Rearrangements and aberrant expression of the retinoic acid receptor alpha gene in acute promyelocytic leukemias.

Although acute promyelocytic leukemias (APLs) are consistently associated with a reciprocal chromosome 15;17 translocation, the gene(s) directly affected by the breakpoints have never been isolated. The chromosome 17 breakpoint maps to near the retinoic acid receptor alpha (RAR alpha) locus. Investigation of 20 APLs and a large series of other neoplastic patients and normal controls revealed RAR alpha gene rearrangements and aberrant transcripts only in the APL cases. These findings suggest that the RAR alpha gene is involved in the APL chromosome 17 breakpoint, is implicated in leukemogenesis, and could be used as a marker for identifying leukemic promyelocytes.

PLZF-mediated control on VLA-4 expression in normal and leukemic myeloid cells.

The promyelocytic leukemia zinc-finger protein (PLZF) is a transcriptional repressor. To investigate the role of PLZF in the regulation of cytoadhesion molecules involved in the mobilization of hemopoietic cells, we have analysed PLZF and very late antigen 4 (VLA-4) expression in normal and leukemic cells. In hematopoiesis, we found a negative correlation between PLZF and VLA-4 expression, except for the megakaryocytic lineage. In contrast, we observed a positive correlation between PLZF and VLA-4 expression in a panel of acute myeloid leukemia (AML) samples. In K562 cells expressing PLZF (K562-PLZF), we found that the expression of VLA-4 and c-kit was downmodulated. We have investigated the possibility for VLA-4 or the c-kit receptor to be direct target genes of PLZF in K562-PLZF cells and identified a PLZF DNA-binding site within the VLA-4 promoter. Furthermore, decrease in VLA-4 expression was associated with loss of adhesion on fibronectin-coated plates, which promotes drug-induced apoptosis of K562-PLZF cells. Our findings indicate that VLA-4 is a potential target gene of PLZF. However, in primary AMLs the control of PLZF on VLA-4 expression is lost. Altogether, we suggest that VLA-4 modulation by PLZF may represent an important step in the control of normal and leukemic cell mobilization.

The PML/RARalpha fusion protein inhibits tumor necrosis factor-alpha-induced apoptosis in U937 cells and acute promyelocytic leukemia blasts.

We investigated the effect of the acute promyelocytic leukemia (APL) specific PML/RARalpha fusion protein on the sensitivity to TNF-alpha-mediated apoptosis. The U937 leukemia cell line was transduced with PML/RARalpha cDNA. PML/RARalpha expression caused a markedly reduced sensitivity to TNF-alpha, even if apoptosis was triggered by agonistic antibodies to TNF-alpha receptors I and II (TNF-alphaRI, II). PML/RARalpha induced a 10-20-fold decrease of the TNF-alpha-binding capacity via downmodulation of both TNF-alphaRI and TNF-alphaRII: this may mediate at least in part the reduced sensitivity to TNF-alpha. Furthermore, the fusion protein did not modify Fas expression (CD95) or sensitivity to Fas-mediated apoptosis. The pathophysiological significance of these findings is supported by two series of observations. (a) Fresh APL blasts exhibit no TNF-alpha binding and are resistant to TNF-alpha-mediated apoptosis. Conversely, normal myeloblasts-promyelocytes show marked TNF-alphaR expression and are moderately sensitive to TNF-alpha-mediated cytotoxicity. Similarly, blasts from other types of acute myeloid leukemia (AML M1, M2, and M4 FAB types) show an elevated TNF-alpha binding. (b) The NB4 APL cell line, which is PML/RARalpha+, shows low TNF-alphaR expression capacity and is resistant to TNF-alpha-triggered apoptosis; conversely a PML/RARalpha- NB4 subclone (NB4.306) exhibits detectable TNF-alpha-binding capacity and is sensitive to TNF-alpha-mediated cytotoxicity. These studies indicate that the PML/RARalpha fusion protein protects against TNF-alpha-induced apoptosis, at least in part via downmodulation of TNF-alphaRI/II: this phenomenon may play a significant role in APL, which is characterized by prolonged survival of leukemic blasts.

Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.

Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed >30 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: <100 copies; five cases: 580-5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.

Identification of a molecular signature for leukemic promyelocytes and their normal counterparts: Focus on DNA repair genes.

