Events In Acute Promyelocytic Leukemia Research Articles

Biochemical risk factors and outcomes of acute promyelocytic leukemia patients with thrombotic events: a matched pair analysis.

Acute promyelocytic leukemia (APL) stands out as a distinctive form of acute leukemia, exhibiting a higher occurrence of thrombotic events when contrasted with other leukemia subtypes. Since thrombosis is a relatively rare but unfavorable condition with poor prognostic implications, it is crucial to determine the risk factors for thrombotic events in APL(thrombosis in large venous or arterial from onset to differentiation therapy in 30d). We performed a retrospective study involving 950 APL patients between January 2000 and October 2022, from which 123 were excluded by younger than 16years of age, 95 were excluded by incomplete data, and 6 were excluded by thrombosis related to CVC or PICC. A total of 23 APL patients with thrombosis for inclusion in our analysis were performed a 1:5 ratio matching based on sex (perfect match) and age (within 5years) to patients without thrombosis. These patients were continuously monitored in the outpatient department over a period of 5years. We meticulously examined clinical and laboratory data to pinpoint the risk factors related to thrombotic events in APL. Our primary clinical endpoints were all-cause mortality and achieving complete remission, while secondary clinical outcomes included APL relapse. Thrombotic events were observed in 2.4% (23/950) of APL patients. Compared to patients without thrombosis, patients with thrombosis had higher lactate dehydrogenase (LDH) [313 (223, 486) vs. 233 (188, 367) U/L, p = 0.020], higher indirect bilirubin [11.2 (7.4, 18.6) vs.8.3 (6.0, 10.7) umol/L, p = 0.004], higher creatinine [72 (62, 85) vs. 63 (54, 74) umol/L, p = 0.026], higher CD2 expression (65.2 vs. 15.2%, p < 0.001), higher CD15 expression (60.9 vs. 24.3%, p = 0.001), and PML/RARαisoforms (p < 0.001). Multivariate-logistic-regression analysis revealed several factors that were markedly related to thrombosis, including LDH (OR≈1.003, CIs≈1.000-1.006, p = 0.021), indirect bilirubin (OR≈1.084, CIs≈1.000-1.188, p = 0.043), CD2 expression positive (OR≈16.629, CIs≈4.001-62.832, p < 0.001), and CD15 expression positive (OR≈7.747, CIs≈2.005-29.941, p = 0.003). The S-type (OR≈0.012, CIs≈0.000-0.310, p = 0.008) and L-type (OR≈0.033, CIs≈0.002-0.609, p = 0.022) PML/RARα isoforms were negatively associated with thrombosis. Kaplan-Meier curves indicated that the survival rates were remarkably varied between APL patients with and without thrombosis (HR:21.34, p < 0.001). LDH and indirect bilirubin are variables significantly associated with thrombosis in APL, S-type and L-type PML/RARαisoforms exhibit a negative association with thrombotic events. The thrombotic events of APL can predict the subsequent survival of thrombosis. The findings of our study have the potential to facilitate early detection of thrombosis and enhance the prognosis for individuals with APL who develop thrombosis. Further validation of our findings will be essential through future prospective or multicenter studies.

Partner Gene Associated Trinary Fusion and Orchestration of Multiple Molecular Events in Acute Promyelocytic Leukemia with Variant Rara Fusion Genes

