods: From 1983 through 1993, 407 pediatric T-lineage ALL patients were enrolled onto two sequential series of risk-adjusted treatment protocols of the Children's Cancer Group. In the current analysis, T-lineage ALL patients were immunophenotypically classified as follows: CD7+ CD2- CD5- pro-thymocyte leukemia (pro-TL), CD7+ (CD2 or CD5)+ CD3- immature TL, and CD7+ CD2+ CD5+ CD3+ mature TL., Results: Similar induction outcomes of 91.4%, 97.1%, and 98.3% were obtained by the pro-, immature, and mature TL groups, respectively. Four-year event-free survival (EFS) was lower for pro-TL patients (57.1%; SD = 8.4%,) compared with immature and mature TL patients (68.5%; SD = 3.5%; and 77.1%; SD = 4.0%, respectively) with an overall significance of .05 (log-rank test) or .04 (log-rank trend test). Relative hazards rates (RHR) were 2.11 and 1.22 for pro-TL and immature TL versus mature TL, respectively. Highly significant differences were found for overall survival (P = .005, log-rank test; P = .009, log-rank trend test). Multivariate analysis confirmed that the prognostic influence of ontogeny grouping was independent of that of other prognostic factors., Conclusion: Leukemic cells of the pro-TL maturation stage identify a small subgroup of T-lineage ALL patients who have a significantly worse EFS outcome than patients whose cells are of a more mature stage of development.

We report the development of a double-cycle elutriation (DCE) technique separating 3 or greater logs of T cells from a stem-cell-enriched marrow fraction and the results of phase I T-cell depletion studies with HLA-disparate related bone marrow transplantation (BMT) donors in two patient groups. In group 1, 10 patients with refractory hematopoietic malignancies received combination chemotherapy, total body irradiation (TBI), and immunosuppression (pre- and post-BMT), and hematopoietic rescue with a marrow transplant, depleted of T cells by elutriation. Potentially to promote engraftment and a graft-versus-leukemia (GVL) effect, 0.5 to 0.75 x 10(5) T cells/kg were added back. All 10 patients engrafted. Five patients developed acute graft-versus-host disease (GVHD; four grade II, one grade III) and two subsequently developed chronic GVHD. Two patients have relapsed (median follow-up, 206 days; range, 46 to 1,035). Four patients died of BMT-related complications (three of infection, one of veno-occlusive disease [VOD]). Four patient are disease-free survivors (median follow-up, 960 days; range, 670 to 1,035). Group 2 included five infants, four with congenital lymphohematopoietic deficiencies and one with refractory acute lymphocytic leukemia (ALL). In these infants, busulfan and increased cyclophosphamide were substituted for TBI. Only the ALL patient received added T cells. Three patients engrafted: one has stable mixed chimerism, one relapsed with ALL, and one rejected the marrow. One patient had primary autologous recovery, while another failed to engraft. None developed GVHD. We conclude that, in this setting of HLA-disparate BMT with post-BMT antithymocyte globulin (ATG) and corticosteroids, DCE significantly depletes T cells from the marrow and that a defined number of T cells can be added without the occurrence of severe GVHD.

Purine nucleoside phosphorylase (PNP) activity has been measured in the lymphoblasts of 22 patients with acute lymphoblastic leukemia (ALL) and correlated with routine immunologic cell surface markers. Fourteen of the 22 patients were considered to have non-T, non-B-cell ALL; 8 patients had T-cell disease. The median PNP activity in 21 control samples of normal peripheral blood mononuclear cells was 83 U. The median PNP activity of the non-T, non-B lymphoblasts was 79 U. No statistical difference in PNP activity between these two groups could be discerned (p < 0.37). In contrast, T-cell lymphoblasts demonstrated diminished PNP activity with a median of 38 U. The differences in activity between T lymphoblasts and both non-T, non-B leukemic cells and normal peripheral blood mononuclear cells were significant (p < 0.001 and p < 0.003, respectively). Evaluation of PNP activity provides further evidence of biochemical heterogeneity among immunologic subclasses of ALL.