Acute promyelocytic leukemia (APL) is a clonal expansion of hematopoietic precursors blocked at the promyelocytic stage. Gene expression profiles of APL cells obtained from 16 patients were compared to eight samples of CD34+-derived normal promyelocytes. Malignant promyelocytes showed widespread changes in transcription in comparison to their normal counterpart and 1020 differentially expressed genes were identified. Discriminating genes include transcriptional regulators (FOS, JUN and HOX genes) and genes involved in cell cycle and DNA repair. The strong upregulation in APL of some transcripts (FLT3, CD33, CD44 and HGF) was also confirmed at protein level. Interestingly, a trend toward a transcriptional repression of genes involved in different DNA repair pathways was found in APL and confirmed by real-time polymerase chain reactor (PCR) in a new set of nine APLs. Our results suggest that both inefficient base excision repair and recombinational repair might play a role in APLs development. To investigate the expression pathways underlying the development of APL occurring as a second malignancy (sAPL), we included in our study eight cases of sAPL. Although both secondary and de novo APL were characterized by a strong homogeneity in expression profiling, we identified a small set of differentially expressed genes that discriminate sAPL from de novo cases.

Characterization of a recurrent translocation t(2;3)(p15-22;q26) occurring in acute myeloid leukaemia.

Six patients with de novo acute myeloid leukemia (AML) and a t(2;3)(p15-21;q26-27) were identified among approximately 1000 cases enrolled in the GIMEMA trial. The t(2;3) was the sole anomaly in three patients, whereas in three cases monosomy 7, trisomy 15 and 22, and trisomy 14 represented additional aberrations. No cryptic chromosome deletions at 5q, 7q, 12p, and 20q were observed. One patient carried a FLT3 D835 mutation; FLT3 internal tandem duplication (ITD) was not detected in three patients tested. Characterization of the translocation breakpoints using a 3q26 BAC contig specific for the PRDM3 locus showed that the breakpoints were located 5' to EVIl as follows: within myelodysplatic syndrome (MDS) intron 1 (# 3), between MDS1 exons 2 and 3 in three patients (# 1, 2, 4) with a 170bp cryptic deletion distal to the breakpoint in one (# 2), and in a more centromeric position spanning from intron 2 to the 5' region of EVI1 (# 6, 5). A set of 2p16-21 BAC probes showed that the breakpoints on chromosome 2p were located within BCL11A in two separate regions (# 1, 4 and # 2-5), within the thyroid adenoma-associated (THADA) gene (# 6) or distal to the ZFP36L2 locus (# 3). Regulatory elements were present in proximity of these breakpoints. RACE PCR studies revealed a chimeric transcript in 1/6 patient analyzed, but no fusion protein. Quantitative PCR showed a 21-58-fold over-expression of the EVIl gene in all cases analyzed. The patients showed dysplasia of at least two myeloid cell lineages in all cases; they had a low-to-normal platelet count and displayed an immature CD34+ CD117+ immunophenotype. Despite intensive chemotherapy and a median age of 43 years (range 36-59), only two patients attained a short-lived response; one patient is alive with active disease at 12 months, five died at 4-14 months. We arrived at the following conclusions: (a) the t(2;3) is a recurrent translocation having an approximate 0.5% incidence in adult AML; (b) breakpoints involve the 5' region of EVIl at 3q26, and the BCL11A, the THADA gene or other regions at 2p16.1-21; (c) cryptic deletions distal to the 3q26 breakpoint may occur in some cases; (d) the juxtaposition of the 5' region of EVIl with regulatory elements normally located on chromosome 2 brings about EVI1 overexpression; (e) clinical outcome in these cases is severe.

Discontinuation of imatinib therapy after achievement of complete molecular response in a Ph(+) CML patient treated while in long lasting complete cytogenetic remission (CCR) induced by interferon.

Imatinib has become the gold standard therapy for Ph(+) CML, as it induces complete cytogenetic remission (CCR) in 75-90% of patients in chronic phase (CP), and up to 40% of these patients obtain at least a 3 log reduction of BCR/ABL transcript [Kantarjian HM, Cortes JE, O'Brien S, Luthra R, Giles F, Verstovsek S, et al. Long-term survival benefit and improved complete cytogenetic and molecular response rates with imatinib mesylate in Philadelphia chromosome-positive chronic-phase chronic myeloid leukemia after failure of interferon-alpha. Blood. 2004;104:1979-1988]. However, it is not yet stated whether continued therapy is required to maintain this response or whether imatinib may be discontinued after confirmation of a prolonged complete molecular remission (CMR). We here report on a Ph(+) CML case in long lasting CCR following interferon-alpha treatment (IFN) which reached CMR with imatinib but soon relapsed at molecular level after this latter drug discontinuation; we considered the present observation also in the light of previously reported data.