Background All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases manifest as APL. So far, 19 RARA fusion partner genes have been reported, with all reported as 5′ partners ( X::RARA). Intriguingly, a significant proportion of X::RARA cases were resistant to ATRA, and mechanisms are yet to be elucidated. Here we report the finding of a novel type of trinary fusion and the orchestrating of multiple molecular events that confer leukemogenesis and ATRA resistance in X::RARA-APL. Methods A total of 23 cases (Fig. 1A) with RNA-seq and WGS data from 10 centers were enrolled. We used Arriba for routine FG calling and manually investigated the nonconventional fusion sequence. RT-PCR, Sanger sequencing, and optical genome mapping (OGM) were used to validate the FGs and to determine whether the RARA 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 17 males and 6 females, aged 3-66 (median 32) years. Morphological and immunophenotype analyses all showed features of APL. We first identified the STAT3::RARA fusion event in an ATRA-resistant case (the index case, #A1). Moreover, we noticed another fusion event of truncated RARA exon 9 and a 3′ partner sequence that did not map to any annotated protein-coding genes. Further analysis determined it was the RC sequence of the transposable element (TE) LTR40a with the annotated loci at 17p11.2. Both OGM (at the genome level) and RT-PCR (Fig. 1A) confirmed that the two RARA fusion events were in the same cistron. Thus, the fusion transcript being STAT3::RARA::LTR40a-RC spliced in tandem, but not separate transcripts of STAT3::RARA and RARA::LTR40a-RC. We named this novel form of tandem splicingtrinary fusion. We, therefore, recruit X::RARA-APL cases to investigate the recurrence of RARA 3′ fusion. We identified 9 RARA 5′ partner genes in 23 cases (Fig. 1B), with TTMV -ORF2, STAT5B, STAT3, IRF2BP1, THRAP3, and TNRC18 in multiple cases. Ten (43.5%) cases also harbor RARA 3′ fusion partners. The partner of case #A1_8 were TEs, and the partner of case #A9_10 was the same gene as their 5′ one. Thirteen cases did not harbor 3′ fusions. Notably, both TNRC18::RARA cases (#15_16) carry mutations on their RARA ligand binding domain (LBD) at the primary onset. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARA 3' fusions confer a poly_A signal sequence, which was essential for a mRNA. In-depth analysis indicated that TEs participate in forming RARA-FGs through a transposition mechanism rather than a translocation mechanism. The molecular events addition to RARA 5′ splicing showed a 5′ partner associated pattern: all of the 4 STAT3::RARA and 4 STAT5B::RARA cases harbor TEs as their 3′ partner, both TNRC18::RARA cases carry LBD mutation, while all 7 TTMV -ORF2::RARA cases did not harbor 3′ splicing or LBD mutation. Remarkably, although the RARA 3' splicing sites varied, they all resulted in partial or complete exon 9 truncation. RARA exon 9 encodes helix 11_12 (H11_12) of its LBD, which is pivotal in allosteric response to ATRA. The RARA trinary fusion will result in LBD-H12 or H11_12 castration due to the 3' splicing, then lead to losing responsiveness to ATRA via an allosteric disability mechanism (Fig. 1C, 1D). Experiments confirmed that cells transfected with STAT3::RARA fusion with intact LBD responded well to ATRA, while both the fusion in case #A1 and artificial fusion with LBD-H12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1E). Conclusions This is the first report of pathological trinary fusion, the TEs and transposition mechanism participating in composing pathological fusion genes, and the RARA LBD castration confers ATRA resistance in X::RARA-APL. Also, there might be another synergistic mechanism of RARA 5' fusion and LBD mutation. The formation of trinary fusion and gain of LBD mutation, in addition to X::RARA fusion, requires more genome splicing or transposition or mutation events, which explains the rarity of X::RARA-APL. This study highlights mechanisms of partner gene-associated trinary fusion and the orchestrating of multiple molecular events in the leukemogenesis in X::RARA-APL cases.

Acute Promyelocytic Leukemia (APL): A Review of the Classic and Emerging Target Therapies towards Molecular Heterogeneity

The occurrence of severe bleeding syndrome because of the PML-RARα fusion protein is a life-threatening event in APL. This protein destabilizes homeostasis, maturation, remodeling, and tissue regeneration in addition to hampering the maintenance and differentiation of hematopoietic cells into different lineages, fixing cells in the promyelocyte stage. APL is a classic example of how effective targeted therapy is and, therefore, how important the use of such therapy is to the overall survival of patients, which in this case is represented by the use of ATRA/ATO. Despite that, about 10% of cases of APL patients demonstrate resistance to treatment. Facing this scenario, we point out promising target therapies such as those recommended by the NCCN and Leukemia Net. Since this is such a heterogeneous molecular disease, it is of great importance to understand how important combined chemotherapy, target therapy, immune-based therapy, and combined therapies are in the survival of these APL patients.

Early Intracranial Hemorrhages in Acute Promyelocytic Leukemia: Analysis of Neuroradiological and Clinico-Biological Parameters

