Nonmyeloablative conditioning, unmanipulated haploidentical SCT and post-infusion CY for advanced lymphomas.

Allo-SCT is regularly performed in advanced lymphoma. Haploidentical family donors are a valuable source of hematopoietic stem cells and transplants from these donors, using T-repleted grafts, has recently been successfully reported. We report on 49 patients with refractory lymphoma who received T-repleted haploidentical SCT with a non-myeloablative regimen and post-transplant CY. The median time to recover ANC >0.5 × 10e9/L and transfusion independent plt count >20 × 10e9/L was 20 days (range 14-38) and 26 days (range 14-395). The probability to reach ANC >0.5 × 10e9/L at 30 days was 87% and transfusion independent plt count >20 × 10e9/L at 100 days was 87%. The cumulative incidence of grade 2-4 acute GVHD (aGVHD) was 25.6% (95% confidence interval (CI): 12.9-38.3%) and the cumulative incidence of chronic GVHD (cGVHD) was 5.2% (95% CI: 0-12.4%). The median follow-up is 20.6 months (range 12-54), and the projected 2-year OS and PFS were 71 and 63%. The relapse rate was 18.7% (95% CI: 7.6-29.8%) and the median time to relapse was 4.4 months (range 1.1-8.3). At 2 years, cumulative incidence of NRM was 16.3% (95% CI: 5.9-26.8%). T-repleted Haploidentical transplantation with post-infusion CY is a feasible and effective therapy in the poor prognosis of advanced lymphoma patients.

T-lymphocyte function after retroviral-mediated thymidine kinase gene transfer and G418 selection.

Generation of an efficient graft-versus-leukemia (GVL) effect in patients with hematological malignancies who relapse after allogeneic bone marrow transplantation depends in part upon the number of infused T lymphocytes. Currently, a GVL reaction cannot be achieved without inducing concomitant graft-versus-host disease (GVHD); thus, one strategy is to try to modulate this GVL/GVHD ratio. We engineered human T lymphocytes with herpes simplex virus-thymidine kinase and neomycin resistance genes, with an LXSN-derived vector that confers a ganciclovir-specific sensitivity to the transduced T cells. We analyzed proliferation, interleukin-2 production, alloreactivity in a mixed lymphocyte culture, and clonogenicity during the different stages of retroviral infection and G418 selection. Our results confirm that a sufficient number of transduced T lymphocytes can be obtained after selection for clinical studies. Their proliferative activity, alloresponsiveness, and ability to produce and respond to interleukin-2 were retained. Compared with control populations, their clonogenicity, as assessed by limiting dilution assays, was reduced after retroviral infection and G418 selection by 1.6 and 2.9 logs, respectively, with both viral supernatant incubation and coculture procedures. This study shows that infection and selection with the thymidine kinase-neomycin resistance gene retroviral vector significantly reduces the number of functional T lymphocytes. This finding should be taken into account when establishing the dose of T lymphocytes necessary to trigger a modulated GVL/GVHD effect.

Retroviral transfer of herpes simplex virus-thymidine kinase and beta-galactosidase genes into U937 cells with bicistronic vector.

In this study we describe a new retroviral vector utilizing an internal ribosome entry site (IRES) from encephalomyocarditis virus to co-express two genes. One is the herpes simplex virus type 1 thymidine kinase gene (HSV-TK) which induces sensitivity to ganciclovir, and the second is the bacterial beta-galactosidase gene (LacZ) which was revealed by an histochemical staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). We engineered the U937 human cell line to co-express both genes and monitored transduced cells using X-Gal staining. Several transduced clones were selected. The clones exhibiting X-Gal positive cells were sensitive to ganciclovir treatment (1 microgram/ml) while X-Gal negative clones were not. Monoclonal cell lines showed a single copy of the provirus integrated in their genome with the TK-IRES-LacZ sequence stably inserted in all clones. The band distribution pattern of the proviral DNA differed only at the long terminal repeat (LTR) level. Northern blot analysis of an X-Gal positive/ganciclovir sensitive clone showed an mRNA band of 6 kb with both LacZ and TK probes. An X-Gal negative/ganciclovir resistant clone was negative with both probes. This report shows: (1) a therapeutic gene can be linked to a marker gene by an IRES element achieving equivalent expression of both proteins; (2) the co-expression of a marker gene makes fluorescein-di-beta-D-galactopyranoside staining possible, and consequently separation of cells expressing the LacZ gene by fluorescence activated cell sorting. Thus the cells expressing the HSV-Tk gene are enriched; (3) the use of a marker gene such as LacZ could open up interesting perspectives in gene therapy protocols because of the opportunity to monitor the transduced cells using a simple cytochemical stain.