Monitoring of treatment outcome in acute promyelocytic leukemia by RT-PCR.

A rearrangement between the PML and RAR-alpha genes underlies the acute promyelocytic leukemia (APL)-specific t(15;17) translocation, leading to the production of a chimeric mRNA. Recent development of a reverse-transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha hybrid has proven useful for rapid diagnosis and monitoring of minimal residual disease (MRD) in APL patients. Preliminary studies in which the prognostic significance of RT-PCR was evaluated indicate that this test may identify patients at high risk of relapse.

Interleukin-3 receptor in acute leukemia.

Recent studies indicate that abnormalities of the interleukin-3 receptor (IL-3R) are frequently observed in acute myeloid leukemias (AMLs) and may contribute to the proliferative advantage of leukemic blasts. This review analyzes the evidences indicating that the IL-3R represents one of the target molecules involved in the stimulation of proliferation of AMLs, and the overexpression of the IL-3Ralpha chain may represent one of the mechanisms contributing to the development of a highly malignant leukemic phenotype. Furthermore, there is evidence that the IL-3Ralpha is a marker of leukemic stem cells, at variance with normal stem cells that are IL-3Ralpha-. Finally, the IL-3R may represent an important target for the development of new antileukemic drugs.

Monitoring of treatment outcome in acute promyelocytic leukemia by RT-PCR.

A rearrangement between the PML and RAR-alpha genes underlies the acute promyelocytic leukemia (APL)-specific t(15;17) translocation, leading to the production of a chimeric mRNA. Recent development of a reverse-transcription polymerase chain reaction (RT-PCR) assay for the PML/RAR-alpha hybrid has proven useful for rapid diagnosis and monitoring of minimal residual disease (MRD) in APL patients. Preliminary studies in which the prognostic significance of RT-PCR was evaluated indicate that this test may identify patients at high risk of relapse.

Polyclonal hemopoiesis in leukemia patients following molecularly documented remission.

We performed clonality studies of hemopoietic reconstitution in 24 female patients (pts) with leukemias characterized by specific tumor markers. Thirteen pts had Acute Promyelocytic Leukemia (APL) with rearrangements of the RAR-a and PML genes, 8 BCR rearranged (BCR+)/Ph+ Chronic Myeloid Leukemia (CML) and 3 BCR+/Ph+ Acute Lymphoid Leukemia (ALL). Bone marrow (BM) DNA samples were obtained at diagnosis and at remission after Southern blot documented suppression of specific markers. The clonal or non-clonal nature of hemopoietic reconstitution was assessed by hybridizing the same DNAs with the M27 beta probe, in order to detect methylation differences at the X-linked DXS255 locus. Twenty four pts showed a polyclonal methylation pattern at remission, whereas in 3 cases an apparently clonal pattern was observed despite no evidence of specific gene rearrangement. In 2 of these 3 cases, however, DNAs derived from non-affected tissues (T lymphocytes, skin and BM fibroblasts) revealed the presence of the same DXS255 unmethylated allele detected at diagnosis, while in the third case we found the same apparently clonal pattern in blood mononuclear cells obtained from her healthy female BM donor. These data indicate that polyclonal hematopoiesis occur in APL and CML pts after therapy induced suppression of specific tumor markers, and that unbalanced or aberrant X chromosome methylation patterns are observed in some cases, most likely reflecting constitutional features.

