Efficacy of decitabine in the treatment of patients with chronic myelomonocytic leukemia (CMML).

Chronic myelomonocytic leukemia (CMML) characterized by cytopenias, bone marrow and peripheral blood cell dysplasia is notoriously hard to treat. Recent reclassification of CMML as a myelodysplastic/myeloproliferative (MDS/MPS) disease rather than a myelodysplastic syndrome (MDS) by the World Health Organisation (WHO) has led to a review of CMML patients treated with decitabine. Overall response rates (ORR) (complete response [CR]+partial response [PR]) in the subset of patients with CMML in one pivotal phase 3 trial (D-0007) and two phase 2 trials (PCH 95-11, PCH 97-19) decitabine were reviewed. For consistency across trials, all decitabine-treated patients were evaluated using the phase 2 response criteria (CR was defined by normocellular bone marrow with <5% blasts and normal Hgb, WBC, and platelet counts, and PR required 50% decrease in blast count, increases in Hgb by >1.5 mmol/L, WBC count by >1000, and platelet count by >50,000). A total of 31 patients diagnosed with CMML are included in this review. Similar demographics and disease characteristics were observed in all three studies, with an average age of 70.2 years and 71% of patients male. Baseline WBC of >20,000 were observed in 8/28 (29%) patients and baseline bone marrow blasts >5% in 11/28 (39%) patients. All clinical responses were centrally reviewed. The ORR was 25% (14% CR+11% PR). Hematologic improvement was observed in 11% of patients and stable disease in 39% of patients. The decitabine adverse event profile seen in CMML patients was similar to observations in other hematologic patient populations, with myelosuppression and related infectious complications. These data demonstrate encouraging activity for decitabine in CMML, and suggest that studies in other myeloproliferative diseases may be warranted.

Relapse of acute promyelocytic leukemia with PML-RARalpha mutant subclones independent of proximate all-trans retinoic acid selection pressure.

Relapse of acute promyelocytic leukemia (APL) following all-trans retinoic acid (ATRA) therapy has been associated with the acquisition of mutations in the high-affinity ATRA binding site in PML-RARalpha, but little information is available about the selection dynamics of the mutation-harboring subclones. In this study, 6/18 patients treated with sequential ATRA and chemotherapy on protocol INT0129 relapsed with complete replacement of the nonmutant pretreatment APL cell population by a PML-RARalpha mutant subclone. Two patients relapsed in proximity of ATRA treatment; however, in four patients there was a 6-48 month hiatus between the last ATRA treatment and relapse. The mutant subclones were not detectable in samples tested > or = 3 months before relapse at > or = 1 in 10(2) (10(-2)) sensitivity. In one patient, a functionally weak mutation was detected at 10(-4) sensitivity before therapy but only limited pre-relapse enrichment of the mutant subclone was observed on subsequent ATRA therapy. These results indicate that proximate ATRA selection pressure is frequently not the main determinant for the emergence of strongly dominant PML-RARalpha mutant subclones and suggest that APL subclones harboring PML-RARalpha mutations are predisposed to the acquisition of secondary genetic/epigenetic alterations that result in a growth/survival advantage.

Use of nonvolume-reduced (unmanipulated after thawing) umbilical cord blood stem cells for allogeneic transplantation results in safe engraftment.

Volume reduction of umbilical cord blood (UCB) units before infusion is standard in most transplant centers. We examined 26 patients who underwent transplantation from May 1997 to December 2001 with unmanipulated (n=18) or volume-reduced (n=8) UCB units for engraftment. Of 18 unmanipulated UCBT patients, 16 achieved ANC >500/mm(3), a median of 26 days (range, 16-104) post-UCBT; two died before engraftment on days +2 and +14. Of 18 unmanipulated UCBT patients, 10 achieved platelet recovery, a median of 60.5 days (range, 41-144) post-UCBT; eight patients died before platelet recovery +2 to +255 days post-UCBT. These results are similar to several reported studies and our series utilizing volume-reduced UCB units for UCBT. At a median follow-up of 29.5 months, the 100-day and 3-year overall survivals of unmanipulated UCBT were 61.1% (95% CI, 38.6-83.6) and 48.6% (95% CI, 24.8-72.4) and of volume-reduced UCBT were 60% (95% CI, 24.4-95.6) and 22.5% (95% CI, 0-58.7). There was no serious toxicity from UCB infusion using unmanipulated UCB units. We conclude that unmanipulated UCB units may be infused safely into UCBT patients with adequate engraftment and survival.

