Comparison of matched unrelated and matched related donor myeloablative hematopoietic cell transplantation for adults with acute myeloid leukemia in first remission.

Hematopoietic cell transplantation (HCT) from a matched related donor (MRD) benefits many adults with acute myeloid leukemia (AML) in first complete remission (CR1). The majority of patients does not have such a donor and will require an alternative donor if HCT is to be undertaken. We retrospectively analyzed 226 adult AML CR1 patients undergoing myeloablative unrelated donor (URD) (10/10 match, n=62; 9/10, n=29) or MRD (n=135) HCT from 1996 to 2007. The 5-year estimates of overall survival, relapse and nonrelapse mortality (NRM) were 57.9, 29.7 and 16.0%, respectively. Failure for each of these outcomes was slightly higher for 10/10 URD than MRD HCT, although statistical significance was not reached for any end point. The adjusted hazard ratios (HRs) were 1.43 (0.89-2.30, P=0.14) for overall mortality, 1.17 (0.66-2.08, P=0.60) for relapse and 1.79 (0.86-3.74, P=0.12) for NRM, respectively, and the adjusted odds ratio for grades 2-4 acute graft-versus-host disease was 1.50 (0.70-3.24, P=0.30). Overall mortality among 9/10 and 10/10 URD recipients was similar (adjusted HR 1.16 (0.52-2.61), P=0.71). These data indicate that URD HCT can provide long-term survival for CR1 AML; outcomes for 10/10 URD HCT, and possibly 9/10 URD HCT, suggest that this modality should be considered in the absence of a suitable MRD.

Nonmyeloablative bone marrow transplantation from partially HLA-mismatched related donors using posttransplantation cyclophosphamide.

Cyclophosphamide (Cy) is a potent immunosuppressive agent that is selectively toxic to lymphocytes proliferating in response to recent antigen stimulation. In animal models, both graft rejection and GVHD after histoincompatible BMT can be inhibited by the posttransplantation administration of high-dose Cy. Therefore, a phase I clinical trial was undertaken to determine the minimal conditioning, including posttransplantation Cy, that permits the stable engraftment of partially HLA-mismatched marrow (up to 3 HLA antigens) from first-degree relatives. Thirteen patients (median age, 53 years) with high-risk hematologic malignancies received conditioning with fludarabine, 30 mg/m2 per day from days -6 to -2, and TBI, 2 Gy on day -1. All patients received Cy, 50 mg/kg on day 3, mycophenolate mofetil from day 4 to day 35, and tacrolimus from day 4 to day > or = 50. Three patients in cohort 1 received no additional conditioning, and 2 experienced graft rejection. Ten patients in cohort 2 received identical conditioning with the addition of Cy 14.5 mg/kg on days -6 and -5. Sustained donor cell engraftment occurred in 8 of these patients, with a median time to absolute neutrophil count > 500/microL of 15 days (range, 13-16 days) and to unsupported platelet count > 20,000/microL of 14 days (range, 0-26 days). All patients with engraftment achieved > or = 95% donor chimerism within 60 days of transplantation. Two patients with myelodysplastic syndrome rejected their grafts but experienced autologous neutrophil recovery at 24 and 44 days. Histologic acute GVHD developed in 6 patients (grade II in 3 patients, grade III in 3 patients) at a median of 99 days (range, 38-143 days) after transplantation and was fatal in 1 patient. At a median follow-up of 191 days (range, 124-423 days), 6 of 10 patients in cohort 2 were alive, and 5 were in complete remission of their disease, including both patients with graft rejection. These data demonstrate that partially HLA-mismatched bone marrow can engraft rapidly and stably after nonmyeloablative conditioning that includes posttransplantation Cy. Clinically significant antitumor responses occur, even among patients who reject their donor grafts.

CD34 selection using three immunoselection devices: comparison of T-cell depleted allografts.