Improved rapid detection of the PML/RARalpha fusion gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tailored treatment. Detection of the PML/RARalpha fusion gene in APL blasts is critical to start promptly the specific therapy with all-trans retinoic acid (ATRA). APL lacking this genetic lesion have been reported as being ATRA resistant. Reverse-transcription polymerase chain reaction (RT-PCR) has been extensively used to detect the PML/RARalpha cDNA. The reported PML/RARalpha amplification techniques are laborious and time consuming, and include conventional RNA extraction, cDNA synthesis and a two-round (nested) PCR. We hereby describe a few variations of the commonly adopted RNA extraction and PML/RARalpha RT-PCR protocols which allow a molecular diagnosis of APL to be carried out in less than 5 h. Processing of small volumes of leukemic cell lysate (0.5 ml) in a microfuge allows extraction of good quality RNA in 1 h. After reverse transcription to obtain cDNA, a 'hot start' PCR procedure was adopted which enabled us to amplify clearly visible and specific products after a single (not nested) amplification round. The PML/RARalpha fusion gene was detected in the blasts of six consecutive APL at diagnosis, and an APL-tailored protocol including ATRA was started in each case within 6 h of admission. On repeated experiments, the assay proved highly specific and sensitive for the rapid detection of all PML/RARalpha transcript types. Our data should encourage the use of this rapid procedure for the diagnosis of both typical APL and, particularly, less typical cases awaiting urgent therapeutic intervention.

Identical utilization of T-cell receptor gene regions in B-lymphoid blast crisis of chronic myeloid leukemia and B-precursor acute lymphoblastic leukemia.

The configuration of the T-cell receptor (TCR) beta, gamma and delta chain genes was analyzed in 16 cases of B-lymphoid blastic crisis of chronic myeloid leukemia (BC-CML) for a better definition of the biological aspects of this cellular population, in comparison with the molecular features of B-precursor acute lymphoblastic leukemia (ALL). All cases displayed B-phenotypic features, were Ph'-positive and had a rearranged configuration of the breakpoint cluster region (bcr) and of the immunoglobulin heavy chain gene region (JH). The TCR beta chain gene was rearranged in four cases (25%), all of which displayed a monoallelic rearrangement involving the J beta 2 region. The TCR gamma chain gene was rearranged in 13 cases (81%); 13 rearranged alleles utilized the J1/2 regions, while the remaining five utilized JP1. The V regions of the group I were mostly involved. The TCR delta chain gene was rearranged or deleted in 15 cases (94%); the 10 rearranged chromosomes displayed exclusively two patterns referable to partial recombinations, a V2-(D)-D3 and a (D)-D3 type. These two configurations are predominant in B-precursor ALL (75% of rearranged chromosomes) and almost absent in T-ALL. Taken together, these results document the close similarities between the genotypic features of B-lymphoid BC-CML and B-precursor ALL, not only in terms of the incidence of rearrangement but more relevantly with regard to the choice of regions involved in the recombinations. This aspect is particularly evident at the TCR delta locus level.

Assessment of minimal residual disease (MRD) in CBFbeta/MYH11-positive acute myeloid leukemias by qualitative and quantitative RT-PCR amplification of fusion transcripts.

The inv(16)(p13q22) chromosomal rearrangement associated with FAB M4Eo acute myeloid leukemia (AML) subtype is characterized by the presence of the CBFbeta/MYH11 fusion transcript that can be used to detect minimal residual disease (MRD). However, qualitative RT-PCR studies of MRD have so far produced conflicting results and seem of limited prognostic value. We have evaluated retrospectively MRD in a large series of CBFbeta/MYH11-positive patients employing both qualitative and quantitative (real-time PCR) approaches. 186 bone marrow samples from 36 patients were examined with a median follow-up of 27.5 months; 15 patients relapsed during follow-up. In qualitative studies, carried out by 'nested' RT-PCR assay, all patients in complete remission (CR) immediately after induction/consolidation therapy were found to be PCR positive. However, follow-up samples at later time points were persistently negative (except one case) in patients remaining in continuous CR (CCR) for more than 12 months. 16 patients were evaluated by quantitative real-time PCR assay: CBFbeta/MYH11 transcript copy number was normalized for expression of the housekeeping gene ABL, expressed as fusion gene copy number per 10(4) copies of ABL. A 2-3 log decline in leukemic transcript copy number was observed after induction/consolidation therapy. After achieving CR, the mean copy number was significantly higher in patients destined to relapse compared to patients remaining in CCR (151 vs 9, P < 0.0001 by Mann-Whitney test). Moreover, in CCR patients, the copy number dropped below the detection threshold after the treatment protocol was completed and remained undetectable in subsequent MRD analysis in accordance with results obtained by qualitative RT-PCR. On the contrary, in the seven patients who relapsed, the copy number in CR never declined below the detection threshold; thus a cut-off value discriminating these two groups of patients could be established. The findings of our study, if confirmed, might confer an important predictive value to quantitative real-time PCR determinations of MRD in patients with inv(16) leukemia.