HLA-DR antigen-negative acute myeloid leukemia.

Human leukocyte antigen (HLA) Class II antigens are variably expressed on acute myeloid leukemia (AML) blasts. The biological and clinical significance of HLA Class II antigen expression by AML cells is not known. Therefore, we sought to characterize cases of AML without detectable HLA-DR expression. Samples from 248 consecutive adult AML patients were immunophenotyped by multiparameter flow cytometry at diagnosis. HLA-DR antigens were not detected on AML cells from 43 patients, including 20 with acute promyelocytic leukemia (APL), and 23 with other subtypes of AML. All APL cases had t(15;17), but there were no characteristic chromosome abnormalities in non-APL cases. No direct expression of other antigens was identified in HLA-DR-negative APL and non-APL cases. Interestingly, cells from three HLA-DR-negative non-APL patients had similar morphology to that of the hypogranular variant of APL. This morphology, however, was not present in any HLA-DR-positive AML cases. Treatment response was similar in the 23 HLA-DR-negative non-APL and the 205 HLA-DR-positive patients. Finally, relapse was infrequently associated with changes in HLA-DR antigen expression, as the HLA-DR antigen was lost at relapse in only 4% of HLA-DR-positive cases, and was gained at relapse in only 17% of HLA-DR-negative cases. We conclude that HLA-DR-negative AML includes approximately equal numbers of APL and non-APL cases, and that the morphology of HLA-DR-negative non-APL cases can mimic the hypogranular variant of APL. The diagnosis of APL cannot be based on morphology and lack of HLA-DR antigen expression; rather, it requires cytogenetic or molecular confirmation.

Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction.

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.

Retrospective multivariate analysis of hepatic veno-occlusive disease after blood or marrow transplantation: possible beneficial use of low molecular weight heparin.

This retrospective cohort study of 462 consecutive adult allogeneic and autologous blood or marrow transplantation (BMT) patients compared the incidence of hepatic veno-occlusive disease (VOD) after BMT with three prophylactic regimens. Patients receiving heparin (Hep), heparin + prostaglandin E1 (Hep + PGE1) or low molecular weight heparin (LMWH) as a prophylactic VOD regimen were compared to a historical cohort receiving no VOD prophylaxis. Of 462 BMT patients, VOD was diagnosed in 22% (31 of 142) of the no prophylaxis group, 11% (11 of 104) of the Hep, 12% (13 of 110) in the Hep + PGE1 and 4% (four of 106) of the LMWH group (P = 0.0002). VOD was the primary cause of death in 20% (12 of 59). By multivariate logistic regression, independent risk factors for developing VOD were: no VOD prophylactic regimen, unrelated allogeneic BMT, Karnofsky performance score (KPS) < 80 and aspartate aminotransferase (AST) > or =50 U/l. There was no increase in the rate of death due to hemorrhagic events or VOD in any prophylaxis group compared to the control group. Prospective randomized trials of Hep vs LMWH vs placebo are warranted to assess the efficacy of heparin compounds in the prevention of VOD.