T-cell immunity and serum levels of thymosin alpha 1, beta 2-microglobulin, circulating immune complexes, serum immunoglobulin levels, antibodies to hepatitis surface or core antigen, and to cytomegalovirus, and Epstein-Barr virus were investigated in 51 patients with haemophilia A ranging in age from 2 to 52 years. All patients had received commercial U.S. lyophilized concentrates of antihaemophilic factor (AHF). The mean helper/cytotoxic-suppressor (OKT4/OKT8) ratio of 11 pre-adolescents (1.6 +/- 0.4 SE) was not significantly different from that of age matched normal controls. In contrast, the mean OKT4/OKT8 ratios of 13 adolescent (1.2 +/- 0.2 SE) and 23 adult (0.8 +/- 0.1 SE) haemophiliacs were significantly reduced. Abnormalities of lymphocyte mitogenic responses were found only in adult haemophiliacs. Nine individuals treated with commercial U.S. prothrombin complex concentrates for antibodies directed against AHF or for haemophilia B had normal mean OKT4/OKT8 values. The mean serum thymosin alpha 1 levels for each age category was similar to that of age matched controls; however, regression analysis revealed a significant relationship between elevated thymosin alpha 1 levels and decreased OKT4/OKT8 ratios in adult haemophiliacs (P = 0.012). Although the mean serum level of beta 2-microglobulin was significantly increased in the adult haemophiliac group, there was no correlation between OKT4/OKT8 ratios and any of the other serologic parameters studied.

Previous studies have documented that the interaction of IgG immune complexes with the FcIgG receptor (Fc-IgGR) of human T lymphocytes induces alterations both in the expression of Fc receptors and in the functional capacities of these cells. After IgG immune complex exposure, Fc-IgGR-bearing T lymphocytes lose their Fc-IgGR and generate Fc-IgMR in subsequent culture. This transition phenomenon is mediated via patching and capping of the Fc-IgGR. Since Colchicine and Cytochalasin B have been demonstrated to interfere with capping of other membrane elements by their known effects on cytoskeletal structures, we investigated the effects of these agents on the expression of Fc receptors of human T cells. Neither Colchicine nor Cytochalasin B directly affects Fc-IgGR or Fc-IgMR expression. However, both agents inhibit the loss of Fc-IgGR subsequent to IgG immune complex exposure as well as the development of Fc-IgMR in culture. Immunofluorescent studies illustrate that this inhibition is mediated in part by interference with capping of the Fc-IgGR-immune complex ligand. This study emphasizes the inverse relationship between Fc-IgGR and Fc-IgMR expression on human T lymphocytes.

We have recently reported that the disialoganglioside GD3 is found in cellular lipid extracts of T-cell acute lymphoblastic malignancies (T-ALL) but is not detectable by resorcinol staining in extracts of non-T acute lymphoblastic leukemia blasts (non-T-ALL). We have now extended this study to assess the detectability of GD3 in T-ALL vs non-T-ALL utilizing an anti-GD3 antibody, R24. Gangliosides isolated from T-ALL and non-T-ALL blasts by two different methods were separated by thin-layer chromatography and stained with anti-GD3 and a control antibody specific for GM3 and sialosylparagloboside (SPG). Anti-GD3 reactivity was observed in extracts from T-ALL cells in all cases, whereas GD3 was not detected in any of the non-T-ALL samples. The anti-GM3/SPG antibody stained GM3 in all of the leukemic samples analyzed as well as SPG in the non-T-ALL samples. Indirect immunofluorescence was used to assess the expression of GD3 at the surface of leukemic blasts. Fluorescence-activated cell sorting analysis with R24 showed that whereas T-ALL blasts were highly reactive with this antibody, non-T-ALL blasts were totally unreactive. In an analysis of a larger number of leukemia patients by fluorescence microscopy, 20 out of 28 samples with the T-ALL phenotype were positive for R24, whereas zero out of 11 non-T-ALL samples were reactive. These results confirm our earlier finding of the specificity of GD3 to the T-ALL subclass of childhood leukemias and furthermore suggest the potential value of anti-GD3 as an immunological tool for the diagnosis and therapy of T-cell ALL.