Analysis of p73 expression pattern in acute myeloid leukemias: lack of DeltaN-p73 expression is a frequent feature of acute promyelocytic leukemia.

p73, the homologue of p53, is a nuclear protein whose ectopic expression, in p53+/+ and p53-/- cells, recapitulates the most well-characterized p53 effects, such as growth arrest, apoptosis and differentiation. Unlike p53, which is mutated in half of human cancers, p73 is rarely mutated. However, altered expression of the p73 gene has been reported in neuroblastoma, lung cancer, prostate cancer and renal cell carcinoma. To investigate the potential involvement of p73 in acute myeloid leukemias (AMLs), we analyzed 71 samples from AML patients for the expression pattern of N-terminal transactivation-p73alpha (TA-p73alpha), its spliced isoforms and N-terminal-deleted-p73 transcripts (DeltaN-p73). We detected p73 gene expression in AML irrespective of FAB (French-American-British) subtypes. Notably, the analysis of DeltaN-p73 expression, which has been reported to inactivate both p53 and p73 antitumor effects, revealed a rather peculiar pattern. In fact, DeltaN-p73 transcript and protein were detectable in 27/28 (96.4%) cases of M0, M1, M2, M4, M5 and M6 AML and in 13/41 (31.7%) cases of PML-RARalpha-positive M3 AML (P<0.01). Thus, the distinct gene expression profile of p73 further supports the notion that acute promyelocytic leukemia is a biologically different subset of AML.

Occurrence of a Ki-1-positive anaplastic large-cell lymphoma in a patient with Ph' positive chronic myelogenous leukemia successfully treated by alpha-interferon.

We describe a patient with Philadelphia-chromosome-positive (Ph' +) chronic myelogenous leukemia (CML), who developed an anaplastic large cell lymphoma (ALCL) with T-phenotype, after 43 months successful treatment with alpha-interferon (IFN). Characterization studies of lymphoma cells showed positivity for Ki-1 monoclonal antibody, T-cell surface markers, T-cell receptor beta chain rearrangement, and germline configuration of the BCR gene. At the time of lymphoma diagnosis, the patient had achieved complete hematologic remission from CML with partial karyotypic conversion (50% Ph' + cells). After twelve weekly courses of polychemotherapy, he obtained complete remission from lymphoma. At present, five years from CML diagnosis, the patient has a remarkably stable disease, being in remission from lymphoma and in well controlled CML chronic phase. Our case thus represents the first well documented description of a T-cell non-Hodgkin's lymphoma developed during the course of CML.

Expression pattern of HOXB6 homeobox gene in myelomonocytic differentiation and acute myeloid leukemia.

Homeobox genes encode transcription factors known to be important morphogenic regulators during embryonic development. An increasing body of work implies a role for homeobox genes in both hematopoiesis and leukemogenesis. In the present study we have analyzed the role of the homeobox gene, HOXB6, in the program of differentiation of the myeloid cell lines, NB4 and HL60. HOXB6 expression is transiently induced during normal granulocytopoiesis and monocytopoiesis, with an initial induction during the early phases of differentiation, followed by a blockade of expression at early maturation. The enforced expression of HOXB6 in promyelocytic NB4 cells or in myeloblastic HL60 cells elicited inhibition of the granulocytic or monocytic maturation, respectively. Furthermore, HOXB6 was frequently expressed (18 out of 49 cases) in AMLs lacking major translocations while it was expressed at very low frequency (two out of 47 cases) in AMLs characterized by PML/RAR-alpha, AML-1/ETO, CBFbeta/MYH11 fusion and rearrangements of the MLL gene at 11q23. According to these observations, we suggest that a regulated pattern of HOXB6 expression is required for normal granulopoiesis and monocytopoiesis. Abnormalities of the HOXB6 expression may contribute to the development of the leukemic phenotype.

Allogeneic stem cell transplantation for advanced acute promyelocytic leukemia: results in patients treated in second molecular remission or with molecularly persistent disease.

