Myb targeted therapeutics for the treatment of human malignancies.
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For the past several years, we have been engaged in developing a therapeutically effective strategy for disrupting gene function with reverse complementary, or so called 'antisense', oligodeoxynucleotides (ODN). This pursuit has focused on finding appropriate diseases in which to apply this approach, and suitable gene targets. Of the genes that we have targeted for disruption using the antisense ODN strategy (Clevenger et al., 1995; Gewirtz and Calabretta, 1988; Ratajczak et al., 1992c; Small et al., 1994) one that has been of particular interest, and one where therapeutically motivated disruptions are now in clinical trial, is the myb gene (reviewed in Lyon et al., 1994). These trials involve treatment of human leukemias. These diseases are a logical choice for developing oncogene targeted therapies because of easy access to tissues, and the abundance of knowledge about the cell and molecular biology of these diseases. Nevertheless, as will be touched on below, other malignancies have also been examined as models for Myb targeted therapy with some surprisingly encouraging results. Finally, while we have focused our efforts on the ODN strategy, I will allude briefly to other strategies for disrupting Myb function with therapeutic intent. (+info)
Isochromosome 17q in blast crisis of chronic myeloid leukemia and in other hematologic malignancies is the result of clustered breakpoints in 17p11 and is not associated with coding TP53 mutations.
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An isochromosome of the long arm of chromosome 17, i(17q), is the most frequent genetic abnormality observed during the disease progression of Philadelphia chromosome-positive chronic myeloid leukemia (CML), and has been described as the sole anomaly in various other hematologic malignancies. The i(17q) hence plays a presumably important pathogenetic role both in leukemia development and progression. This notwithstanding, the molecular consequences of this abnormality have not been investigated in detail. We have analyzed 21 hematologic malignancies (8 CML in blast crisis, 8 myelodysplastic syndromes [MDS], 2 acute myeloid leukemias, 2 chronic lymphocytic leukemias, and 1 acute lymphoblastic leukemia) with i(17q) by fluorescence in situ hybridization (FISH). Using a yeast artificial chromosome (YAC) contig, derived from the short arm of chromosome 17, all cases were shown to have a breakpoint in 17p. In 12 cases, the breaks occurred within the Smith-Magenis Syndrome (SMS) common deletion region in 17p11, a gene-rich region which is genetically unstable. In 10 of these 12 cases, we were able to further map the breakpoints to specific markers localized within a single YAC clone. Six other cases showed breakpoints located proximally to the SMS common deletion region, but still within 17p11, and yet another case had a breakpoint distal to this region. Furthermore, using chromosome 17 centromere-specific probes, it could be shown that the majority of the i(17q) chromosomes (11 of 15 investigated cases) were dicentric, ie, they contained two centromeres, strongly suggesting that i(17q) is formed through an intrachromosomal recombination event, and also implicating that the i(17q), in a formal sense, should be designated idic(17)(p11). Because i(17q) formation results in loss of 17p material, potentially uncovering the effect of a tumor suppressor on the remaining 17p, the occurrence of TP53 mutations was studied in 17 cases by sequencing the entire coding region. In 16 cases, no TP53 mutations were found, whereas one MDS displayed a homozygous deletion of TP53. Thus, our data suggest that there is no association between i(17q) and coding TP53 mutations, and that another tumor suppressor gene(s), located in proximity of the SMS common deletion region, or in a more distal location, is of pathogenetic importance in i(17q)-associated leukemia. (+info)
Endogenous CD8+ T cell expansion during regression of monoclonal EBV-associated posttransplant lymphoproliferative disorder.
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There are experimental data which suggest that the primary immune effector cell responsible for maintaining immune surveillance against the outgrowth of EBV-transformed B cells in humans is the CTL, but in vivo proof of this is lacking. In this study we perform a series of cellular and molecular assays to characterize an autologous, endogenous immune response against a transplantation-associated, monoclonal, EBV+ posttransplant lymphoproliferative disorder (PTLD). Following allogeneic bone marrow transplantation, a patient developed a monoclonal PTLD of donor B cell origin. With a decrease in immune suppression, we document the emergence of endogenous, donor-derived CD3+CD8+ CTLs, followed by regression of the PTLD. The TCR Vbeta repertoire went from a polyclonal pattern prior to the development of PTLD to a restricted TCR Vbeta pattern during the outgrowth and regression of PTLD. Donor-derived CD3+CD8+ T lymphocytes displayed MHC class I-restricted cytolytic activity against the autologous EBV+ B cells ex vivo without additional in vitro sensitization. The striking temporal relationship between the endogenous expansion of a TCR Vbeta-restricted, CD3+CD8+ population of MHC class I-restricted CTL, and the regression of an autologous monoclonal PTLD, provides direct evidence in humans that endogenous CD3+CD8+ CTLs can be responsible for effective immune surveillance against malignant transformation of EBV+ B cells. (+info)
Long-term survival and late deaths after allogeneic bone marrow transplantation. Late Effects Working Committee of the International Bone Marrow Transplant Registry.
