Biomedical subjects

J H Frederik Falkenburg

Publications and source records attributed to J H Frederik Falkenburg.

At least 19 recordsLinked to original sources

PubMed2006

Efficiency of T-cell receptor expression in dual-specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 complex.

Genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T-cell immunity toward viral infections and malignancies. We previously demonstrated redirected antileukemic reactivity of cytomegalovirus (CMV)-specific T cells by transfer of minor histocompatibility antigen HA-2-specific T-cell receptors (TCRs). HA-2-TCR-transferred CMV-specific T cells were potent effectors against HA-2-expressing leukemic cells, as well as CMV-expressing cells. Functional activity of these T cells correlated with TCR cell-surface expression. In the present study we analyzed which properties of transferred and endogenous TCRs are crucial for efficient cell-surface expression. We demonstrate that expression of the introduced TCR is not a random process but is determined by characteristics of both the introduced and the endogenously expressed TCR. The efficiency of TCR cell-surface expression is controlled by the intrinsic quality of the TCR complex. In addition, we demonstrate that chimeric TCRs can be formed and that efficiency of TCR expression is independent of whether TCRs are retrovirally introduced or naturally expressed. In conclusion, introduced, endogenous, and chimeric TCRs compete for cell-surface expression in favor of the TCR-CD3 complex with best-pairing properties.

Amino Acid Sequence↗

PubMed2006

Detection and functional analysis of CD8+ T cells specific for PRAME: a target for T-cell therapy.

PURPOSE: Preferentially expressed antigen on melanomas (PRAME) is an interesting antigen for T-cell therapy because it is frequently expressed in melanomas (95%) and other tumor types. Moreover, due to its role in oncogenic transformation, PRAME-negative tumor cells are not expected to easily arise and escape from T-cell immunity. The purpose of this study is to investigate the usefulness of PRAME as target for anticancer T-cell therapies. EXPERIMENTAL DESIGN: HLA-A*0201-subtyped healthy individuals and advanced melanoma patients were screened for CD8+ T cells directed against previously identified HLA-A*0201-binding PRAME peptides by IFN-gamma enzyme-linked immunosorbent spot assays and tetramer staining. PRAME-specific T-cell clones were isolated and tested for recognition of melanoma and acute lymphoid leukemia (ALL) cell lines. PRAME mRNA expression was determined by quantitative real-time reverse transcription-PCR. RESULTS: In 30% to 40% of healthy individuals and patients, PRA(100-108)-specific CD8+ T cells were detected both after in vitro stimulation and directly ex vivo after isolation by magnetic microbeads. Although CD45RA- memory PRA(100-108)-specific T cells were found in some individuals, the majority of PRA(100-108)-tetramer+ T cells expressed CD45RA, suggesting a naive phenotype. PRA(100-108)-tetramer+ T-cell clones were shown to recognize and lyse HLA-A*0201+ and PRAME+ melanoma but not ALL cell lines. Quantitative real-time reverse transcription-PCR showed significantly lower PRAME mRNA levels in ALL than in melanoma cell lines, suggesting that PRAME expression in ALL is below the recognition threshold of our PRA(100-108)-tetramer+ T cells. CONCLUSION: These data support the usefulness of PRAME and in particular the PRA(100-108) epitope as target for T-cell therapy of PRAME-overexpressing cancers.

Antigens, Neoplasm↗

PubMed2006

Alphabeta T-cell receptor engineered gammadelta T cells mediate effective antileukemic reactivity.