In all, 17 consecutive patients in hematological complete remission (HCR) of acute promyelocytic leukemia (APL) received allogeneic stem cell transplantation (SCT) from an HLA-identical sibling and were monitored by reverse transcriptase polymerase chain reaction of PML/RARalpha prior and after transplant. Median age was 31 years (range 3-50 years). At 10 years, the actuarial probabilities of nonrelapse mortality, relapse and disease-free survival were 32% (95% CI: 8-56%), 33% (95% CI: 6-60%) and 46% (95% CI: 22-70%). Six patients tested PCR +ve (1st HCR n=2; 2nd HCR n=3; 3rd HCR n=1) and 11 PCR -ve (2nd HCR n=11) pre-SCT. Of the six patients PCR +ve, two showed early persistence of PCR positivity and converted to sustained PCR negativity after CSA withdrawal (one died of secondary tumor in molecular remission and one is alive in relapse), while four converted to PCR -ve rapidly (one died of the underlying disease and three are in molecular remission). Of the 11 patients PCR -ve pre-SCT, six died (four of transplant-related mortality, one of relapse and one after heart transplantation) and five are alive, four in molecular remission and one is in relapse. Allogeneic SCT seems a valid option for advanced APL, particularly for the poor prognostic group of patients with pre-SCT molecularly persistent disease.

Hypermethylation of GpG islands in the promoter region of p15(INK4b) in acute promyelocytic leukemia represses p15(INK4b) expression and correlates with poor prognosis.

We evaluated the methylation status of p15 gene in a series of 65 patients with newly diagnosed acute promyelocytic leukemia (APL) receiving homogeneous treatment. Moreover, in 32 of them, the methylation status of p15 gene was correlated to the p15 m-RNA expression. In total, 31 patients had no p15 methylation (U group). An abnormal methylation pattern was found in 34 patients: in seven of these patients only methylated DNA was detected (M group), while in the remaining 27 patients (M/U group), both methylated and unmethylated DNA were amplified. Patients from M group showed a higher incidence of relapses and a lower disease-free survival (DSF) with respect to patients from U and M/U groups (29, 64 and 79% at 5 years for M, U/M and U patients, respectively, P=0.03), while p15 methylation had no impact on overall survival. The p15 expression was detectable in all patients with unmethylated DNA, in none of patients with fully methylated DNA and in 60% of patients with partially methylated DNA. The DFS estimate at 5 years for p15-negative patients was significantly lower than that of p15-positive patients (P=0.03). These data confirm that the presence of p15 methylation negatively influences the prognosis of APL, mainly when it represses the p15 gene transcription.

C-fms expression correlates with monocytic differentiation in PML-RAR alpha+ acute promyelocytic leukemia.

We have investigated the expression of the M-CSF receptor (c-fms) in 16 freshly isolated acute promyelocytic leukemias (APL) expressing the PML/RAR alpha fusion protein. In parallel, we evaluated the capacity of these cells to differentiate along the granulocytic and monocytic pathways. c-fms was constitutively and constantly expressed in all cases sensitive in vivo to all-trans retinoic acid (ATRA) and its expression was further potentiated following in vitro induction with ATRA. Furthermore, gel-shift analysis of APL cells showed elevated levels of PU.1 binding activity to the M-CSF receptor promoter, particularly after ATRA stimulation. Interestingly, the rise of PU.1 binding activity as well as of PU.1 levels after ATRA treatment was significantly higher in APL patients exhibiting monocytic maturation, as compared to those that did not undergo monocytic differentiation. A variable proportion of ATRA-induced APL cells exhibited monocyte-like morphology and immunophenotype: the proportion of monocytic cells was consistently increased by combined treatment with ATRA and diverse hematopoietic growth factors cocktails, which always comprised M-CSF. Monocytic cells originating from in vitro ATRA-induced maturation of APL cells derive from the leukemic clone as suggested by two lines of evidence: (1) monocytic cells harbor the 15;17 translocation; (2) monocytic cells possess Auer bodies. The c-fms(bright) leukemic blasts preferentially showed the capacity for monocytic differentiation as compared to the c-fms(dim/-) subset: indeed, enforced expression of c-fms into NB4, a PML/RAR alpha+ cell line, favored the onset of monocytic maturation. Finally, low c-fms expression was observed in an APL relapsing patient resistant to ATRA, as well as in an APL case with t(11;17), PLZF/RAR alpha+. These observations indicate that PML/RAR alpha+ APL blasts are bipotent for differentiation through both neutrophilic and monocytic lineages, whereby monocytic differentiation is linked to c-fms expression and stimulation.