Granulocyte colony-stimulating factor (G-CSF) prevents dose-limiting neutropenia in lymphoma patients receiving standard dose chemotherapy.

In this study, nine patients with non-Hodgkin's lymphoma (n = 6) and Hodgkin's disease (n = 3) receiving different cytotoxic chemotherapy regimens were given granulocyte colony-stimulating factor (G-CSF) (5 micrograms/kg/day) from 48 hours after the end of chemotherapy to 48 hours before the next chemotherapy administration. The decrease in mean absolute neutrophil counts (ANC) and in mean platelet (Plt) counts was not significant when pre-therapy counts were compared with post-therapy ones (p < 0.375 and p > 0.4, respectively). The mean actual dose intensity was 92% (range 68-100%). G-CSF treatment after chemotherapy reduces neutropenia and permits administration of the full chemotherapy program. A wash-out period between G-CSF treatment and chemotherapy administration is needed to prevent the detrimental effect of chemotherapy on leukocyte and platelet recovery when repeated cycles of cytotoxic drugs and G-CSF are administered.

Regulation of early hematopoiesis in serum-deprived cultures of mafosfamide-treated and untreated CD34-enriched bone marrow cells.

Our experiments have addressed the regulation of early hematopoietic progenitor cell expansion by interleukin 3 (IL-3) and interleukin 1 beta (IL-1 beta) and its modulation by bone marrow fibroblasts in vitro. In a two-stage assay utilizing serum-deprived (SD) presuspension cultures of CD34-enriched bone marrow (BM) cells followed by clonal cultures, absolute numbers of granulocyte-macrophage progenitor cells (day-14 granulocyte-macrophage colony-forming units [CFU-GM]) increased progressively to 164% and 204% of input levels after 12 days of culture in the presence of IL-3 alone or in combination with IL-1 beta, respectively. Multilineage (granulocyte erythrocyte macrophage megakaryocyte colony-forming units, CFU-GEMM) and erythroid (erythroid burst-forming units, BFU-E) progenitor cell numbers increased above or were maintained at input levels after 4 and 7 days of liquid culture in the presence of IL-3 and IL-3 plus IL-1 beta, respectively, but in contrast to granulocyte-macrophage colony-forming units (CFU-GM) they were essentially undetectable after 12 days of culture. Progenitors more primitive than colony-forming cells (pre-CFU) were assessed in SD-presuspension cultures of CD34-enriched BM cells purged with mafosfamide to eliminate base-line CFU-GM, CFU-GEMM, and BFU-E. Under these conditions and in the absence of stromal elements, CFU-GM but neither CFU-GEMM nor BFU-E developed in response to cytokines alone. In the additional presence of passaged bone marrow fibroblasts, however, IL-3 plus IL-1 beta and to a lesser degree IL-3 alone induced a pronounced amplification of BFU-E and CFU-GEMM, indicating that their development from a more primitive progenitor compartment requires growth activities in addition to IL-3 and IL-1 beta that are provided by marrow-derived stromal cells such as fibroblasts.

Establishment and characterization of a B-cell line derived from a patient with a myelodysplastic syndrome which expresses myelomonocytic and lymphoid markers.

We describe a novel continuous B-cell line (PV-90) derived from a patient with myelodysplastic syndrome (MDS) and originating from spontaneous infection with the Epstein-Barr virus (EBV). The patient progressed to acute myeloblastic leukaemia (AML) 5 months after clinical onset of MDS. PV-90 is of clonal origin as indicated by the presence of immunoglobulin (Ig) gene rearrangements, monoclonal surface immunoglobulins, and a single DNA restriction fragment corresponding to the EBV genomic termini. PV-90 cells also express a number of myelomonocytic markers, including alpha-naphthyl acetate esterase (ANAE), coagulation factor XIII, and CD68 antigen. Moreover, PV-90 cells constitutively express the c-fms proto-oncogene mRNA as the patient's blast cells did. Whereas a trisomy 11 (+11) was found in the patient's bone marrow cells, PV-90 cells had a normal karyotype initially, but at 4 months showed two different and independent chromosomal abnormalities: 90, XX, -Y, -Y, t(9;16) (q11;p13), and 90, XX, -Y, -Y, t(17;18) (p13;q21), the latter possibly involving the p53 (17,p13) and bcl-2 (18, q21) proto-oncogenes. The early development of these chromosomal aberrations is consistent with a genetic instability of PV-90 cells. Expression of bi-lineage markers and genetic instability may suggest that PV-90 cells originated from transformation of a myelodysplastic progenitor cell capable of both myeloid and B-cell differentiation. The PV-90 cell line might be useful in a number of studies, including the possible role of c-fms in cell differentiation, pathogenetic mechanisms of human preleukaemia and lineage promiscuity in acute leukaemia.