Introduction In the last decades, survival outcomes tremendously improved in acute promyelocytic leukemia (APL) due to all‐trans retinoic acid (ATRA)/anthracycline‐based chemotherapy, and more recently ATRA/arsenic trioxide (ATO) combinations. Despite the excellent results, with response rates exceeding 90%, early death (ED), mainly due to hemorrhagic complications occurring during the first 30 days after diagnosis, remains an unsolved issue. The prevalence of ED is largely underestimated due to discrepancies between data deriving from clinical trials, reporting 3-10% rates, and real-life, where it ranges from 10 to 30% of cases, and still remains the dominant cause of poor outcome. The most common site of bleeding is the brain (65%), particularly in fatal cases. Many theories have been proposed to elucidate the pathophysiology of this complication, considering the coagulopathy of APL and the perturbed homeostasis as primum movens, but no specific neuroradiologic studies have been conducted so far. Moreover, there is no specific predictor for bleeding events in APL, with exception of increased white blood cell counts (WBC). Patient and Methods We retrospectively identified 38 patients with APL (23 males and 15 females with a median age of 51.83 years, range 11-82), consecutively diagnosed at our Institution between 2004 and 2019, and treated with standard ATRA-based induction (Table 1). Occurrence of ED and ICH was then correlated with clinico-biological parameters, selecting for commonly reported variables predictors of these complications. In addition, CT scans of the 5 patients who experienced an intracranial hemorrhage (ICH) were revised by an expert neuroradiologist, looking for radiographic predictors of poor outcome (Al-Mufti et al 2018). Results Approximately 13% (5/38) of our APL patients experienced an ICH during induction therapy, which was fatal in 3 cases, while the overall incidence of ED was about 10% (4/38, including 1 case of death due to a differentiation syndrome). Looking at clinico-biological variables, the only predictor of ICH was a lower albumin level at baseline (3.8 vs 4.2 gr/dl; p=0.022), as compared to patients who did not experience ICH. A high Sanz-risk score was present in 2 of 3 pts with a fatal outcome of ICH (67% of cases), together with a shorter activated partial thromboplastin time (aPTT, 0.75 vs 0.86 ratio)(Table 1). Looking at neuroradiological findings (Figure 1) the three fatal cases showed a wider ICH volume, with perilesional edema and, interestingly, a positive "swirl" sign, which is defined as a marker of ongoing extravasation of blood within a hematoma. Both cases with a favourable outcome had a cerebellar involvement, without perilesional edema, and no major involvement of basal ganglia or thalamus. None of the patients had radiological signs of arteriovenous malformations. Conclusions Our data remark the impact of lower albumin levels at baseline and of a high Sanz-risk category on ED in patients with APL. In APL cases with fatal ICH, the thrombo-hemorrhagic imbalance is more profound than in other AMLs and results in a more devastating clinical picture. Taking into account the limitations of the small sample size, we found that a wide hemorrhage volume with perilesional edema, and the presence of the "swirl" sign are characteristic of fatal cases, while arteriovenous malformations did not seem to play a predisposing role. Further studies including large series of patients are warranted to identify the characteristics and the possible strategies to ameliorate the outcome of ICH in APL. Disclosures Venditti: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees.

PB1774 ACUTE PROMYELOCYTIC LEUKEMIA PRESENTING AS VERTEBROBASILAR ISCHEMIC STROKE IN A YOUNG WOMAN

Background:Acute Promyelocytic Leukemia (APL) carries a high risk of early death due to coagulopathy triggered by the leukemic cells, which initiate a cascade of disseminated intravascular coagulation and hyperfibrinolysis. Patients are at risk for hemorrhagic and thrombotic events, but bleeding dominates the clinical picture and is responsible for most of the early deaths.Arterial thrombosis is rare in APL, and its mortality is extremely high. The best management strategy when occurring in APL is unknown, since anticoagulants or fibrinolytics may aggravate the CID but without them the thrombosis may be fatal.Aims:We present a rare case of APL presenting as a vertebrobasilar ischemic stroke, which evolved favorably with treatment of the subjacent leukemia alone, without use of anticoagulation or antiaggregation.Methods:Case report.Results:A 36‐year old previously healthy woman presented with sudden onset aphasia and right‐sided hemiparesis with central facial paralysis, vertical gaze palsy and loss of horizontal gaze to the left.Blood work showed anemia (Hb 6.6 g/dL), a white blood cell (WBC) count of 7900x109/L of which 62% were atypical promyelocytes, and 19,000 x109/L platelets (Figure 1‐A). A concomitant disseminated intravascular coagulation was diagnosed based on thrombocytopenia, hypofibrinogenemia (100 mg/dL), prolonged prothrombin time (&gt; seconds) and elevated D‐dimers (10,98 μg/mL). These findings raised the suspicion of Acute Promyelocytic Leukemia, an entity usually associated with bleeding but also rarely with thrombosis.Emergent head CT and angio‐CT scan showed no lesions. Based on the neurological findings, a presumptive diagnosis of vertebrobasilar ischemic stroke was established, and thrombolysis with alteplase was indicated. However, the risks of aggravating the bleeding predisposition of the patient and precipitating hemorrhagic transformation of the stroke outweighed the benefits, and both alteplase and anticoagulants were withheld.Instead, management focused on treating the leukemia. Oral all‐trans retinoic acid was immediately initiated, and idarrubicin was added after confirming major infiltration of the bone marrow with atypical promyelocytes. Surprisingly, the hallmark translocation t(15;17) was not detected by fluorescence in‐situ hybridization (FISH), but the fusion transcript PML‐RARα was identified by reverse transcription with polymerase chain reaction (RT‐PCR), thus confirming a diagnosis of cryptic APL.A magnetic resonance of the head performed 24 hours later showed hyperintense lesions in the left thalamus, caudate and midbrain, and right parietal operculum (Figure 1).The patient achieved hematologic complete remission after induction therapy and recovered to a mild right hemiplegia after intensive physical rehabilitation. She is currently in molecular complete remission and is undergoing maintenance treatment in the outpatient setting, medicated with dipyridamole as secondary stroke prevention.Summary/Conclusion:The management of thrombotic events in APL is a challenge, since the increased bleeding risk associated with anticoagulation must be weighed against the potential lethality of the thrombosis if it is left untreated. Central nervous system arterial thrombosis has a particularly poor prognosis.imageWe present a case that illustrates the importance of early APL treatment with ATRA and supportive measures in reverting the coagulopathy of the disease.

