Modified LSA2-L2 treatment in 53 children with E-rosette-positive T- cell leukemia: results and prognostic factors (a Pediatric Oncology Group Study)

In an attempt to improve the poor outlook for children with T-cell leukemia (T-ALL), the Southwest Oncology Group, Pediatric Division, used a modified LSA2-L2 multidrug regimen to treat 53 patients with E- rosette-positive T-ALL. This regimen was chosen because of its demonstrated efficacy in T-cell (mediastinal) non-Hodgkin's lymphoma. Complete remission (CR) rate was 88%. Range of follow-up for those patients remaining in CR is 24-49 mo (median 39 mo). Life table analysis estimates that 40% (SE 8.3%) of all patients who started induction therapy will remain failure-free at 3 yr. For patients achieving CR, 46% (SE 9%) are projected to remain in both marrow and extramedullary CR at 3 yr. Median failure-free duration was 13 mo, but only 1 patient has relapsed beyond 16 mo. Twenty-nine percent of initial relapses were isolated CNS relapses. The following presenting factors did not relate significantly to outcome: hemoglobin, platelet count, uric acid, race, and mediastinal mass. Age greater than 10 yr was a poor prognosis indicator only in the less than 50,000/microliter WBC group. Sex was not a significant factor after adjusting for WBC. WBC was the most important prognostic factor: 19% (SE 8%) of patients with WBC greater than 50,000/microliter are projected to remain failure- free at 3 yr as compared to 67% (SE 11%) of patients with WBC less than 50,000/microliter. Although the overall results are better than those previously reported for pediatric patients with T-ALL, the long-term failure-free rate remains low for patients presenting with greater than 50,000/microliter WBC.     

Prothrombin complex concentrates and a specific antidote to dabigatran are effective ex-vivo in reversing the effects of dabigatran in an anticoagulation/liver trauma experimental model

Introduction

New oral anticoagulants are effective alternatives to warfarin. However, no specific reversal agents are available for life-threatening bleeding or emergency surgery. Using a porcine model of trauma, this study assessed the ability of prothrombin complex concentrate (PCC), activated PCC (aPCC), recombinant FVIIa (rFVIIa) and a specific antidote to dabigatran (aDabi-Fab) to reverse the anticoagulant effects of dabigatran.

Methods

Dabigatran etexilate (DE) was given orally for 3 days (30 mg/kg bid) and intravenously on day 4 to achieve consistent, supratherapeutic concentrations of dabigatran. Blood samples were collected at baseline, after oral DE, after intravenous dabigatran, and 60 minutes post-injury. PCC (30 and 60 U/kg), aPCC (30 and 60 U/kg), rFVIIa (90 and 180 μg/kg) and antidote (60 and 120 mg/kg) were added to blood samples ex-vivo. Coagulation was assessed by thromboelastometry, global coagulation assays and diluted thrombin time.

Results

Plasma concentrations of dabigatran were 380 ± 106 ng/ml and 1423 ± 432 ng/ml after oral and intravenous administration, respectively, and all coagulation parameters were affected by dabigatran. Both PCCs and aDabi-Fab, but not rFVIIa, reversed the effects of dabigatran on thromboelastometry parameters and prothrombin time. In contrast, aPTT was only normalised by aDabi-Fab. Plasma concentration (activity) of dabigatran remained elevated after PCC and rFVIIa therapy, but was not measureable after aDabi-Fab.

Conclusion

In conclusion, PCC and aPCC were effective in reducing the anticoagulant effects of dabigatran under different conditions, while aDabi-Fab fully corrected all coagulation measures and decreased the plasma concentration of dabigatran below the limit of detection. No significant effects were observed with rFVIIa.     

Multiple tumor-suppressor gene 1 inactivation is the most frequent genetic alteration in T-cell acute lymphoblastic leukemia