[Effect of tumor necrosis factor on human hematopoietic precursor cells]. / Untersuchungen über die Wirkung von Tumornekrosefaktor auf humane hämatopoetische Vorläuferzellen.

The effect of human recombinant tumor necrosis factor (rhTNF-alpha) on the in vitro colony growth of normal hematopoietic progenitor cells was investigated. In a clonal colony-assay a dose-dependent inhibition of erythroid BFU-E, granulocyte/monocyte CFU-GM and megakaryocyte CFU-Mk was demonstrated. CFU-Mk were completely inhibited by low doses of TNF-alpha (3 U-300 U/ml). 50% inhibition occurred at 10 U/ml of TNF for CFU-Mk, 100% inhibition at 300 U/ml of TNF. 50% inhibition occurred at 233 U/ml of TNF for CFU-GM, and 100% inhibition for CFU-GM was not observed. For inhibition of BFU-E higher doses of TNF-alpha were necessary (100 U-1,000 U/ml). The growth inhibitory effect could selectively be abolished by antibodies against TNF-alpha. Removal of adherent cells and T-lymphocytes from the bone marrow cells had no significant influence of the suppressive effect of TNF-alpha. The inhibitory effect of TNF-alpha is due to a direct action on hematopoietic progenitor cells and not mediated by accessory cells.

Effects of recombinant human H-subunit and L-subunit ferritins on in vitro growth of human granulocyte-monocyte progenitors.

We have studied the influence of purified recombinant human H-subunit (rHF, acidic) and L-subunit (rLF, basic) ferritins on in vitro colony formation by normal human granulocyte-macrophage progenitor cells (CFU-GM). Whereas rLF had no significant effect, rHF produced significant decrease in colony formation: mean inhibition of CFU-GM was 38% +/- 13% at 10(-8) M and 22% +/- 13% at 10(-9) M. The inhibitory activity of rHF was lost at 10(-10) M, and was inactivated with a monoclonal antibody recognizing the H subunit, but not with a monoclonal antibody recognizing the L subunit. Although H-type isoferritins were found in normal and leukaemic cells, their concentration in peripheral blood plasma and bone marrow plasma from normal subjects and patients with different haematological disorders including acute leukaemia were 10(-11) M or lower, i.e. levels showing no activity in vitro. We conclude that: (i) acidic H-subunit-rich isoferritins have inhibitory effects on in vitro growth of granulocyte-macrophage progenitors; (ii) levels of these isoferritins in peripheral blood and bone marrow plasma are 2-3 orders of magnitude lower than the effective concentrations in vitro, indicating that these molecules do not behave as circulating regulatory or suppressive factors in vivo.

Defective in vitro growth of the hemopoietic progenitor cells in the acquired immunodeficiency syndrome.

In addition to immunologic derangement, hematological abnormalities have been reported in the majority of patients with acquired immunodeficiency syndrome (AIDS). In this study 15 patients with AIDS or AIDS-related complex (ARC) were evaluated for the in vitro growth of hemopoietic progenitor cells. In all patients a significant reduction of growth (mean +/- SEM) of colony-forming unit-granulocyte, erythrocyte, macrophage, (megakaryocyte) (CFU-GEM) (1.2 +/- 0.3), burst-forming unit-erythroid (BFU-E) (17 +/- 10), CFU-megakaryocyte (CFU-Mk) (1.7 +/- 0.6), and CFU-granulocyte-macrophage (CFU-GM) (35 +/- 10) was observed in comparison with normal controls. Depletion of T cells from the bone marrow before culture led to a significant increase in colony growth, which indicated an imbalance of the normally modulating T cell subsets. This increase was reversed by readdition of autologous T cells causing a decrease in colony growth to a degree, dependent on the T4 to T8 ratio. A decreased number of hemopoietic progenitor cells and/or a defective modulation of progenitor cell growth, normally carried out by T lymphocyte subsets, might be the cause of the hematological abnormalities in AIDS patients.

Lymphoproliferative disorder of large granular lymphocytes: reversal of lymphocyte proliferation, anemia and neutropenia with chlorambucil.

A 54-year-old woman presented with hepatosplenomegaly, anemia, neutropenia and lymphocytosis. Most peripheral blood lymphocytes had the surface antigens T3+, Leu11+ and were morphologically large granular lymphocytes. Bone marrow presented 60% lymphoid infiltration. Treatment with chlorambucil produced complete reversal of hepatosplenomegaly, anemia, neutropenia and lymphocytosis, and reduction of marrow infiltration. The patient is well 12 months after discontinuation of therapy.