BACKGROUND: T-cell depletion of allografts markedly reduces the incidence of GvHD following BMT. The approach taken at our Center has utilized the physical separation method of counterflow centrifugal elutriation (CCE), augmented by recovery of stem cells from lymphocyte-rich fractions by immunoaffinity selection of CD34(+) stem cells. We wanted to compare the performance characteristics of three commercially available selection devices, as well as the clinical outcomes of patients who received allografts engineered by the different devices. METHODS: BM allografts were prepared for patients undergoing BMT for hematologic malignancies. BM cells were separated into lymphocyte-rich and lymphocyte-depleted fractions using CCE, followed by recovery of CD34(+) cells from the lymphocyte-rich fraction using one of three immunoselection devices [CellPro CEPRATE, Nexell Isolex 300i (software version 2.5) and AmCell CliniMACS]. Allografts consisted of the lymphocyte-depleted fraction plus the CD34-selected fraction. RESULTS: Yields of CD34(+) cells were comparable for the three devices. However, there were significant differences in purity (CEPRATE < Isolex 300i < CliniMACS) and time from start of fractionation to infusion (CEPRATE < CliniMACS < Isolex 300i). More technical problems were encountered with the Isolex 300i device. Allograft compositions were comparable. Transplant outcomes (engraftment and incidence of GvHD) also were comparable. DISCUSSION: Qualitatively and quantitatively, allografts prepared with the CEPRATE, Isolex 300i (v 2.5) and CliniMACS devices should be considered comparable for use in this setting and probably also for direct T-cell depletion of BM.

Immunotherapy with rituximab during peripheral blood stem cell transplantation for non-Hodgkin's lymphoma.

Peripheral blood stem cell grafts from patients with lymphoma are often contaminated with neoplastic cells. Administration of a lymphoma-specific monoclonal antibody before collecting stem cells may be one way of reducing the contamination. Similarly, an antibody after transplantation at a time of minimal residual disease may increase the efficacy of the procedure. The objective of this study was to determine the safety of using rituximab as both an in vivo purging agent and a posttransplantation adjuvant. Eligible patients with lymphoma received 375 mg/m2 rituximab intravenously IV) on day 1, 2.5 g/m2 cyclophosphamide IV on day 4, and 10 microg/kg per day filgrastim starting on day 5 and continuing until completion of leukapheresis. Patients subsequently received a standard preparative regimen and then received 375 mg/m2 rituximab IV 7 days after platelet independence was achieved. Twenty-five patients (14 men, 11 women; median age, 51 years) were enrolled. Of the 25 patients, 23 received transplants after at least 2.0 x 10(6) CD34+ cells/kg were harvested. As determined with a sensitive polymerase chain reaction assay, 6 of 7 stem cell products tested were free of tumor contamination. All patients engrafted promptly, and the rituximab infusions were well tolerated. Transient neutropenia of uncertain etiology occurred in 6 patients a median of 99.5 days post-transplantation. An additional patient developed progressive pancytopenia. Rituximab used as an in vivo purging agent and adjuvant immunotherapy with peripheral blood stem cell transplantation for non-Hodgkin's lymphoma is a well-tolerated regimen. However, the ultimate determination of efficacy will require the results of ongoing studies.

CD34+ stem cell augmentation of elutriated allogeneic bone marrow grafts: results of a phase II clinical trial of engraftment and graft-versus-host disease prophylaxis in high-risk hematologic malignancies.

Although T cell depletion of allografts used in BMT has reduced GVHD, it has been associated with inferior engraftment and an increased risk of relapse. We have found that T cell depletion by counterflow centrifugal elutriation (CCE) also results in depletion of CD34+ stem cells. In order to determine if the discarded CD34+ cells would improve engraftment, we undertook a phase II trial of allogeneic BMT in which 110 patients (median age 43) with a variety of hematologic malignancies received CD34+ stem cell augmented, elutriated marrow grafts. The T cell-depleted grafts were tightly controlled and contained a mean of 4.3 x 10(7) mononuclear cells/kg, 3.3 x 10(6) CD34+ cells/kg, 1.5 x 10(5) CFU-GM/kg and 5.5 x 10(5) CD3+ T cells/kg. Median time to engraftment of granulocytes (>500/microl) was 16 days and of platelets (>50000/microl) was 25 days, comparable to that seen with unmanipulated marrow. No mixed hematopoietic chimerism was observed that was not associated with disease relapse. The four patients (3.6%) who failed to engraft were all at high risk because of prior donor transfusions or underlying marrow disorders. The incidence of GVHD was dependent on the duration of cyclosporin A (CsA) immunosuppression. In patients who received CsA for > or = 80 days, the incidence of clinically significant acute GVHD (>stage 1) and extensive, chronic GVHD was 5% and 11%, respectively. Peritransplant (< or = 100 day post-BMT) mortality for this group of patients was 15%. Event-free survival in selected subsets of patients compared favorably to previous studies in which patients received unmanipulated marrow allografts.

The Mlvi-1 locus involved in the induction of rat T-cell lymphomas and the pvt-1/Mis-1 locus are identical.