No constant genetic alteration has yet been unravelled in T-cell acute lymphoblastic leukemia (T-ALL), and, to date, the most frequent alteration, the SIL-TAL1 deletion, is found in approximately 20% of cases. Recently, two genes have been identified, the multiple tumor- suppressor gene 1 (MTS1) and multiple tumor-suppressor gene 2 (MTS2), whose products inhibit cell cycle progression. A characterization of the MTS locus organization allowed to determine the incidence of MTS1 and MTS2 inactivation in T-ALL. MTS1 and MTS2 configurations were determined by Southern blotting using 8 probes in 59 patients with T- ALL (40 children and 19 adults). Biallelic MTS1 inactivation by deletions and/or rearrangements was observed in 45 cases (76%). Monoallelic alterations were found in 6 cases (10%). The second MTS1 allele was studied in the 4 cases with available material. A point mutation was found in 2 cases. The lack of MTS1 mRNA expression was observed by Northern blot analysis in a third case. A normal single- strand conformation polymorphism pattern of MTS1 exons 1alpha and 2 was found and MTS1 RNA was detected in the fourth case, but a rearrangement occurring 5' to MTS1 exon 1 alpha deleting MTS1 exon 1Beta was documented. One case presented a complex rearrangement. Germline configuration for MTS1 and MTS2 was found in only 7 cases. The localization of the 17 breakpoints occurring in the MTS locus were determined. Ten of them (59%) are clustered in a 6-kb region located 5 kb downstream to the newly identified MTS1 exon 1Beta. No rearrangement disrupting MTS2 was detected and more rearrangements spared MTS2 than MTS1 (P<.01). MTS1 but not MTS2 RNA was detected by Northern blotting in the human thymus. These data strongly suggest that MTS1 is the functional target of rearrangements in T-ALL. MTS1 inactivation, observed in at least 80% of T-ALL, is the most consistent genetic defect found in this disease to date.     

Expression of pertussis toxin adenosine diphosphate-ribosyltransferase in a T-cell hybridoma reduces metastatic capacity

T-cell hybridomas are highly metastatic, and their in vitro invasiveness correlates with metastatic capacity. Invasion is blocked by pertussis toxin (PT), which adenosine diphosphate (ADP)-ribosylates G1-proteins, and we have provided evidence that the PT-sensitive signal stimulates leukocyte function-associated antigen-1 (LFA-1)-mediated adhesion required for invasion. PT pretreatment of TAM2D2 T-cell hybridoma cells reduced metastasis, but only to a limited extent. In the present study, we have transfected the cDNA of the PT ADP- ribosyltransferase S1 subunit into TAM2D2 cells to abrogate G1-protein function permanently. We report here a substantial reduction in the metastatic capacity of two transfectants, S05 and S09, in which 88% and 95% of the G1-proteins was ADP-ribosylated. Two-thirds of the mice injected with S09 cells were tumor-free. Metastasis to the liver was almost completely prevented and less metastases were formed in the spleen and kidneys. Metastasis formation by S05 cells in liver and spleen was much reduced, but in lymph nodes and peritoneal tissues, metastases occurred with a frequency similar to that of controls. We conclude that G1-proteins play an important role in T-cell hybridoma metastasis. We propose that the reduction in metastasis is due to diminished entry of tumor cells from the blood into tissues.     

Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease

Infusions of large numbers (> 10(8)/kg) of donor leukocytes can induce remissions in patients with chronic myeloid leukemia (CML) who relapse after marrow transplantation. We wanted to determine if substantially lower numbers of donor leukocytes could induce remissions and, if so, whether this would reduce the 90% incidence of graft-versus-host disease (GVHD) associated with this therapy. Twenty-two patients with relapsed CML were studied: 2 in molecular relapse, 6 in cytogenetic relapse, 10 in chronic phase, and 4 in accelerated phase. Each patient received escalating doses of donor leukocytes at 4- to 33-week intervals. Leukocyte doses were calculated as T cells per kilogram of recipient weight. There were 8 dose levels between 1 x 10(5) and 5 x 10(8). Lineage-specific chimerism and residual leukemia detection were assessed using sensitive polymerase chain reaction (PCR) methodologies. Nineteen of the 22 patients achieved remission. Remissions were achieved at the following T-cell doses: 1 x 10(7) (n = 8), 5 x 10(7) (n = 4), 1 x 10(8) (n = 3), and 5 x 10(8) (n = 4). To date, 15 of the 17 evaluable patients have become BCR-ABL negative by PCR. The incidence of GVHD was correlated with the dose of T cells administered. Only 1 of the 8 patients who achieved remission at a T-cell dose of 1 x 10(7)/kg developed GVHD, whereas this complication developed in 8 of the 11 responders who received a T-cell dose of > or = 5 x 10(7)/kg. Three patients died in remission, 1 secondary to marrow aplasia, 1 of respiratory failure and 1 of complications of chronic GVHD. Sixteen patients who were mixed T-cell chimeras before treatment became full donor T-cell chimeras at the time of remission. Donor leukocytes with a T-cell content as low as 1 x 10(7)/kg can result in complete donor chimerism together with a potent graft-versus-leukemia (GVL) effect. The dose of donor leukocytes or T cells used may be important in determining both the GVL response and the incidence of GVHD. In many patients, this potent GVL effect can occur in the absence of clinical GVHD.     