An integrative approach reveals genetic complexity and epigenetic perturbation in acute promyelocytic leukemia: a single institution experience

Acute promyelocytic leukemia (APL) is a distinct type of acute myeloid leukemia that is defined by the presence of the translocations that mostly involve the RARA gene. The most frequent translocation is the t(15;17), which fuses the RARA gene with the PML gene. Previous studies have shown that other cooperative mutations are required for the development of APL after the initiating event of the t(15;17). In this study, we combined cytogenetics with next-generation sequencing and single-nucleotide polymorphism array to study the genetic complexity in 20 APL cases diagnosed in our institution. All but 3 cases had additional genetic aberrations. Our study demonstrated that somatic mutations are frequent events in APL. In addition to the previously reported recurrent cooperative mutations in the FLT3, WT1, and RAS genes, we identified mutations in several epigenetic modifiers, including TET2, EZH2, and DNMT3A, co-occurring with either FLT3 or WT1 mutations. Mutations of the WT1 gene and chromosome 11p copy neutral loss of heterozygosity affecting WT1 are present in a third of the cases in our series. Two-thirds of APL cases in our study demonstrated a global reduction but focal accumulation of H3K27 methylase (H3K27me) expression, indicating a disorganized chromatin methylation pattern with generally more accessible chromatin status. Our study confirmed genetic complexity of APL and revealed that epigenetic aberrations are more common than previously expected. Although epigenetic modulation is not a common treatment strategy in APL, targeting this pathway may have some clinical utility in refractory or relapsed APL cases.

Progressive Hyperleukocytosis during Initial Therapy Is a Predictive Marker for Differentiation Syndrome and Early Mortality in Adult Patients with Acute Promyelocytic Leukemia

Background: Acute promyelocytic leukemia (APL) is classified into a favorable-risk group and long-term overall survival (OS) is estimated at around 80%. Relapse rate of APL is lower than another acute myeloid leukemia (AML) subtypes, but we confront higher incidence of early deaths caused by fatal complications including bleeding events and differentiation syndromes (DS) during initial therapy. Recently, although arsenic trioxide (ATO) is introduced with a better survival outcome, the results were from data of low to intermediate-risk group. Thus, patients in high-risk group still show poor survival outcome with high probability of early complications and deaths. We calculated the incidence of DS and early deaths, and tried to find out affecting factors for those early events.Methods: In this single center retrospective study, 259 APL patients (median 42 years old (16-72), follow-up was 65.4 months (11.1 - 170.5) from 2002 to 2014 were analyzed. APL was diagnosed by RT-PCR method for detection of PML-RARa and all patients were available with cytogenetic results. All except 5 patients with normal karyotype was identified with t(15;17)(q22;q21) and 77 showed combination of additional karyotypes. All patients were supported with sufficient transfusion and received ATRA. Our treatment protocol was based on the modified AIDA protocol using ATRA and idarubicin monotherapy (Sanz et al. Blood. 1999; 94: 3015-21) but some patients with comorbidity were treated with ATO, low-dose cytarabine, and ATRA alone for remission induction. For hyperleukocytosis, we conducted leukapheresis when leukocyte counts exceeded 50 (x109/L) and some were treated with hydroxyurea, cytarabine and prophylactic dexamethasone. High-risk group was determined according to the Sanz criteria which presented leukocyte count > 10 (x109/L) at diagnosis. For leukocyte count, we checked diagnostic level (WBCdx) and the maximal level (WBCmax) during initial therapy and identified a group which showed a meaningful increment of WBCmax compared to WBCdx.Results: ATRA was applied in 258 patients and 217 (84.1%) were treated with idarubicin, 13 (5.0%) were with ATO, 3 (1.2%) were with low-dose cytarabine. Eight-week cumulative incidence of early death and DS was 13.5% and 17.8%, and hematological CR was identified in 222 (86.0%) patients. Five-year OS and EFS was 76.8% and 69.8%, and CIR rate was 15.7%. Six patients showed clonal evolution to therapy-related AML and 3 patients died in CR. FLT3-TKD and FLT3-ITD mutation was identified in 12 (7.3%) and 34 (20.7%) patients, and PML-RARa BCR3 and BCR1 subtype was identified in 70 (36.8%) and 120 (63.2%) patients, respectively. For leukocyte counts, except for WBCdx higher than 43 (x109/L), which showed significantly higher rate of early death and DS, patient groups with WBCdx <10 (x109/L) vs. 10 to 43 (x109/L) showed no differences regarding early death or DS. We identified that the significance of WBCdx has been changed with increment during initial therapy which revealed WBCmax was more influential. Among the patients with WBCdx <43 (x109/L), WBCmax increased higher than 43 (x109/L) was related with higher incidence of early death (35.5%) and DS (30.6%), while more DS (40%) was identified in patients with higher increment ratio from WBDdx <10 (x109/L). Multivariate analysis revealed WBCmax > 43 (x109/L) and low antithrombin III were significant for DS, while old age, WBCmax, and high D-dimer were associated with early death. In our data, dexamethasone prophylaxis did not show a preventive effect for DS or early death, while leukapheresis in patients with WBCmax >43 (x109/L) showed marginally decreased early death rate ‘resulting superior OS without significant bleeding complications.Conclusion: Our data revealed WBCmax with higher increment ratio was a significant predictive factor for early death and DS compared to WBCdx even in the low Sanz-risk group. The role of dexamethasone, transfusion support including antithrombin III, leukapheresis or cytoreduction should be evaluated in the specific patient subset for reducing early events in APL. DisclosuresKim:ILYANG: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Lee:Alexion Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