Retroviral transfer of T-cell receptors (TCR) to peripheral blood-derived T cells generates large numbers of T cells with the same antigen specificity, potentially useful for adoptive immunotherapy. One drawback of this procedure is the formation of mixed TCR dimers with unknown specificities due to pairing of endogenous and introduced TCR chains. We investigated whether gammadelta T cells can be an alternative effector population for TCR gene transfer because the gammadeltaTCR is not able to form dimers with the alphabetaTCR. Peripheral blood-derived gammadelta T cells were transduced with human leukocyte antigen (HLA) class I- or HLA class II-restricted minor histocompatibility antigen (mHag) or virus-specific TCRs. Because most gammadelta T cells do not express CD4 and CD8, we subsequently transferred these coreceptors. The TCR-transduced gammadelta T cells exerted high levels of antigen-specific cytotoxicity and produced IFN-gamma and IL-4, particularly in the presence of the relevant coreceptor. gammadelta T cells transferred with a TCR specific for the hematopoiesis-specific mHag HA-2 in combination with CD8 displayed high antileukemic reactivity against HA-2-expressing leukemic cells. These data show that transfer of alphabetaTCRs to gammadelta T cells generated potent effector cells for immunotherapy of leukemia, without the expression of potentially hazardous mixed TCR dimers.

CD3 Complex↗

PubMed2006

Identification of the angiogenic endothelial-cell growth factor-1/thymidine phosphorylase as a potential target for immunotherapy of cancer.

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched hematopoietic stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated tumor-reactive cytotoxic CD8(+) T-cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma after allogeneic HLA-matched SCT. Using cDNA expression cloning, the target molecule of an HLA-B7-restricted CTL clone was identified. The CTL clone recognized a minor histocompatibility antigen produced by a single nucleotide polymorphism (SNP) in the angiogenic endothelial-cell growth factor-1 (ECGF1) gene also known as thymidine phosphorylase. The SNP leads to an Arg-to-His substitution in an alternatively translated peptide that is recognized by the CTL. The ECGF1 gene is predominantly expressed in hematopoietic cells, although low expression can also be detected in other tissues. The patient from whom this CTL clone was isolated had mild graft-versus-host disease despite high numbers of circulating ECGF-1-specific T cells as detected by tetramer staining. Because solid tumors expressing ECGF-1 could also be lysed by the CTL, ECGF-1 is an interesting target for immunotherapy of both hematologic and solid tumors.

Amino Acid Substitution↗

PubMed2006

Minor histocompatibility antigen DDX3Y induces HLA-DQ5-restricted T cell responses with limited TCR-Vbeta usage both in vivo and in vitro.

In vitro stimulation of human female T cells with male HLA-identical dendritic cells resulted in the generation of HLA-DQB1*0501/0502-restricted minor histocompatibility H-Y antigen-specific CD4(+) T cell clones. Two clones generated from different HLA-identical pairs were analyzed. Use of HLA-DQ5-expressing female Epstein-Barr virus transformed B lymphoblastoid cell lines transfected with various H-Y genes and loaded with overlapping peptides demonstrated that both T cell clones are specific for a peptide encoded by DDX3Y. Previously, an HLA-DQ5-restricted T cell clone specific for the same peptide was isolated from a patient with graft-versus-host disease. Thus, we compared the T cell receptor (TCR) rearrangements of the 2 in vitro generated T cell clones and the ex vivo isolated T cell clone. All 3 clones shared the same TCRBV5-4* gene segment and 2 of 3 clones also used similar TCR-Valpha segments. Our results suggest that T cells recognizing the HLA-DQ5/DDX3Y T cell epitope might be characterized by a relatively limited TCR-beta repertoire. The differences in the junctional TCR-beta region had no effect on the antigen specificity, but altered the capacity of the TCR to distinguish the HLA-DQ5/DDX3Y complex from its allelic counterpart. The results also demonstrate that in vitro stimulation of T cells with allogeneic HLA-identical dendritic cells may facilitate the characterization of in vivo, potentially relevant HLA class II-restricted minor H epitopes.

CD4-Positive T-Lymphocytes↗

PubMed2006

The mechanisms of Ara-C-induced apoptosis of resting B-chronic lymphocytic leukemia cells.