Current issues in the management of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is caused by one of four genetic lesions that disrupt the alpha receptor for retinoic acid, RAR alpha. The fusion protein responsible for greater than 99% of APL cases, PML-RAR alpha, inhibits PML-dependent apoptotic pathways in a dominant negative fashion and blocks myeloid differentiation by direct transcriptional inhibition of retinoic acid target genes. This transcriptional inhibition is mediated by recruitment of co-repressor proteins and resultant deacetylation of histones in the promoter regions of genes (yet to be identified) that control promyelocyte development. In the presence of high levels of all-trans retinoic acid (ATRA), both PML-dependent apoptotic mechanisms and myeloid-specific gene expression programs are reactivated. In the clinic, the combination of anthracycline-based chemotherapy plus ATRA cures approximately 80% of APL patients, and a high percentage of relapsed patients can achieve second remissions with arsenic trioxide. With the publication of results from the European APL 93 trial, the 'standard-of-care' for induction treatment of APL now includes ATRA plus concurrent anthracycline-based chemotherapy. The amount and type of consolidation therapy necessary for an individual APL patient remains somewhat of an open question, but at present should include at least two cycles of chemotherapy. Based on recent trials that demonstrate a benefit of maintenance ATRA (+/- low-dose chemotherapy), all APL patients should probably receive some type of maintenance therapy. While the above approach currently cures the majority of APL patients, future improvements in the treatment of this disease will require risk-adapted protocols that incorporate real-time molecular monitoring and appropriate introduction of novel therapeutic agents.

Functional analysis of the domain structure of tumor necrosis factor-alpha converting enzyme.

Many membrane-bound proteins, including cytokines, receptors, and growth factors, are proteolytically cleaved to release a soluble form of their extracellular domain. The tumor necrosis factor (TNF)-alpha converting enzyme (TACE/ADAM-17) is a transmembrane metalloproteinase responsible for the proteolytic release or "shedding" of several cell-surface proteins, including TNF and p75 TNFR. We established a TACE-reconstitution system using TACE-deficient cells co-transfected with TACE and substrate cDNAs to study TACE function and regulation. Using the TACE-reconstitution system, we identified two additional substrates of TACE, interleukin (IL)-1R-II and p55 TNFR. Using truncations and chimeric constructs of TACE and another ADAM family member, ADAM-10, we studied the function of the different domains of TACE in three shedding activities. We found that TACE must be expressed with its membrane-anchoring domain for phorbol ester-stimulated shedding of TNF, p75 TNFR, and IL-1R-II, but that the cytoplasmic domain is not required for the shedding of these substrates. The catalytic domain of ADAM-10 could not be functionally substituted for that of TACE. IL-1R-II shedding required the cysteine-rich domain of TACE as well as the catalytic domain, whereas TNF and p75 TNFR shedding required only the tethered TACE catalytic domain.

Identification of B94 (TNFAIP2) as a potential retinoic acid target gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by a block to myeloid differentiation caused by expression of the fusion oncoprotein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha). The purpose of this study was to identify genes that are regulated in a PML-RARalpha-dependent fashion by retinoic acid (RA), because such genes may be integrally involved in APL pathogenesis and/or myeloid differentiation. A cDNA microarray approach was used to identify genes induced in response to RA in TF1 myeloid leukemia cells expressing PML-RARalpha (TF1-PR cells). The B94 gene (TNFAIP2; Unigene Hs.101382), originally identified as a tumor necrosis factor alpha-inducible gene in endothelial cells, was one of several genes found to be induced by RA specifically in TF1-PR cells, but not in TF1-neo (control) cells. The induction of B94 was most pronounced in cells expressing the PML-RARalpha short isoform and was negligible in cells that expressed a mutant PML-RARalpha protein containing a deletion of the PML coiled-coil domain. B94 induction by RA occurred within 1 h, did not require new protein synthesis, and was inhibited by actinomycin D, suggesting rapid transcriptional activation. B94 was also induced by RA in NB4, UF1, and HL-60 cells, but not in other hematopoietic cell lines tested, suggesting that its up-regulation by RA may be specific to cells that express PML-RARalpha or are at the late myeloblast or promyelocyte stage of myeloid development. A screen of bone marrow cells from normal donors or patients with acute myelogenous leukemia showed that B94 was highly expressed in normal marrow and in marrow from patients with acute myelogenous leukemia French-American-British subtypes M0-M2, but was repressed in marrow cells from APL patients. Treatment of APL blasts in vitro with all-trans-RA resulted in up-regulation of B94 mRNA. These results suggest that B94 plays a role in myeloid development and support the hypothesis that B94 is a target gene of PML-RARalpha in APL.