PML-Rara/Nrf2-Regulated Mir-125b Targets CEBPA and Influences Acute Promyelocytic Leukemia Cell Proliferation

Acute promyelocytic leukemia (APL) is characterized by the presence of PML-RARA fusion protein. The PML-RARA oncoprotein is known to be the initiating factor for APL development. We have previously found that miR-125b is highly expressed in APL and upregulated by PML-RARA. However, the molecular mechanisms by which PML-RARA controls miR-125b expression had been uncharacterized. In the present study, we further confirmed that expression of PML-RARA resulted in a significant increase in primary miR-125b expression, and we found that the nuclear transcription factor E2-related factor 2 (Nrf2) targeting genes (HO1) was also upregulated (Figure 1A). Consistently, HO1 and miR-125b are transcriptionally upregulated by proteasome inhibitor (MG132) which has been showed to activate Nrf2 (Figure 1B). Knockdown of Nrf2 can inhibit the effect of PML-RARA expression and MG132 (Figure 1C). Thus, these results indicated that Nrf2 may mediate PML-RARA-induced miR-125b expression. Furthermore, we demonstrated the importance of miR-125b in APL cell proliferation (Figure 1D), we showed that promoting leukemic cell grow is associated with AKT pathway and MAPK signaling (Figure 1E). Subsequent bioinformatic and RNA-seq data analysis (Figure 1F and G) identified CEBPA as a putative target of miR-125b, and we further experimentally verified using luciferase reporter constructs (Figure H). In conclusion, we identified the mechanisms responsible for the high expression and the function of miR-125b in APL, suggesting that the expression of miR-125b could be a primary oncogenic event in APL leukemogenesis.This study was supported by grants from the National Natural Science Foundation of China (No.81400102), the China Postdoctoral Science Foundation (No.2015M570751), and the Medical Scientific Research Foundation of Guangdong Province, China (A2015420). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thrombotic events in acute promyelocytic leukemia

IntroductionThrombotic events (TE) appear to be more common in acute promyelocytic leukemia (APL) than in other acute leukemias, with reported prevalence ranging from 2 to10-15%. Materials and MethodsWe retrospectively analyzed the data on TE appearance in 63 APL patients. ResultsTE occured in 13 (20.6%) cases, four arterial (6.3%) and nine venous (14.3%). TE were more frequently diagnosed after initiation of weekly D-dimer monitoring (7 TE during 20months vs 6 during 76months, P=0.032). Patients with and without venous thrombosis were significantly different regarding female/male ratio (P=0.046), PT (P=0.022), aPTT (P=0.044), ISTH DIC score (P=0.001), bcr3 (P=0.02) and FLT3-ITD (P=0.028) mutation. The most significant risk factor for venous TE occurrence in multivariate analysis was FLT3-ITD mutation (P=0.034). PAI-1 4G/4G polymorphism was five times more frequent in patients with venous TE than without it (P=0.05). Regarding risk factors for arterial TE we failed to identify any. ConclusionsWe have demonstrated that APL-related TE rate is higher than previously reported and that weekly D-dimer monitoring might help to identify patients with silent thrombosis. Moreover, our study suggests a possible relationship between venous TE occurrence and several laboratory findings (PT, aPTT, ISTH DIC score, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G). Prophylactic use of heparin might be considered in patients with ISTH DIC score<5, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G.