Mlvi-1 defines a locus of proviral integration in rat thymomas induced by Moloney murine leukemia virus. pvt-1/Mis-1 represents an independently identified locus which becomes rearranged either by chromosomal translocation in murine plasmacytomas or by provirus insertion in retrovirus-induced murine and rat thymic lymphomas. Although it had been claimed that pvt-1/Mis-1 and Mlvi-1 represent two different loci, we present here evidence showing that they are identical. This finding demonstrates the need for rigorous characterization of any newly identified common regions of integration in retrovirus-induced neoplasms.

Influence of enhancer sequences on thymotropism and leukemogenicity of mink cell focus-forming viruses.

Oncogenic mink cell focus-forming (MCF) viruses, such as MCF 247, show a positive correlation between the ability to replicate efficiently in the thymus and a leukemogenic phenotype. Other MCF viruses, such as MCF 30-2, replicate to high titers in thymocytes and do not accelerate the onset of leukemia. We used these two MCF viruses with different biological phenotypes to distinguish the effect of specific viral genes and genetic determinants on thymotropism and leukemogenicity. Our goal was to identify the viral sequences that distinguish thymotropic, nonleukemogenic viruses such as MCF 30-2 from thymotropic, leukemogenic viruses such as MCF 247. We cloned MCF 30-2, compared the genetic hallmarks of MCF 30-2 with those of MCF 247, constructed a series of recombinants, and tested the ability of recombinant viruses to replicate in the thymus and to induce leukemia. The results established that (i) MCF 30-2 and MCF 247 differ in the numbers of copies of the enhancer sequences in the long terminal repeats. (ii) The thymotropic phenotype of both viruses is independent of the number of copies of the enhancer sequences. (iii) The oncogenic phenotype of MCF 247 is correlated with the presence in the virus of duplicated enhancer sequences or with the presence of an enhancer with a specific sequence. These results show that the pathogenic phenotypes of MCF viruses are dissociable from the thymotropic phenotype and depend, at least in part, upon the enhancer sequences. On the basis of these results, we suggest that the molecular mechanisms by which the enhancer sequences determine thymotropism are different from those that determine oncogenicity.

Biological effects of a murine retrovirus carrying an activated N-ras gene of human origin.

We have introduced a genomic DNA clone of a mutated human N-ras gene from a T-cell leukemia cell line into a retroviral vector equipped with a neo resistance gene and with SV40 and pBR322 origins of replication. The helper free N-ras virus, which was recovered after transfection of the construction in the psi 2 packaging cell line, contained a correctly spliced N-ras gene. Proviral DNA was amplified in cos cells and subsequently cloned in bacteria. Nucleic acid sequence analysis of the activated N-ras gene revealed a point mutation at codon 12 resulting in a glycine to aspartic acid substitution. The N-ras virus was able to transform mouse fibroblastic cell lines, but failed to fully transform mouse primary embryo fibroblasts. MoMuLV or amphotropic 4070A pseudotypes of the virus were injected intraperitoneally into newborn mice. The MoMuLV pseudotype produced only helper-virus-induced leukemias. The amphotropic pseudotype caused fibrosarcomas after a long latent period. The results of these and other in vivo experiments are discussed in relation to known pathogenic effects of other retroviruses carrying H-ras or K-ras genes.

Deregulation of the c-myc oncogene in virus-induced thymic lymphomas of AKR/J mice.

A high frequency (greater than or equal to 65%) of thymomas induced by mink cell focus-forming virus 69L1 in AKR/J mice contain proviral integrations which are clustered 0.7-kilobase upstream of the c-myc oncogene predominantly in the opposite transcriptional orientation. Blot hybridization experiments showed that on the average there was only a twofold elevation of steady-state c-myc RNA in the thymomas as compared with levels in normal AKR/J thymocytes. Such an increase would not appear to be sufficient as a mechanism of oncogene activation in this system. In contrast, S1 nuclease analysis of transcripts initiated from the two known c-myc promoters indicated a strong shift in promoter usage in virtually all thymomas tested. In normal thymus the ratio of transcripts initiated from the proximal promoter P1 to the distal promoter P2 was 0.2 to 0.3. In contrast, most of the thymomas tested (18 of 23) showed an average P1/P2 ratio of 1.2 regardless of whether or not proviral integrations could be detected within a 21-kilobase EcoRI fragment containing the three c-myc exons. We conclude that alterations in P1/P2 ratios are good indicators of c-myc deregulation in thymic lymphomas.

Changes in thymocyte proliferation at different stages of viral leukemogenesis in AKR mice.