We examined alpha-, beta-, and gamma-T cell receptor (TCR) gene activation within acute lymphoblastic leukemias (ALLs) that represent early stages of B and T cell development. We wished to determine if TCR rearrangement and expression was lineage restricted, showed any developmental hierarchy, or could identify new subsets of T cells. Rearrangement of gamma and beta TCR genes occurred early in development but in no set order, and most T-ALLs (22/26) were of sufficient maturity to have rearranged both genes. T-ALLs preferentially rearranged C gamma 2 versus the C gamma 1 complex; no preference within the beta locus was apparent. Once rearranged, the beta TCR continued to be expressed (11/13), whereas the gamma TCR was rarely expressed (3/14). The alpha TCR was expressed only in more mature T-ALLs (8/14) that usually displayed T3. The 3A-1 T cell associated antigen appeared earliest in development followed by T11 and T3. Within pre-B cell ALL a higher incidence of lineage spillover was noted for gamma TCR rearrangements (8/17) than for beta rearrangements (3/17). This also contrasts with the only occasional rearrangement of immunoglobulin (Ig) heavy chains (3/25) in T-ALL. However, in pre-B ALL the pattern of gamma TCR usage was distinct from that of T cells, with the C gamma 1 complex utilized more frequently. Almost all ALLs could be classified as pre-B or T cell in type by combining Ig and TCR genes with monoclonal antibodies recognizing surface antigens, although examples of lineage duality were noted. Unique subpopulations of cells were discovered including two genetically uncommitted ALLs that failed to rearrange either Ig or TCR loci. Moreover, two T lymphoblasts were identified that possessed the T3 molecule but failed to express alpha plus beta TCR genes. These T-ALLs may represent a fortuitous transformation of T cell subsets with alternative T3-Ti complexes.

We examined immunoglobulin (Ig) heavy chain, K light chain, and T cell receptor (TCR) gamma and beta gene configuration in the leukemic cells from a series of infants aged less than 1 year with acute lymphoblastic leukemia (ALL). Each of these 11 cases demonstrated leukemic cell surface antigens that have been correlated with a B cell precursor phenotype. Of the 11, lymphoblasts of 4 retained the germline configuration of both Ig and TCR loci, whereas 7 had rearranged the Ig heavy chain gene. Two of these seven showed light chain gene rearrangement. TCB beta chain rearrangement had occurred in only one of the 11 patients' tumors. No TCR gamma chain rearrangements were identified. These results are in contrast to earlier studies of B cell precursor ALL in children in which Ig heavy chain gene rearrangements were evident in every case and approximately 40% showed Ig light chain rearrangement as well. In addition, 45% of cases of B cell precursor ALL of children had rearranged their gamma TCR genes, and 20% had rearranged beta. These data suggest that ALL in infancy represents an earlier stage of B cell development than is found in B cell precursor ALL of children. ALL in the infant age group has been associated with the worst prognosis of all patients with ALL. This study suggests that the disease in infants differs not only clinically, but also at the molecular genetic level, from the disease in children.

Lymphoblasts from seven children with T-cell lymphoblastic malignancies and three children with non-T, non-B acute lymphoblastic leukemia (ALL) were analyzed for ganglioside content. Nonmalignant T-cells from thymus served as controls. Both ganglioside and glycoprotein sialic acid were increased approximately 3-3.5-fold in T-cell disease compared to thymic tissue when expressed on a per cell basis, but not on a per milligram protein basis. Thin-layer chromatography of the isolated ganglioside fraction from T-cell lymphoblasts revealed two major resorcinol-positive bands. One ganglioside comigrated with II3-alpha-N-acetylneuraminosyllactosylceramide (GM3), the major ganglioside in normal lymphoid tissue, and the other ganglioside comigrated with authentic II3-alpha-N-acetylneuraminosyl-alpha 2----8-N-acetylneuraminosyllactosylceramide (GD3) in three different solvent systems. Neuraminidase treatment of the latter ganglioside yielded GM3 and lactosyl ceramide, hydrolysis products of GD3. Scanning densitometry revealed that whereas thymus cells contained 97% GM3 and 3% GD3, T-cell lymphoblasts contained from 22 to 86% GD3 and a corresponding decrease in GM3. The shift to increased GD3 was observed in the blasts from all seven T-cell patients, but not in the blasts from the non-T, non-B patients studied. Only trace quantities of GD3 were detected from two continuous T-ALL cell lines, HSB2 and RPMI 8402. The results demonstrate a consistently significant increase in ganglioside GD3 in uncultured, patient-derived T-cell ALL lymphoblasts when compared to non-T-cell ALL and normal lymphoid tissue. Therefore, GD3 may represent a tumor-associated antigen for the T-cell subclass of childhood lymphoblastic malignancy.