Cerebral venous and sinus thrombosis in a patient with acute promyelocytic leukemia during all-trans retinoic acid induction treatment

Cerebral venous and sinus thrombosis is a rare cerebrovascular disorder, which seldom represents a complication of acute promyelocytic leukemia (APL). As a part of the coagulopathy of APL, thrombosis is a less recognized and underestimated life-threatening manifestation and is overshadowed by the more obvious bleeding complications. Here, we described a 28-year-old woman with APL who developed massive thrombosis of the cerebral sinuses while on induction treatment with all-trans retinoic acid. On the basis of this report, the potential pathogenic mechanisms and the diagnosis based on magnetic resonance imaging (MRI) combined with magnetic resonance venogram (MRV) are discussed. Early anticoagulant therapy contributed to the progressive dissolution of the thrombosis, as documented by MRI, with the complete disappearance of neurological signs without sequelae. Given the increasing recognition of thromboembolic events in APL, the use of prophylactic anticoagulation during induction therapy may need to be redefined.

The limited contribution of DNA methylation to PML-RARα induced leukemia

The limited contribution of DNA methylation to PML-RARα induced leukemia

DNA methylation changes are a late event in acute promyelocytic leukemia and coincide with loss of transcription factor binding

AbstractThe origin of aberrant DNA methylation in cancer remains largely unknown. In the present study, we elucidated the DNA methylome in primary acute promyelocytic leukemia (APL) and the role of promyelocytic leukemia–retinoic acid receptor α (PML-RARα) in establishing these patterns. Cells from APL patients showed increased genome-wide DNA methylation with higher variability than healthy CD34+ cells, promyelocytes, and remission BM cells. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score, and Flt3-mutation status characterized methylation subtypes. Transcription factor–binding sites (eg, the c-myc–binding sites) were associated with low methylation. However, SUZ12- and REST-binding sites identified in embryonic stem cells were preferentially DNA hypermethylated in APL cells. Unexpectedly, PML-RARα–binding sites were also protected from aberrant DNA methylation in APL cells. Consistent with this, myeloid cells from preleukemic PML-RARα knock-in mice did not show altered DNA methylation and the expression of PML-RARα in hematopoietic progenitor cells prevented differentiation without affecting DNA methylation. Treatment of APL blasts with all-trans retinoic acid also did not result in immediate DNA methylation changes. The results of the present study suggest that aberrant DNA methylation is associated with leukemia phenotype but is not required for PML-RARα–mediated initiation of leukemogenesis.

Evidence of functional interaction between NuMA-RARα and RXRα in an in vivo model of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by reciprocal balanced chromosomal translocations involving retinoic acid receptor-alpha (RARalpha). RARalpha heterodimerizes with the retinoid X receptor-alpha (RXRalpha) and transcriptionally regulates myeloid differentiation in response to ATRA (all-trans retinoic acid). Several lines of evidence suggest that APL fusion proteins interact with RXRalpha. To elucidate the role of RXRalpha in APL, we conditionally knocked out RXRalpha in the hCG-NuMA-RARalpha APL mouse model. Phenotype analysis of NuMA-RARalpha+ mice demonstrated that these mice developed a myeloproliferative disease-like myeloid leukemia within 4 months of birth. While hemizygous and homozygous RXRalpha conditional knockout mice were phenotypically normal as late as 12 months of age, we observed that the leukemic phenotype in NuMA-RARalpha+ mice was dependent on the presence of functional RXRalpha. Bone marrow promyelocyte counts were significantly reduced in NuMA-RARalpha+ mice with RXRalpha knocked down. Significant differences in the accumulations of Gr-1+ and Mac-1+ cells were also seen. We further observed that genes previously identified to be cooperating events in APL were also regulated in an RXRalpha-dependent manner. We therefore propose that the APL fusion protein NuMA-RARalpha cooperates with RXRalpha in the development of leukemia in hCG-NuMA-RARalpha transgenic mice and suggest a novel role for RXRalpha in the pathogenesis of APL.

Comparative Analysis of Downstream Genetic Targets of the Variant Acute Promyelocytic Leukemia Fusion Proteins NPM-RARA and NuMA-RARA.