Proliferation in total populations of thymocytes from control AKR mice or AKR mice injected intrathymically with MCF 69L1 virus was measured by flow cytometry of acridine orange-stained cells. Cell sorting experiments showed that the majority subpopulations of small cortical and medullary thymocytes in control mice were noncycling and were predominantly in the Go phase of the cell cycle. Of the 15 to 20% cycling thymic lymphoblasts, approximately 50% were in the G1 phase, 35% were in the S phase, and 15% were in the G2 + M phases of the cell cycle. Cycling cells appeared to consist of a major subpopulation with low RNA content and a minority subpopulation with high RNA content. In virus-injected mice, no changes in cell cycling were observed at stage I of leukemogenesis (30 to 40 days postinjection), at which time infection of thymocytes by MCF virus is maximum and constant but no clonality is evident. Thus, MCF virus infection of thymocytes per se does not appear to alter cell proliferation. Increased cell cycling and a shift in cell cycle distribution to more cells in G1 was observed at stage II of leukemogenesis (50 to 60 days postinjection), at which time a clonally expanded cell population is known to emerge in thymuses of injected mice. Acridine orange staining resolved these novel cycling cells from subpopulations of normal thymic lymphoblasts on the basis of intermediate RNA content. The transition from stage II to stage III (50 to 60 days postinjection) was accompanied by the outgrowth of a major cycling population with a distinct, often increased, RNA content. As a result, the residual "normal" background of cycling cells often observed in stage II was markedly reduced or completely absent by stage III. Populations of cycling blasts from mice with frank leukemia differed from those at stage III by a variability in mean RNA content and in cell cycle distribution indicative of individual tumor heterogeneity. In addition, thymomas often contained multiple populations of cycling blasts that could be resolved by their discrete RNA distributions. Simultaneous staining of DNA and RNA by acridine orange appears particularly well-suited for studying a heterogeneous population of cycling and noncycling cells represented by mouse thymus. This method has permitted a rapid and quantitative analysis of cell cycle parameters at progressive stages of viral leukemogenesis in AKR mice.

Early clonality and high-frequency proviral integration into the c-myc locus in AKR leukemias.

Blot hybridization of thymocyte DNA from AKR/J mice was used to detect new proviral junction fragments as markers of clonality at different stages of viral leukemogenesis and to detect DNA rearrangements at the c-myc locus due to proviral insertion. Clonal populations of thymocytes were observed in mink cell focus-forming virus-injected mice as early as 35 days postinjection, at a stage distinguishable from frank leukemia by flow cytometric analysis and transplantation bioassay. Specific proviral integrations in the c-myc locus were detected in 15% of these early clones and in up to 65% of late-developing thymomas and frank leukemias. Thus, in this system c-myc activation appears to be a common mechanism in T-cell leukemogenesis.

Stages in development of mink cell focus-inducing (MCF) virus-accelerated leukemia in AKR mice.

Flow cytometric techniques involving correlated dual parameter analysis of fluorescence and light scatter and transplantation bioassays were used to describe a series of cellular changes in thymus of young (1-4 mo old) AKR mice during development of mink cell focus-inducing (MCF) virus-accelerated leukemia. Three stages of leukemogenesis were defined before appearance of frankly leukemic mice. Stage 1, apparent 28-40 d after injection of MCF 69L1 virus, represented steady-state infection of thymocytes by MCF virus without apparent change in light scatter properties of the cells or in expression of alloantigens Thy-1, Lyt-1, Lyt-2, L3T4a, B2A2, or H-2K on the major thymocyte subpopulations. Expression of MCF virus was highest in the population of small cortical thymocytes. Stage II was observed at highest frequency 50-60 d postinjection and represented the emergence of a clonal population of cells with transformed properties which could be resolved from normal thymocytes by light scatter and expression of B2A2, H-2K, and gp70 antigens. Stage III was observed at highest frequency at 70 d postinjection, when considerable enlargement of thymus had occurred, and appeared to represent the outgrowth of fully transformed cells that replaced the normal thymocyte subpopulations. The alloantigen phenotype of blast cells from frankly leukemic mice did not differ qualitatively from that of stage II or stage III cells but displayed considerable heterogeneity with respect to quantitative expression of alloantigens and gp70. At least two populations of leukemic blasts could be resolved in the majority of primary thymomas analyzed. It is unclear whether these populations represent the outgrowth of independent clones of transformed cells or if they are related in some way. Our data are consistent with MCF virus-induced transformation of cells in the lineage to small peanut agglutinin-positive, cortisone-sensitive thymocytes, a subpopulation that predominates in the thymus and which is thought to be destined for cell death in situ.