Identification of two major sites in the type I interleukin-1 receptor cytoplasmic region responsible for coupling to pro-inflammatory signaling pathways.

Type I interleukin-1 receptor is the prototype for a family of proteins, which play a central role in early responses to injury and infection. The similarity of function across the family is reflected in similarity in signaling: all members tested couple to activation of NFkappaB and stress kinases. The coupling to these pathways is mediated by a 200-residue intracellular domain (the Toll/interleukin-1 receptor domain), in which sequence conservation is primarily confined to three short motifs (boxes 1, 2, and 3) located at amino acid residue positions 10 (box 1), 60 (box 2), and 170 (box 3). We have analyzed the contribution of these motifs to function by alanine scanning mutagenesis of the human interleukin-1 receptor type I. Mutant receptors were tested for expression, ligand binding, activation of receptor-associated kinase(s), NFkappaB, stress kinases, and transcription. Mutations in all three motifs led to low cell surface expression. Mutants in box 3 were, however, wild type for signaling, whereas mutants in boxes 1 and 2 were defective. We conclude that the conserved motifs box 1 and box 2 mediate the coupling of molecules in the family to inflammation signaling pathways.

Molecular analysis and clinical outcome of adult APL patients with the type V PML-RARalpha isoform: results from intergroup protocol 0129.

The type V (for variable) promyelocytic leukemia retinoic acid receptor (PML-RAR)alpha transcript, found in approximately 8% of adult patients with acute promyelocytic leukemia (APL), is defined molecularly by truncation of PML exon 6 and frequent insertion of genetic material from RARalpha intron 2. To more fully characterize the molecular features of PML-RARalpha V-type transcripts and to determine whether V-form APL patients have a distinct clinical presentation or prognosis, we analyzed 18 adult V-form APL patients enrolled on Intergroup protocol 0129 (INT-0129). Truncations in PML exon 6 ranged from 8 to 146 nucleotides, and 3 to 127 extra nucleotides (1 to 42 extra amino acids) were inserted at the PML exon 6/RARalpha exon 3 junction in 13 cases. No distinguishing morphologic, cytogenetic, or immunophenotypic features of V-form blasts were identified. A total of 5 of 7 patients induced with ATRA and 8 of 11 patients who received chemotherapy for induction achieved complete remission (CR). Six patients have relapsed, 4 after chemotherapy induction and 2 after ATRA. Nine patients (50%) are alive, 6 in continuous CR, 2 after salvage therapy for relapsed or refractory disease, and 1 after alternative treatment following early removal from protocol. Although the failure rate for V-form APL patients was high (61%), the low power of the current study to detect clinically significant differences precludes a meaningful comparison of clinical outcomes between the 18 V-form cases and non-V-form adult APL patients enrolled on INT-0129. (Blood. 2000;95:398-403)

Thiotepa-associated cardiomyopathy during blood or marrow transplantation: association with the female sex and cardiac risk factors.

Thiotepa (TT) has not been reported to cause cardiomyopathy, whereas cyclophosphamide (Cy)-related cardiomyopathy is well characterized. To search for cases of acute onset cardiomyopathy associated with TT, we retrospectively reviewed 171 patients who received TT-containing conditioning regimens for blood or marrow transplantation (BMT). Nine of 171 patients (5.3%) developed clinical congestive heart failure in the post-BMT period. The median time to onset of heart failure was 15 days after BMT (range 5-30). The median pre-BMT left ventricular ejection fraction (LVEF) was 50% (range 42-65%) as determined by two-dimensional echocardiogram, or gated blood pool scan. At the time of cardiomyopathy onset, LVEF was 30%. Six patients died of causes unrelated to heart failure. All affected patients who developed congestive heart failure following administration of TT had some evidence of cardiac dysfunction prior to transplantation. Significant risk factors for the development of cardiomyopathy included low pre-BMT-LVEF and female sex--particularly in females receiving allogeneic transplantation. The incidence of congestive heart failure with TT-containing regimens was similar to the incidence using other regimens with and without Cy. The mean time to clinical evidence of TT-associated cardiomyopathy was longer than the mean time reported with Cy. We recommend caution in using high-dose TT-containing regimens for patients with histories of cardiac dysfunction.