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BACKGROUND AND METHODS: It is uncertain whether mortality rates among patients who have undergone bone marrow transplantation return to the level of the mortality rates of the general population. We analyzed the characteristics of 6691 patients listed in the International Bone Marrow Transplant Registry. All the patients were free of their original disease two years after allogeneic bone marrow transplantation. Mortality rates in this cohort were compared with those of an age-, sex-, and nationality-matched general population. Cox proportional-hazards regression was used to identify risk factors for death more than two years after transplantation (late death). RESULTS: Among patients who were free of disease two years after transplantation, the probability of living for five more years was 89 percent (95 percent confidence interval, 88 to 90 percent). Among patients who underwent transplantation for aplastic anemia, the risk of death by the sixth year after transplantation did not differ significantly from that of a normal population. Mortality remained significantly higher than normal throughout the study among patients who underwent transplantation for acute lymphoblastic leukemia or chronic myelogenous leukemia and through the ninth year among those who underwent transplantation for acute myelogenous leukemia. Recurrent leukemia was the chief cause of death among patients who received a transplant for leukemia, whereas chronic graft-versus-host disease was the chief cause among those who received a transplant for aplastic anemia. Advanced, long-standing disease before transplantation and active chronic graft-versus-host disease were important risk factors for late death. CONCLUSIONS: In patients who receive an allogeneic bone marrow transplant as treatment for acute myelogenous or lymphoblastic leukemia, chronic myelogenous leukemia, or aplastic anemia and who are free of their original disease two years later, the disease is probably cured. However, for many years after transplantation, the mortality among these patients is higher than that in a normal population. (+info)
LightCycler technology for the quantitation of bcr/abl fusion transcripts.
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Quantifying bcr/abl fusion transcripts in chronic myelogenous leukemia is thought to serve as a powerful parameter for monitoring the kinetic nature of this clonal disease in vivo and in vitro. Recently, we demonstrated the technical advantages as well as the clinical relevance of quantitating bcr/abl fusion mRNA using the 5-nuclease assay and a real-time fluorescence reverse transcriptase-PCR (RT-PCR) detection system (ABI PRISM 7700 SDS). Meanwhile, another technique was introduced (LightCycler technology) that may be used for the same purpose. To investigate whether this method may be an appropriate alternative to the described procedure, we have established bcr/abl LightCycler RT-PCR for major and minor bcr/abl fusion transcripts. We found that, with only minor modifications, TaqMan RT-PCR and fluorescent probe design can be used to obtain comparable results in the LightCycler system. The developed method could quantitate as little as 10 bcr/abl copies per 100 ng cDNA and was as safe and reproducible as the previously described technique. Because reaction efficiency was identical within different bcr/abl major fusions, one single RT-PCR could be established that simultaneously detects b2a3, b2a2, b3a2, and b3a3 fusion RNA with equal specificity and sensitivity. Compared to results generated by the ABI PRISM 7700 SDS, absolute amounts of bcr/abl did not differ significantly, and there was a linear correlation between the respective values. We conclude that TaqMan chemistry can be used in the LightCycler and that both real-time fluorescence PCR detection systems equally fulfill the criteria for the safe and reliable quantitation of bcr/abl fusion RNA in clinical samples. This may be of help for further standardization of quantitative bcr/abl RT-PCR, which, again, is necessary for the comparison of results generated by different investigators. (+info)
The risk of residual molecular and cytogenetic disease in patients with Philadelphia-chromosome positive first chronic phase chronic myelogenous leukemia is reduced after transplantation of allogeneic peripheral blood stem cells compared with bone marrow.