Leukemogenesis by Gross passage A murine leukemia virus: expression of viruses with recombinant env genes in transformed cells.

Gross passage A murine leukemia virus (MuLV) derived from extracts of C3Hf/Bi mouse leukemias has been shown to be a virus complex consisting of ecotropic, xenotropic, and recombinant, dualtropic MuLV components. The three virus components were distinguished biochemically by differences in the molecular weights and peptide maps of their primary env gene products synthesized in infected cells in vivo and in vitro. Virus expression was studied in primary leukemias induced in C3Hf/Bi mice by Gross passage A virus extracts and by the individual ecotropic and recombinant MuLV components that were isolated in vitro. Our findings suggest that expression of the recombinant MuLV component of the Gross passage A virus complex is necessary and sufficient for the induction of leukemias in C3Hf/Bi mice. In contrast, induction of leukemias by the ecotropic virus component appears to involve generation of a second virus with characteristics of recombinant, dualtropic MuLV.

Dimethylsulfoxide-induced alterations in the growth properties and protein composition of in vitro-propagated murine hepatoma cells.

The effects of the differentiation-inducing agent dimethylsulfoxide (DMSO) on the growth properties and protein synthetic capacity of BW77-1 mouse hepatoma cells were compared at various media concentrations of the polar solvent. DMSO produced a dose-dependent reduction in final population density, reduced or eliminated cell piling and stimulated synthesis of albumin. The rising albumin content reflected dose-related DMSO-induced increases in total cellular protein and in the albumin contribution to total cellular protein. In order to determine whether viral gene expression was associated with DMSO-induced stimulation of albumin synthesis, BW77-1 cultures were examined for the production of ecotropic and xenotropic type C virus. The BW77-1 hepatic tumor cell line was determined to be a nonproducer of type C virus by assays designed to measure extracellular reverse transcriptase, viral env gene product and infectivity on mouse and mink indicator cells. Type C virus could not be induced in BW77-1 cultures by treatment with DMSO under conditions which lead to a reduced proliferative capacity and enhanced expression liver-specific genes.

A cell surface antigen of the mouse related to xenotropic MuLv defined by naturally occurring antibody and monoclonal antibody. Relation to Gix G(rada1), G(aksl2) systems of MuLV-related antigens.

A new cell surface antigen of the mouse related to xenotropic murine leukemia virus (MuLV) is described. The antigen, designated G(erld), is defined by cytotoxic tests with the B6-x-ray-induced ERLD and naturally occurring antibody. G(erld) is distinct from all previously defined cell surface antigens. Monoclonal antibody with the same specificity has been developed. Inbred mouse strains are classified as G(erld)+ or G(erld)- according to the presence of absence of the antigen on lymphoid cells. G(erld)+ strains differ with regard to quantitative expression of G(erld) on normal thymocytes. The emergence of G(erld)+ tumors in G(erld)- strains indicates the presence of genes coding for the antigen even in strains not normally expressing the antigen. G(erld) has the characteristic of a differentiation antigen in normal mice. In G(erld)+ strains, high levels of the antigen are found on thymocytes with lower levels being detected on cells of spleen, lymph nodes and bone marrow. No G(erld) was detected in brain or kidney or on erythrocytes. The segregation ratios for G(erld) expression on thymocytes in backcross and F2 mice of crosses between G(erld)+ (B6, 129, and B6-Gix+) and G(erld)- (BALB/c) strains suggest that G(erld) expression is controlled by a single locus in B6, by two unlinked loci in 129, and by three unlinked loci in B6-Gix+ mice. Induction of the antigen by MuLV infection of permissive cells in vitro indicates that G(erld) is closely related to xenotropic and dualtropic MuLV; all xenotropic and dualtropic MuLV tested induced the antigen, whereas the majority of ecotropic and the two amphotropic MuLV failed to do so. As dualtropic MuLV are thought to be recombinants between ecotropic and xenotropic MuLV sequences, G(erld) coding by dualtropic MuLV may signify the contribution of the xenotropic part in the recombinational event. Serological and biochemical characterization indicates that G(erld) is related to the gp 70 component of the MuLV envelope. The relation of G(erld) to the previously defined gp 70-related cell surface antigens (Gix, G(rada), and G(aksl2) is discussed, particularly with regard to their characteristics as differentiation antigens, the genetic origin of dualtropic MuLV, and the leukemogenicity of MuLV.