BACKGROUND AND OBJECTIVES: Cytarabine (Ara-C) is commonly used for the treatment of acute leukemia. Incorporation of Ara-C into DNA is a key event in the mechanism of killing of proliferating leukemic cells. Previously, we demonstrated that Ara-C was cytotoxic to proliferating but not to resting (G(O)) malignant cells from patients with acute leukemia. In contrast, here we show unexpected apoptosis of G(O) B-chronic lymphocytic leukemia (CLL) cells by Ara-C in a dose-dependent manner. In this study we analyzed which cellular processes were involved in Ara-C-mediated killing of G(O)-B-CLL cells. DESIGN AND METHODS: Using primary B-CLL cells (>98% in G(O)), we examined the mechanisms of Ara-C-mediated apoptosis in resting G(O) cells. CFSE-based cytotoxicity assays combined with cell cycle analysis were used to perform a long-term analysis of Ara-C-mediated killing of B-CLL cells. The effects of Ara-C on DNA and RNA synthesis were studied using various 3H-incorporation experiments. RESULTS: Ara-C-mediated cell death of B-CLL cells showed the characteristics of normal apoptosis, such as phosphatidyl serine exposure and caspase activation. The mechanism of killing of quiescent B-CLL cells by Ara-C was shown not to be dependent on DNA replication. In contrast, CD40L-activated B-CLL cells showed S-phase-specific depletion of proliferating CLL cells. We demonstrated that Ara-C was converted into its active triphosphate by G(O)-B-CLL cells, coinciding with a 30% inhibition of RNA synthesis. INTERPRETATION AND CONCLUSIONS: In conclusion, our data indicate that Ara-C can induce apoptosis in resting G(O)-B-CLL cells using a mechanism independent of cell proliferation and DNA replication but associated with inhibition of RNA synthesis and downregulation of Mcl-1.

Apoptosis↗

PubMed2006

Outcomes after myeloablative unrelated donor stem cell transplantation using both in vitro and in vivo T-cell depletion with alemtuzumab.

HLA-matched unrelated donor (MUD) stem cell transplantation (MUD) is complicated by a high incidence of graft-versus-host-disease (GVHD) resulting in significant morbidity and mortality. To circumvent this problem we included alemtuzumab for in vivo and in vitro T-cell depletion in a myeloablative MUD-SCT regimen. After SCT, no severe acute GVHD was observed in the 30 transplanted patients. Donor lymphocyte infusion administered at a later time point resulted in sustained anti-tumor responses in most patients with chronic myeloid leukemia. After donor lymphocyte infusion three patients developed severe acute GVHD. Due to good responsiveness to immunosuppressive therapy only two patients developed persistent chronic GVHD. The main advantage of the transplantation regimen including alemtuzumab is that not only mortality due to GVHD is limited but also extensive chronic GVHD, which potentially leads to chronic morbidity and diminished quality of life, is hardly observed.

Adolescent↗

PubMed2005

Cytokine-dependent proliferation of human CD34+ progenitor cells in the absence of serum is suppressed by their progeny's production of serine proteinases.

In this study, we demonstrate that the synthesis and release of serine proteinases by hematopoietic cells affects the in vitro proliferation of hematopoietic progenitor cells (HPCs) in response to proteins, including hematopoietic growth factors (HGFs), transferrin, insulin, and albumin in serum-free cultures. In serum-free cultures, bone marrow mononuclear cells or the CD34- progeny of the CD34+ cells were shown to release the serine proteinases human neutrophil elastase (HNE), cathepsin G (Cath G), and proteinase 3 (Pr3). In the absence of serum, we showed that HNE, Cath G, and Pr3 rapidly and dose-dependently degraded HGF and other proteins present in the medium, resulting in decreased proliferation of HPCs. Addition of the serine proteinase inhibitors alpha1-proteinase inhibitor (alpha1-PI) or the secretory leukocyte proteinase inhibitor (SLPI), but not leupeptin, aprotinin, or AEBSF (4-[2-aminoethyl]-benzenesulfonylfluoride hydrochloride), could completely prevent the degradation of proteins relevant to the growth of hematopoietic cells. Thus, the addition of serine proteinase inhibitors like alpha1-PI or SLPI may be critical for the expansion of CD34+ cells or gene transfer into CD34+ cells or other hematopoietic cells in vitro using serum-free media under good manufacturing practice conditions.

Animals↗

PubMed2005

Up-regulated expression in nonhematopoietic tissues of the BCL2A1-derived minor histocompatibility antigens in response to inflammatory cytokines: relevance for allogeneic immunotherapy of leukemia.