Acute promyelocytic leukemia (APL) is characterized by accumulation of abnormal promyelocytes in the bone marrow and peripheral blood, and sensitivity to treatment with all-trans retinoic acid. APL cases have a balanced chromosomal translocation involving retinoic acid receptor alpha (RARA) on chromosome 17. The resulting fusion proteins (X-RARA) are aberrant transcription factors and block ATRA-induced neutrophil differentiation. Loss of RARA signalling impairs granulopoiesis, but is not sufficient to cause a leukemic phenotype. We elucidated the identities of additional signalling pathways, which can potentially cooperate with X-RARA in APL, that are commonly modulated by multiple X-RARA. We used the U-937 hematopoetic cell line retrovirally transduced with NPM-RARA (Kamel-Reid et al, 2003), and NuMA-RARA, in addition to NB4 cells (expressing PML-RARA) to determine the common genes and pathways deregulated in APL. Gene expression analysis was carried out on RNA harvested in triplicate from control and X-RARA expressing cell lines, using the Affymetrix U133Plus2 array platform. Gene expression and pathways analysis of array data was carried out using a suite of analysis tools. Array data were validated in an independent sample set by real-time quantitative PCR. We observed a total of 311 genes deregulated at least 2-fold by NuMA-RARA (192 up-regulated, 119 down-regulated), 393 genes deregulated by NPM-RARA (292 up-regulated, 101 down-regulated), and 2056 genes deregulated by PML-RARA (1097 up-regulated, 959 down-regulated). A total of 65 genes, in 5 major interaction networks, were commonly deregulated by all three X-RARA (42/65 up-regulated, 23/65 down-regulated). The majority of these genes are involved in cellular signalling (14 genes, p-value 1.57E-07–7.77E-3), transcription (13 genes, p-value 5.68E-7–3.91E-3), cell proliferation (25 genes, p-value 9.73E-7–7.77E-3), apoptosis (26 genes, p-value 9.96E-7–7.71E-3), and cell movement (17 genes, p-value 1.43E-6–7.60E-3). Genes involved in the CEBPA interaction network (GFI1, TRIB2, ELA2), as well as other genes that we anticipated to be deregulated in APL including ID1, MMP9, and JUN were found through this analysis. NF-kB (p-value 2.10E-3), AHR (p-value 2.12E-3), IL-6 (p-value 5.40E-3), and G-protein coupled receptor (p-value 7.45E-3) signalling were among the top canonical pathways determined to be altered by X-RARA. Over-expression of a number of NF-kB downstream transcriptional targets, including VEGF, IL8, MMP9, cIAP2, and TNFAIP3, were also observed in multiple X-RARA expressing cell lines. In addition, in vitro results were compared to NuMA-RARA gene targets identified in primary bone marrow cultures derived from the hCG-NuMA-RARA transgenic mouse model (Sukhai et al, 2004). We observed that pathways involved in cell signalling, cell death, gene expression, proliferation, and cell cycle were significantly deregulated in both mouse and human datasets, indicating that these pathways may be important cooperating events in APL. Our data represent the first comparison of the genetic profiles of the variant fusion proteins NPM-RARA and NuMA-RARA in a haematopoietic cell system. Our studies are a significant step in identifying key targets that cooperate with X-RARA in the development of APL.

Pattern of extramedullary relapse in latin patients with acute promyelocytic leukemia (APL) treated with all-trans-retinoic acid (ATRA) and chemotherapy

17520 Background: Extramedullary relapse (EMR) is a rare event in APL and reports have suggested a possible rol of ATRA in its pathogenesis. Our study intent to determine the pattern of EMR in Latin patients treated whit trans-retinoic acid (ATRA) and chemotherapy (CT). Methods: We evaluated 87 patients diagnosed of APL from January 2001 to December 2005. They were treated with ATRA and chemotherapy. Results: 74/87 received ATRA plus chemotherapy. Median age was 28 (5–68). 39/74 (53%) patients were female. Subtype M3v constituted 21/74 (28%). 76% presented disseminated intravascular coagulation (DIC). Extramedullary deposit was observed in 7 cases, more frequent was central nervous system (CNS). According the Sanz Index, 32/74 (32%) were High Risk, 40/74 (54%) Intermediate Risk, 2 Low Risk. 31/55 (56%) presented t(15,17). 52/74 (70%) patients had complete response. EMR was documented in 8 (15%) (CNS: 5, External Auditory Foramen: 3), 4 were High Risk. Median time to EMR was 11.5 months (7.03–27.3). 55% are alive at date. The following median was 36 months, 41 months of Disease Free Survival (DFS), rate was 62 % to 3 years. Overall survival was 44 months; rate was 83% to 3 years. Conclusions: EMR incidence was 8%, higher report that other series, this event was presented in CNS and external auditory foramen. It suggest to establish EMR risk factors and to include CNS prophylactic treatment. No significant financial relationships to disclose.