Biology and treatment of acute progranulocytic leukemia.

Acute progranulocytic leukemia (APL) is one of the most curable of all human cancers. Combination treatment with retinoic acid (RA) and anthracycline-based chemotherapy is safe and effective for the vast majority of patients, and several novel treatment approaches are under investigation for high-risk or relapsed patients. The APL-specific oncogenes PML-RAR alpha and PLZF-RAR alpha both bind nuclear corepressors and recruit histone deacetylase activity to promoters of RA target genes. The differential sensitivity of binding of these oncogenes to nuclear corepressors in the presence of RA appears to explain the resistance of PLZF-RAR alpha-related APL to RA and at the same time explains the effectiveness of RA in PML-RAR alpha-positive APL. Transcriptional repression of RA target genes, mediated by histone deacetylase activity, may thus be a key pathogenetic event in APL. Cure of the minority of resistant patients requires further refinement of current treatment approaches and appropriately timed incorporation of novel therapies, such as arsenic trioxide or histone deacetylase inhibitors.

The molecular biology of acute promyelocytic leukemia.

The preceding two years have witnessed an explosion in the accumulation of knowledge relating to the molecular pathogenesis of APL. Critical advances include: The molecular delineation of atypical APL cases with alternative RAR alpha fusion partners, and the demonstration that cells from 2 of the 3 types of 'atypical' APL retain sensitivity to ATRA. Perhaps the key question is why such cases are so rare. However, at a minimum, the presence of such cases argues persuasively that disruption of the retinoid signaling pathway is a (perhaps the) key pathogenetic feature of APL. Although certainly not 'passive' partners, it is likely that PML, PLZF, NPM, and NuMA serve similar functions in the pathogenesis of APL. The demonstration, in transgenic mice, that PML-RAR alpha (and PLZF-RAR alpha) can disrupt normal hematopoiesis and, given sufficient time, cause an APL-like syndrome. the variation in phenotype of the mice, which appears to be a consequence of the specific expression vector used, emphasizes the cell-type-specific nature of PML-RAR alpha function. Continuing functional analysis of PML, PLZF, and RAR alpha. In particular, the demonstration that PML and PLZF can form heterodimers provides a critical functional link between these proteins and offers a tantalizing glimpse at how both, when linked with RAR alpha, can cause APL. The demonstration that PML-RAR alpha is degraded, perhaps via a ubiquitin-dependent pathway, in response to ATRA. This result offers a unifying, if not yet proven, hypothesis to explain the sensitivity of leukemic promyelocytes to ATRA. Unfortunately, it is not known if ATRA can also cause degradation of NPM-RAR alpha or NuMA-RAR alpha (atypical cytogenetic APL variants that retain ATRA responsiveness). Whether PML-RAR alpha degradation is a cause, or consequence, of promyelocytic maturation remains unclear. Continuing insight into retinoid resistance, including the first demonstration of mutations in the PML-RAR alpha molecule from ATRA-resistant patients. The definitive demonstration that the two major PML-RAR alpha isoforms, while having subtle differences in biological activity and producing slightly different APL phenotypes, nevertheless do not, in and of themselves, have prognostic significance in patients treated with ATRA/chemotherapy combinations. Further functional analysis of PML-RAR alpha in vitro. The fascinating finding that PML-RAR alpha is cytotoxic to most cell types suggests that it must function as an oncogene in a very specialized milieu. In addition, the demonstration that both the DBD (from RAR alpha) and dimerization interface (from PML) are required for full in vitro functional activity, coupled with the finding that PML itself is a strong transcriptional suppressor, suggests that PML-RAR alpha may directly repress transcription of RA target genes. The challenge in APL research now is to integrate the above findings into a cohesive, unifying model that explains the biology of APL at a molecular level. The creation and validation of such a model will clarity whether APL is a fortunate medical curiosity or whether it will serve as a paradigm for the development of effective differentiation therapies in other types of human cancers.