T cells directed against hematopoietic-restricted minor histocompatibility antigens (mHags) may mediate graft-versus-leukemia (GVL) reactivity without graft-versus-host disease (GVHD). Recently, the HLA-A24-restricted mHag ACC-1 and the HLA-B44-restricted mHag ACC-2 encoded by separate polymorphisms within the BCL2A1 gene were characterized. Hematopoietic-restricted expression was suggested for these mHags. We demonstrate BCL2-related protein A1 (BCL2A1) mRNA expression in mesenchymal stromal cells (MSCs) that was up-regulated by the inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and/or interferon gamma (IFN-gamma). Analysis of cytotoxicity and IFN-gamma production illustrated that ACC-2-specific T cells did not recognize untreated MSCs or IFN-gamma-treated MSCs but showed specific recognition and killing of MSCs treated with TNF-alpha plus IFN-gamma. We hypothesize that under steady-state circumstances BCL2A1-specific T cells may exhibit relative specificity for hematopoietic tissue, but reactivity against nonhematopoietic cells may occur when inflammatory infiltrates are present. Thus, the role of BCL2A1-specific T cells in differential induction of GVL reactivity and GVHD may depend on the presence of inflammatory responses that may occur during GVHD.

Cytokines↗

PubMed2005

Primary allogeneic T-cell responses against mantle cell lymphoma antigen-presenting cells for adoptive immunotherapy after stem cell transplantation.

PURPOSE: In patients treated with allogeneic stem cell transplantation for advanced mantle cell lymphoma (MCL), complete sustained remissions have been observed illustrating susceptibility of MCL cells to a graft-versus-lymphoma effect. To potentiate this graft-versus-lymphoma effect, adoptive transfer of in vitro selected MCL-specific CTL can be an attractive approach. The lack of expression of costimulatory molecules on MCL cells hampers the generation of MCL-reactive T-cell responses. The purpose of this study was to modify MCL cells into antigen-presenting cells (APC) and to use these MCL-APCs to induce allogeneic MCL-reactive T-cell responses. EXPERIMENTAL DESIGN: Interleukin (IL)-4, IL-10, CpG, and CD40 activation were tested for their capacity to up-regulate costimulatory molecules on MCL cells. Primary MCL cells or the modified MCL-APCs were then used to evaluate the induction of MCL-reactive T-cell responses in HLA-matched donors. RESULTS: Ligation of CD40 on MCL cells was essential to up-regulate costimulatory molecules and to induce production of high amounts of IL-12. In contrast to primary MCL cells, MCL-APC cells as stimulators were capable of inducing CD8+ CTL lines from HLA class I-matched donors. High numbers of CTL clones could be generated capable of efficiently killing the primary MCL cells and MCL-APC but not donor-specific targets. CONCLUSION: These results show the feasibility to generate primary allogeneic T-cell responses against MCL-APC, and may provide new immunotherapeutic tools to further exploit the graft-versus-lymphoma effect following allogeneic stem cell transplantation in patients with MCL.

Adult↗

PubMed2005

Responses to donor lymphocyte infusion for acute lymphoblastic leukemia may be determined by both qualitative and quantitative limitations of antileukemic T-cell responses as observed in an animal model for human leukemia.

OBJECTIVE: Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation is largely unsuccessful in the treatment of acute lymphoblastic leukemia (ALL). To allow identification of the causes of this failure, we established an animal model of DLI in human ALL. METHODS: NOD/scid mice were inoculated with primary human ALL cells. Cells from five different patients were studied. After engraftment, DLI was performed by infusion of human leukocyte antigen (HLA)-identical donor T cells or HLA-disparate donor T cells. RESULTS: DLI resulted in expansion of activated, leukemia-reactive T cells in all donor-patient combinations. After 40 days of expansion, T cells abruptly declined in numbers and displayed loss of cytotoxicity. At this moment, remissions were observed in three of five donor-patient combinations. In animals engrafted with the two unresponsive ALL, remissions could be achieved when HLA-disparate DLI was given. CONCLUSION: Our results indicate that the inefficacy of DLI in ALL may be due to the limitation of the proliferative capacity of ALL-reactive T cells and that the antileukemic efficacy during the limited time span of proliferation depends on the antigenic disparity between the donor and the patient. The model can be used to study whether alternative strategies may result in more sustained antileukemic responses after DLI in ALL.