A point mutation in the integrin beta 3 cytoplasmic domain (S752-->P) impairs bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-IIIa)

Agonist-induced inside-out signaling results in an increased affinity of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) for soluble ligands (fibrinogen [Fg] and PAC1). Ligand binding to integrins initiates outside-in signaling that leads to cellular responses such as cell spreading and focal adhesion formation. A point mutation in the beta 3 cytoplasmic domain (S752-->P) is associated with blocked inside- out alpha IIb beta 3 signaling in a variant Glanzmann's thrombasthenia. This mutation was introduced into beta 3 and cotransfected into Chinese hamster ovary cells with a chimeric alpha subunit consisting of the alpha IIb extracellular and transmembrane domains and the alpha 6B cytoplasmic domain. The substitution of the alpha IIb cytoplasmic domain with that of alpha 6 led to activation of alpha IIb beta 3 to bind PAC1, mimicking inside-out signaling. This effect was reversed by the S752-->P mutation, indicating a disruption of inside-out signaling by the mutation. In addition, transfectants expressing this beta 3 variant showed reduced alpha IIb beta 3-mediated cell spreading on immobilized Fg, focal adhesion, and fibrin clot retraction, suggesting an impairment in outside-in alpha IIb beta 3 signaling. Therefore, a single point mutation in the beta 3 cytoplasmic domain impaired bidirectional signaling through alpha IIb beta 3.     

Molecular basis of the heterogeneity of expression of glycosyl phosphatidylinositol anchored proteins in paroxysmal nocturnal hemoglobinuria

The purpose of these studies was to determine the molecular basis of the phenotypic mosaicism that is a defining feature of paroxysmal nocturnal hemoglobinuria (PNH). Analysis of T cell clones from a female patient revealed four distinct phenotypes based on surface expression of glycosyl phosphatidylinositol-anchored proteins (GPI-AP). When PIG-A (the gene that is mutant in PNH) from these clones was analyzed, four discrete somatic mutations were identified. Analysis of X chromosomal inactivation among the abnormal T cell clones was consistent with polyclonality. Together, these studies demonstrate that the phenotypic mosaicism that is characteristic of PNH is a consequence of genotypic mosaicism and that, at least in this case, PNH is a polyclonal rather than a monoclonal disease. That four distinct somatic mutations were present in a single patient suggests that in conditions that predispose to PNH PIG-A may be hypermutable.     

P53 gene mutations in acute myeloid leukemia with 17p monosomy

We looked for mutations of exons 5 to 8 of the P53 gene in 10 patients with acute myeloid leukemia (AML) and 17p monosomy, and 36 patients with AML and no cytogenetic abnormalities of 17p. DNA was analyzed by polymerase chain reaction, single-strand conformation polymorphism analysis, and nucleotide sequencing. Four of the 10 patients with 17p monosomy showed point mutation, single-nucleotide deletion, or insertion in exons 7 or 8. By contrast, only 1 of the 36 patients with AML and no cytogenetic abnormalities of 17p showed a mutation of the P53 gene in exons 5 to 8 (P less than .01). These results suggest that alterations of the P53 gene may have a role in leukemogenesis in some cases of AML. The fact that P53 gene mutations occurred more often in patients with 17p monosomy seems to support the "recessive" model of tumor suppressive activity of the P53 gene rather than the "dominant" model, in which alteration of only one allele is sufficient for the development of malignancy.     

Prevalence and predictors of Toxoplasma seropositivity in women with and at risk for human immunodeficiency virus infection

We assessed the prevalence and predictors of latent Toxoplasma infection in a large group of human immunodeficiency virus (HIV)-infected and HIV-uninfected at-risk US women. The prevalence of latent Toxoplasma infection was 15% (380 of 2525 persons) and did not differ by HIV infection status. HIV-infected women aged > or =50 years and those born outside of the United States were more likely to have latent Toxoplasma infection, with prevalences of 32% and 41%, respectively.