The biology and treatment of acute progranulocytic leukemia.

Acute progranulocytic leukemia (APL) is one of the most curable of all human cancers. Combination treatment with retinoic acid (RA) and anthracycline-based chemotherapy is safe and effective for the vast majority of patients, and several novel treatment approaches are under investigation for high-risk or relapsed patients. The APL-specific oncogenes PML-RAR alpha and PLZF-RAR alpha both bind nuclear corepressors and recruit histone deacetylase activity to promoters of RA target genes. The differential sensitivity of binding of these oncogenes to nuclear corepressors in the presence of RA appears to explain the resistance of PLZF-RAR alpha-related APL to RA and at the same time explains the effectiveness of RA in PML-RAR alpha-positive APL. Transcriptional repression of RA target genes, mediated by histone deacetylase activity, may thus be a key pathogenetic event in APL. Cure of the minority of resistant patients requires further refinement of current treatment approaches and appropriately timed incorporation of novel therapies, such as arsenic trioxide or histone deacetylase inhibitors.

An essential role for ectodomain shedding in mammalian development.

The ectodomains of numerous proteins are released from cells by proteolysis to yield soluble intercellular regulators. The responsible protease, tumor necrosis factor-alpha converting enzyme (TACE), has been identified only in the case when tumor necrosis factor-alpha (TNFalpha) is released. Analyses of cells lacking this metalloproteinase-disintegrin revealed an expanded role for TACE in the processing of other cell surface proteins, including a TNF receptor, the L-selectin adhesion molecule, and transforming growth factor-alpha (TGFalpha). The phenotype of mice lacking TACE suggests an essential role for soluble TGFalpha in normal development and emphasizes the importance of protein ectodomain shedding in vivo.

Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor.

The amyloid protein, Abeta, which accumulates in the brains of Alzheimer patients, is derived by proteolysis of the amyloid protein precursor (APP). APP can undergo endoproteolytic processing at three sites, one at the amino terminus of the Abeta domain (beta-cleavage), one within the Abeta domain (alpha-cleavage), and one at the carboxyl terminus of the Abeta domain (gamma-cleavage). The enzymes responsible for these activities have not been unambiguously identified. By the use of gene disruption (knockout), we now demonstrate that TACE (tumor necrosis factor alpha converting enzyme), a member of the ADAM family (a disintegrin and metalloprotease-family) of proteases, plays a central role in regulated alpha-cleavage of APP. Our data suggest that TACE may be the alpha-secretase responsible for the majority of regulated alpha-cleavage in cultured cells. Furthermore, we show that inhibiting this enzyme affects both APP secretion and Abeta formation in cultured cells.

Expression and function of the megakaryocyte growth and development factor receptor in acute myeloid leukemia blasts.

The receptor for megakaryocyte growth and development factor (MGDF), also known as thrombopoietin, has recently been cloned. MGDF stimulates platelet production and maturation both in vitro and in vivo. MGDF may thus have a role in attenuating the thrombocytopenia associated with acute myeloid leukemia (AML) and its therapy. However, there is concern that MGDF might induce AML blast proliferation and thereby increase the risk of treatment failure. To address this concern, we studied the expression of c-mpl mRNA and c-Mpl protein by blasts from AML patients. In addition we examined the in vitro effect of MGDF as well as the combined effect of MGDF and granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF) on leukemic blast proliferation, recruitment into S-phase, induction of programmed cell death and activation of signal transducers and activators of transcription (STAT) proteins. Our results demonstrate that blasts from a substantial proportion of cases of AML express the receptor at either the mRNA or protein level. Moreover, the function of the MGDF receptor was demonstrated by activation of STAT proteins following exposure to MGDF. Nevertheless, blast proliferation in response to MGDF was rare, and the proliferative effect of MGDF was less than that of G-CSF or SCF. Furthermore, MGDF did not prevent programmed cell death induced by cytarabine. Finally, there appeared to be no correlation between receptor expression by AML blasts and functional response to MGDF. Based on these data, it would appear that clinical trials of MGDF may be undertaken safely in patients with AML.