Adult↗

PubMed2005

A novel approach to identify antigens recognized by CD4 T cells using complement-opsonized bacteria expressing a cDNA library.

In patients with hematological malignancies receiving HLA-matched stem cell transplantation, T cells specific for minor histocompatibility antigens play a major role in graft rejection, induction of graft-versus-host disease and beneficial graft-versus-leukemia reactivity. Several human minor histocompatibility antigens recognized by T cells have been identified, but only two are presented by HLA class II molecules. In search of an efficient approach to identify antigenic peptides processed through the HLA class II pathway, we constructed a cDNA library in bacteria that were induced to express proteins. Bacteria were opsonized with complement to enforce receptor-mediated uptake by Epstein-Barr virus immortalized B cells that were subsequently used as antigen-presenting cells. This approach was validated with an HLA class II-restricted antigen encoded by gene DBY. We were able to identify bacteria expressing DBY diluted into a 300-fold excess of bacteria expressing a nonrelevant gene. Screening of a bacterial library using a DBY-specific CD4 T cell clone resulted in the isolation of several DBY cDNAs. We propose this strategy for a rapid identification of HLA class II-restricted antigenic peptides recognized by CD4 T cells.

Bacteria↗

PubMed2005

Characterization of the human CD34+ hematopoietic progenitor cell compartment during the second trimester of pregnancy.

BACKGROUND AND OBJECTIVES: Characterization of the different sites of fetal hematopoiesis during the second trimester of pregnancy can provide important information for the timing of in utero stem cell transplantation (SCT), as an experimental treatment for congenital hematologic disorders. DESIGN AND METHODS: We analyzed the distribution of the different hematopoietic precursor cells in fetal blood, liver, bone marrow (BM), spleen and thymus from 66 fetuses between the ages of 13 to 23 weeks of gestation by flow cytometry and culture of hematopoietic progenitor cells (HPC) in semi-solid media. RESULTS: During the second trimester the percentages of CD34+ cells did not change and were 4.0% (1.0-12.0%) (median [min.-max.]) in blood, 16.5% (3.0-32.0%) in BM, 6.0% (2.0-16.0%) in liver, 5.0% (2.0-14.0%) in spleen, and 1.1% (0.9-3.0%) in the thymus. Each tissue contained all subsets of CD34+ cells at various levels. Within the CD34+ population, in BM the main sub-population was CD34+CD19+ (38% (11-67%)), in thymus CD34+ CD7+ (83% [45-98%]), and in blood and liver CD34+ CD33+ (57% (30-80%) and 48% (20-82%), respectively). In all tissues approximately 1 % of nucleated cells were non-committed CD34+ CD38- cells. The frequencies of both committed CD34+ cells and non-committed CD34+ CD38- cells were constant from 13 to 23 weeks in fetal blood, BM, liver and spleen. The frequencies of cultured HPC were high in fetal liver, low in fetal BM, and increasing in fetal blood. INTERPRETATION AND CONCLUSIONS: During the second trimester of gestation, all CD34+ subsets were present in each hematopoietic compartment at different levels. An exchange of stem cells between organs is likely, but no major shift of the hematopoietic stem cell compartment from the liver to other hematopoietic organs was found during the mid-trimester. No arguments for a specific time window for performing in utero SCT were found, but if engraftment of donor stem cells in the human fetus is influenced by competition of endogenous stem cells or fetal immune competence, in utero SCT should be performed as early as possible during fetal development.

Antigens, CD19↗

PubMed2004

Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer.

