A restricted signature of miRNAs distinguishes APL blasts from normal promyelocytes.

MicroRNAs (miRNAs) are small non-coding RNAs involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. A small set of miRNAs is differentially expressed in hematopoietic cells and seemingly has an important role in granulopoiesis and lineage differentiation. In this study, we analysed, using a quantitative real-time PCR approach, the expression of 12 granulocytic differentiation signature miRNAs in a cohort of acute promyelocytic leukemia (APL) patients. We found nine miRNAs overexpressed and three miRNAs (miR-107, -342 and let-7c) downregulated in APL blasts as compared with normal promyelocytes differentiated in vitro from CD34+ progenitors. Patients successfully treated with all-trans-retinoic acid (ATRA) and chemotherapy showed downregulation of miR-181b and upregulation of miR-15b, -16, -107, -223, -342 and let-7c. We further investigated whether the APL-associated oncogene, promyelocytic leukemia gene (PML)/retinoic acid receptor alpha (RARalpha), might be involved in the transcriptional repression of miR-107, -342 and let-7c. We found that PML/RARalpha binds the regulatory sequences of the intragenic miR-342 and let-7c. In addition, we observed, in response to ATRA, the release of PML/RARalpha paralleled by their transcriptional activation, together with their host genes, EVL and C21orf34alpha. In conclusion, we show that a small subset of miRNAs is differentially expressed in APL and modulated by ATRA-based treatment.

PLZF-mediated control on c-kit expression in CD34(+) cells and early erythropoiesis.

The promyelocytic leukemia zinc-finger protein (PLZF) is a transcription factor and c-kit is a receptor tyrosine kinase associated with human disease, particularly in hematopoietic cells. MicroRNAs (miRs) are post-transcriptional regulators of gene expression, and c-kit has been described as a target of miRs-221 and -222 in erythropoiesis. In the present study, we identified c-kit as a target of PLZF in normal and leukemic cells. Particularly, in erythropoietic (E) culture of CD34(+) progenitors, PLZF is downregulated, whereas c-kit expression at both the mRNA and protein levels inversely increases during the first days of E differentiation. In functional experiments, PLZF transfection induces c-kit downregulation, inhibits E proliferation and delays differentiation, whereas PLZF knockdown induces opposite effects, independently of miRs-221 and -222 expression. The inverse correlation between PLZF and c-kit expression was found in normal CD34(+)38(+/-) hematopoietic progenitor/stem cells and in acute myeloid leukemias of M0/M1 French-American-British subtypes, suggesting that the control of PLZF on c-kit expression may be crucial at the level of the stem cell/progenitor compartment. Altogether, our data indicate a new mechanism of regulation of c-kit expression that involves a transcriptional control by PLZF in CD34(+) cells and early erythropoiesis.

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.

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.

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.

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.

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.

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.

Alterations of the FLT3 gene in acute promyelocytic leukemia: association with diagnostic characteristics and analysis of clinical outcome in patients treated with the Italian AIDA protocol.

Alterations in the FLT3 gene, including internal tandem duplications (ITDs) and D835 mutations occur frequently in acute myelogenous leukemia. We investigated the prevalence and clinico-biological correlations of FLT3 ITDs and D835 mutations in 90 patients with acute promyelocytic leukemia (APL) receiving the AIDA protocol. Twenty patients in which both presentation and relapse material was available were analyzed sequentially. Thirty-three patients (37%) harbored the ITD, and seven (7.7%) the D835 mutation in blasts obtained at diagnosis. Presence of ITDs was strongly associated with high WBC count (P = 0.0001), M3 variant (P = 0.0004), and the short (BCR3) PML/RARalpha isoform (P = 0.003). There was no difference in response to induction in the two ITD+ve and ITD-ve groups, while a trend towards inferior outcome was observed for ITD+ve cases when analyzing disease-free survival (DFS) and relapse risk (RR). These differences, however, did not reach statistical significance. Sequential studies showed variable patterns in diagnostic and relapse material, ie ITD (-ve/-ve, +ve/+ve, +ve/-ve, -ve/+ve) and D835 (-ve/-ve, +ve/-ve, -ve/+ve). Our results indicate that FLT3 alterations are associated in APL with more aggressive clinical features and suggest that these lesions may not play a major role in leukemia progression.

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.

Ear involvement in acute promyelocytic leukemia at relapse: a disease-associated 'sanctuary'?

Extramedullary (EM) involvement occurs infrequently in acute promyelocytic leukemia (APL) and usually involves skin and CNS. We describe seven patients (four observed at a single institution) who relapsed in various sites of the auditory apparatus, including the external canal and middle ear (temporal bone). Front-line treatment included ATRA and chemotherapy (six patients) or chemotherapy alone (one patient). Three patients had concomitant hematologic relapse, two had molecular relapse and two were in hematologic and molecular remission when ear localization was documented. Local symptoms that stimulated further diagnostic studies included ear bleeding/discharge in the first patient, but were mild in the others (hypoacusia, five patients; earache, two patients). Ear involvement by leukemia was documented by histological and/or molecular studies after local surgery in five cases, and by CT scan or NMR in the remaining patients. We suggest that the ear might represent a specific sanctuary for disease involvement in APL.