Constitutive expression of the promyelocytic leukemia-associated oncogene PML-RARalpha in TF1 cells: isoform-specific and retinoic acid-dependent effects on growth, bcl-2 expression, and apoptosis.

Two major isoforms of PML-RARalpha are associated with (15;17)-positive acute promyelocytic leukemia (APL); however, functional differences between these isoforms have been difficult to define, and the molecular mechanism by which each isoform contributes to the pathogenesis of APL is not fully understood. To address these issues, the 'short' (S) and 'long' (L) isoforms of PML-RARalpha were constitutively expressed in the factor-dependent human erythroleukemia cell line, TF1. Expression of the L, but not the S, isoform inhibited growth of these cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). In the absence of GM-CSF, the S isoform partially protected against apoptosis, while the L isoform accelerated cell death. Treatment with all-trans retinoic acid (ATRA) inhibited cell growth and caused apoptosis only in PML-RARalpha-expressing cells, and these effects of ATRA were more marked in cells expressing the L isoform. ATRA treatment also led to downregulation of bcl-2 and endogenous RARalpha in PML-RARalpha-expressing cells, but had little effect on the level of exogenously expressed PML-RARalpha. We conclude that (1) subtle differences exist in the biologic activities of the L and S isoforms of PML-RARalpha, and (2) both isoforms are capable of transducing an ATRA-mediated signal that leads to downregulation of bcl-2 and induction of programmed cell death.

Association of PML-RAR alpha fusion mRNA type with pretreatment hematologic characteristics but not treatment outcome in acute promyelocytic leukemia: an intergroup molecular study.

In each case of acute promyelocytic leukemia (APL) one of three PML-RAR alpha mRNA types is produced, depending on the break/fusion site in the PML gene that is linked to a common RAR alpha gene segment: a short (S)-form type, PML exon 3 RAR alpha exon 3; a long (L)-form type, PML exon 6 RAR alpha exon 3; or a variable (V)-form type, variably deleted PML exon 6 RAR alpha exon 3. We evaluated whether PML-RAR alpha mRNA type is associated with distinct pretreatment clinical characteristics and therapeutic outcome in previously untreated adult APL patients registered to protocol INT 0129 by the Eastern Cooperative Oncology Group, the Southwest Oncology Group, and the Cancer and Leukemia Group B. Of 279 clinically eligible cases, 230 were molecularly evaluable, and of these, 111 were randomized to receive remission induction therapy with all-trans retinoic acid (ATRA) and 119 with conventional chemotherapy. Nine cases not excluded by central pathology review were PML-RAR alpha negative, and notably, none of five of these cases treated with ATRA achieved complete remission (CR). Among 221 PML-RAR alpha-positive cases, there were 82 S-form cases (37%), 121 L-form cases (55%), and 18 V-form cases (8%). Before any antileukemic therapy, the S-form type, compared with the L-form type, was associated with higher values for the white blood cell (WBC) count (median 2,500/microL v 1,600/microL; P = .009), the percentage of blood blasts plus promyelocytes (median 29% v 8.5%; P = .03), and the absolute blood blasts plus promyelocytes (884/microL v 126/microL; P = .019). Also, an increased percentage of S-form versus L-form cases had the M3 variant phenotype, 24% v 12% (P = .036). There were no differences between S-form and L-form cases in either CR rate (79% v 69%; P = .14) or disease free survival distribution (multivariate analysis adjusting for the association of S-form type and higher WBC count; P = .40). We conclude that the S-form type is associated with previously-identified adverse risk WBC parameters but that the identification of the S-form or L-form type of PML-RAR alpha mRNA, per se, does not predict clinical outcome or add to the value of an increased WBC count as a negative prognostic indicator in APL patients.