T cells directed against minor histocompatibility antigens (mHags) might be responsible for eradication of hematological malignancies after allogeneic stem cell transplantation. We investigated whether transfer of T cell receptors (TCRs) directed against mHags, exclusively expressed on hematopoietic cells, could redirect virus-specific T cells toward antileukemic reactivity, without the loss of their original specificity. Generation of T cells with dual specificity may lead to survival of these TCR-transferred T cells for prolonged periods of time in vivo due to transactivation of the endogenous TCR of the tumor-reactive T cells by the latent presence of viral antigens. Furthermore, TCR transfer into restricted T cell populations, which are nonself reactive, will minimize the risk of autoimmunity. We demonstrate that cytomegalovirus (CMV)-specific T cells can be efficiently reprogrammed into leukemia-reactive T cells by transfer of TCRs directed against the mHag HA-2. HA-2-TCR-transferred CMV-specific T cells derived from human histocompatibility leukocyte antigen (HLA)-A2+ or HLA-A2- individuals exerted potent antileukemic as well as CMV reactivity, without signs of anti-HLA-A2 alloreactivity. The dual specificity of these mHag-specific, TCR-redirected virus-specific T cells opens new possibilities for the treatment of hematological malignancies of HLA-A2+ HA-2-expressing patients transplanted with HLA-A2-matched or -mismatched donors.

Amino Acid Sequence↗

PubMed2004

Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen-specific cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) specific for hematopoietic-restricted minor histocompatibility antigens (mHags) are important reagents for adoptive immunotherapy of relapsed leukemia after allogeneic stem cell transplantation. However, expansion of these CTLs to therapeutic numbers is often hampered by the limited supply of antigen-presenting cells (APCs). Therefore, we evaluated whether cell-sized latex beads coated with HLA/mHag complexes HLA-A2/HA-1 or HLA-A2/HA-2 and recombinant CD80 and CD54 molecules can replace professional APCs. The artificial antigen-presenting constructs (aAPCs) effectively stimulated HA-1- and HA-2-specific CTL clones as shown by ligand-specific expansion, cytokine production, and maintenance of cytotoxic activity, without alteration of CTL phenotype. Furthermore, HA-1-specific polyclonal CTL lines were enriched as efficiently by aAPCs as by autologous HA-1 peptide-pulsed dendritic cells. Thus, aAPCs coated with HLA/mHag complexes, CD80, and CD54 may serve as tools for in vitro enrichment of immunotherapeutic mHag-specific CTL lines.

Antigen Presentation↗

PubMed2004

Randomized comparison of low-dose versus high-dose interferon-alfa in chronic myeloid leukemia: prospective collaboration of 3 joint trials by the MRC and HOVON groups.

The optimal dose of interferon-alfa (IFN) for chronic myeloid leukemia (CML) is unknown. Retrospective analyses suggest that low doses are as effective as high doses, with less toxicity and fewer patients abandoning the drug. The Dutch Hemato-Oncology Association (HOVON) and British Medical Research Council (MRC) cooperative groups jointly performed randomized trials in newly diagnosed CML patients, comparing high-dose IFN (5 MIU/m(2) daily) with low-dose (3 MIU, 5 times a week). Both arms allowed additional hydroxyurea to keep the white blood cell count lower than 5 x 10(9)/L. Quality of life data were collected in a subset of patients. Between 1993 and 2001, 407 patients were randomized. At a median follow-up of 53 months, there were no significant differences in overall survival (odds ratio = 1.09, 95% confidence interval, 0.81-1.46), progression-free survival, and complete hematologic or major cytogenetic responses. Fewer patients in the low-dose group abandoned IFN for reasons other than transplant or progressive disease (P =.002, 58% vs 72% at 5 years). Quality of life data showed comparable results in both arms for most factors. There is no evidence of benefit for high-dose IFN compared with low-dose for the treatment of CML. Therefore, when IFN is combined with other drugs, low-dose IFN is advised, to minimize toxicity and costs.

Adolescent↗

PubMed2004

Identification of a novel RAS GTPase-activating protein (RASGAP) gene at 9q34 as an MLL fusion partner in a patient with de novo acute myeloid leukemia.