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The detection of residual molecular and cytogenetic disease was prospectively compared in patients with Philadelphia-chromosome (Ph1) positive first chronic phase chronic myelogenous leukemia (CML) who underwent allogeneic transplantation of unmanipulated peripheral blood stem cells (PBSCT) (n = 29) or bone marrow (BM) (n = 62) using genotypically HLA-identical sibling donors or partially HLA-matched extended family donors. A molecular relapse (MR), as defined by two consecutive positive polymerase chain reaction (PCR) assays for the detection of M-bcr-abl transcripts in a 4-week interval, was found in two of 29 (7%) patients after PBSCT compared with 20 of 62 (32%) patients after bone marrow transplantation (BMT). This corresponds to a 4-year molecular relapse estimate (+/- standard error) of 7% +/- 5% after PBSCT and of 44% +/- 8% after BMT (P <.009). With identical follow-up periods of survivors in both patient subsets between 6 and 55 months (median, 28 months), 14 of the 20 patients with MR after BMT progressed to an isolated cytogenetic (n = 10) or a hematologic (n = 4) disease recurrence, resulting in a 4-year cytogenetic relapse estimate of 47% +/- 11%, while none of the patients after PBSCT has so far relapsed (P <.006). Multivariate analysis including all potential influencial factors of posttransplant disease recurrence identified the source of stem cells (P <.02) as the only independent predictor of molecular relapse. In conclusion, this prospective comparison of molecular and cytogenetic residual disease demonstrates that peripheral blood stem cell transplants have a more pronounced activity against residual CML cells than bone marrow transplants. Prospective randomized trials comparing PBSCT and BMT in patients with first chronic phase Ph1-positive CML are strictly required to further substantiate differences in the antileukemic activity of the two stem cell sources. (+info)
T-cell depletion plus salvage immunotherapy with donor leukocyte infusions as a strategy to treat chronic-phase chronic myelogenous leukemia patients undergoing HLA-identical sibling marrow transplantation.
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T-cell depletion (TCD) of the donor marrow graft has been shown to reduce the severity of graft-versus-host disease (GVHD) in patients with chronic-phase (CP) chronic myelogenous leukemia (CML) undergoing HLA-identical sibling allogeneic marrow transplantation. However, there has been a corresponding reduction in the graft-versus-leukemia effect so that any decrease in GVHD-related mortality has been offset by an increased rate of disease relapse. Therapy of recurrent disease with donor leukocyte infusions (DLI) has been proven to be effective salvage therapy for the majority of patients who relapse after allogeneic BMT with CP CML. However, the overall impact of salvage DLI therapy on the survival of CP CML patients initially transplanted with TCD marrow grafts is not defined. To address this question, we have evaluated a clinical strategy of TCD followed by targeted adoptive immunotherapy with DLI in 25 CP CML patients undergoing allogeneic BMT from HLA-identical siblings. All patients received a standardized preparative regimen along with ex vivo TCD and posttransplant cyclosporine as GVHD prophylaxis. Durable engraftment was observed in all 25 patients. The incidence of grade II to IV acute GVHD was 8%. The cumulative incidence of transplant-related mortality (TRM) was 4%, and the 1-year probability of overall survival was 96%. The 3-year cumulative relapse incidence was 49%. All relapsed patients received DLI to reinduce remission. The total T-cell dose administered to these patients varied from 0.1 to 5.0 x 10(8) T cells/kg. Complete responses were observed in 12 of 14 patients, with 1 additional patient still too early to evaluate. Three patients died of GVHD after DLI, and 1 relapsed into blast crisis after a transient cytogenetic remission. Of the remaining 10 patients, 8 are in molecular remission, 1 is alive in relapse, and 1 is receiving DLI treatment. The median follow-up after infusion of surviving DLI patients in remission is 5.3 years. The probability of overall 5-year survival for the entire population is 80%, with a median follow-up of 6.4 years. We conclude that the clinical strategy of TCD followed by targeted adoptive immunotherapy with DLI for those patients with evidence of recurrent disease is a viable transplant strategy for CP CML, resulting in 80% survival and a low risk of acute GVHD and transplant-related mortality. (+info)
Clinical use of streptolysin-O to facilitate antisense oligodeoxyribonucleotide delivery for purging autografts in chronic myeloid leukaemia.
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Antisense oligodeoxyribonucleotides (ODN) targeted against the breakpoint in BCR-ABL mRNA will specifically decrease BCR-ABL mRNA, provided cells are first permeabilised with streptolysin-O (SL-O). We used 18-mer chimeric methylphosphonodiester: phosphodiester linked (4-9-4) ODN complementary to 9 bases either side of the BCR-ABL junction to purge harvests ex vivo in three CML patients who remained completely Ph positive after multiple chemotherapy courses. After CD34+ cell selection and SL-O permeabilisation, harvests were purged with 20 microM ODN. After purging, all individual CFU-GM colonies grown from the two b3a2 breakpoint cases remained positive for BCR-ABL mRNA. In contrast, all 24 colonies grown from the b2a2 breakpoint case were BCR-ABL mRNA negative. Patients were conditioned with busulphan 16 mg/kg. The initial post-transplant course was uneventful, although the time to return to 0.5 x 10(9)/l neutrophils was slow at 25-51 days. Both chronic phase patients remain in haematological remission at +724 and +610 days, although each has cytogenetic evidence of relapse. The b2a2 accelerated phase patient died of myeloid blast transformation at day +91. The present SL-O-facilitated ODN purging strategy appears to be without significant toxicity, and offers considerable improvements in ODN delivery to the cytosol. (+info)