The t(9;11) has been described in patients with acute myeloid leukemia (AML), and two genes [AF9 (at 9p21) and FBP17 (at 9q34)] have been cloned as fusion partners of the MLL gene. From an AML-M5 with a t(9;11)(q34;q23), we identified a novel MLL fusion partner, AF9Q34. The AF9Q34 protein shows high homology with nGAP, a RAS GTPase-activating protein (RASGAP), and contains the highly conserved GRD and FLR motifs characteristic of RASGAPs. Recently, the rat homologue (DAB2IP) also was identified and reported to act as a RASGAP both in vivo and in vitro. RASGAPs negatively regulate the activity of RAS proteins that modulate diverse cellular processes by cycling between an inactive GDP-bound and an active GTP-bound state. In addition, the NH(2) terminus harbors an amino acid stretch with homology to the pleckstrin homology (PH) domain implicated in regulating the interaction between RAS and the catalytic domain of RASGAP. As a result of the breakpoint in the AF9Q34-MLL fusion protein, this PH domain is disrupted. This suggests that because of the translocation, the normal function of the AF9Q34 gene is aborted. Thus, AF9Q34 encodes a novel RASGAP gene that appears to be deregulated as a result of the translocation. The identification of this RASGAP protein in a novel MLL fusion implies that an indirect RAS-deregulating mechanism could be involved in leukemic transformation.

Amino Acid Sequence↗

PubMed2004

Alpha-interferon with very-low-dose donor lymphocyte infusion for hematologic or cytogenetic relapse of chronic myeloid leukemia induces rapid and durable complete remissions and is associated with acceptable graft-versus-host disease.

Donor lymphocyte infusion (DLI) results in complete cytogenetic remission (CCR) of relapsed chronic-phase chronic myeloid leukemia (CML-CP) after allogeneic stem cell transplantation (SCT) in up to 80% of patients. The main complication of DLI is graft-versus-host disease (GVHD). Decreasing the dose of DLI is associated with less GVHD but also with a longer interval between treatment and CCR. We postulated that combining alpha-interferon (alpha-IFN) with DLI would enable us to decrease the dose of DLI, thereby limiting GVHD, and at the same time to decrease the interval between DLI and CCR for patients with either a hematologic or cytogenetic relapse. For molecular relapses, we hypothesized that because of a lower tumor load, very low doses of DLI without alpha-IFN could be an effective treatment. Two groups of CML-CP patients treated with DLI at a very low dose of 0.5 to 1.0 x 10(7) mononuclear cells per kilogram, containing 2 to 6 x 10(6) CD3+ T cells per kilogram, were analyzed: 13 patients with a cytogenetic or a hematologic relapse after allogeneic SCT (group A) were treated with additional alpha-IFN therapy at a dose of 3 x 10(6) U 5 d/wk, and 8 patients with a molecular relapse were treated without alpha-IFN (group B). Twelve patients from group A reached a CCR. The median interval between DLI and CCR was 7 weeks (range, 5-18 weeks) for group A. All patients with a CCR reached complete donor chimerism at a median of 10 weeks after DLI (range, 6-121 weeks). Eleven patients reached molecular remission at a median of 15 weeks after DLI (range, 8-34 weeks). In group B, all patients reached a molecular remission at a median of 14 weeks (range, 12-29 weeks). Five patients from group A developed acute GVHD grade II to IV and extensive chronic GVHD. In group B, 1 patient developed acute GVHD grade II to IV and subsequently developed extensive chronic GVHD. With a median follow-up of 62 months, 10 patients in group A are alive and in continuous CCR. One patient had a molecular relapse, for which she successfully received additional DLI; another patient reached molecular remission only after 5 doses of DLI. Two patients from group A died of a gram-negative sepsis, and 1 died of an acute myocardial infection. In group B, all patients are alive and in molecular remission with a median follow-up of 20 months. One patient's disease progressed but was successfully treated with DLI plus alpha-IFN. In conclusion, very-low-dose DLI in combination with alpha-IFN as treatment for cytogenetic or hematologic relapses of CML-CP after allogeneic SCT reduced the interval to obtain a CCR with acceptable GVHD when compared with the literature. Patients with a CCR also reached complete donor chimerism and complete molecular remissions. For patients with a molecular relapse, very-low-dose DLI alone is sufficient to induce molecular remissions in most patients and is associated with limited GVHD.

Adult↗