Occurrence of thrombotic events in acute promyelocytic leukemia correlates with consistent immunophenotypic and molecular features

Although the occurrence of thrombosis in acute promyelocytic leukemia (APL) has been reported during retinoic acid treatment, no studies carried out in large clinical cohorts have specifically addressed this issue. We analyzed 124 APL patients treated with the all-trans retinoic acid and idarubicin protocol and compared clinico-biologic characteristics of 11 patients who developed thrombosis with those of 113 patients who had no thrombosis. In seven patients, the events were recorded during induction, whereas in four patients deep vein thrombosis occurred in the post-induction phase. Comparison of clinico-biological characteristics of patients with and without thrombosis revealed in the former group higher median white blood cell (WBC) count (17 x 10(9)/l, range 1.2-56, P=0.002), prevalence of the bcr3 transcript type (72 vs 48%, P=0.01), of FLT3-ITD (64 vs 28%, P=0.02), CD2 (P=0.0001) and CD15 (P=0.01) expression. No correlation was found with sex, age, French-American-British subtype, all-trans-retinoic acid syndrome or with thrombophilic state that was investigated in 5/11 patients. Our findings suggest that, in APL patients consistent biologic features of leukemia cells may predict increased risk of developing thrombosis.

Cytogenetics, FISH and RT-PCR Analysis of Acute Promyelocytic Leukemia: Structure of the Fusion Point in a Case Lacking Classic t(15;17) Translocation

The chimeric gene product PML-RAR α, the result of a reciprocal t(15;17) translocation, plays an important role in the pathogenesis of acute promyelocytic leukemia (APL). In the present study on clinical effects of cytogenetics and molecular events in APL, we performed chromosome analysis, fluorescence in situ hybridization (FISH) using gene-specific probe, and reverse transcription-polymerase chain reaction (RT-PCR) analysis in 10 patients with APL. Patients were treated with all- trans retinoic acid (ATRA) and/or chemotherapy, and all achieved complete remission. Cytogenetic analysis revealed the classic translocation t(15;17) in nine of 10 patients, and a remaining patient had an apparently normal karyotype. Interphase FISH was performed in nine patients, and revealed the presence of PML-RAR α fusion gene in all patients. RT-PCR analysis in 10 patients showed that eight expressed long (L)-form type, one expressed a short (S)-form type, and the other expressed a variable (V)-form type. Metaphase FISH of the patient with normal karyotype revealed a juxtaposed PML-RAR α fusion signal on one chromosome 17 homologue, an RAR α signal on the other chromosome 17 homologue, and one PML signal on each chromosome 15 homologue. Moreover, V-form of the PML-RAR α transcript was detected, and a portion of RAR α intron 2 was found inserted in the breakpoint region. ATRA differentiation induction therapy was effective in treating this patient, a result infrequently reported in cytogenetic and molecular investigations of APL.

Cryptic translocation of PML/RARA on 17q. A rare event in acute promyelocytic leukemia

Cryptic translocations in acute promyelocytic leukemia are rare. Usually the gene fusion PML/RARA is located on chromosome 15. Combined cytogenetic, fluorescence in situ hybridization (FISH), and molecular (polymerase chain reaction [PCR]) analysis were employed for the diagnosis and precise localization of the fusion gene. Conventional cytogenetics showed a normal karyotype; PCR showed a typical PML/RARA rearrangement in exon 1. FISH analysis revealed that a submicroscopic part of chromosome 15 had been inserted into 17q. This case adds further information on alternative ways of rearrangement of the PML/RARA genes, possibly correlated with all-trans retinoic acid resistance.

Retinoic Acid Receptor α (RARα) Mutations in Human Leukemia

The retinoic acid receptor α (RARα) plays a central role in the biology of the myeloid cellular compartment. Chromosomal translocations involving the RARα locus probably represent the malignant initiating events in acute promyelocytic leukemia (APL). Recent studies that identify novel interactions between RARα and the nuclear receptor co-activators and co-repressors. new functions of the oncogenic RARα fusion proteins and their catabolism in retinoic acid-induced differentiation, and the availability of new transgenic mice models have provided important insights into our understanding of the mechanisms by which mutant forms of RARα can be implicated in the development of leukemia. Novel alterations of the RARα gene identified in hematopoietic malignant disorders other than APL, such as myelody-splastic syndromes, non-APL acute myeloid leukemias and B-chronic lymphocytic leukemias, suggest that disruption of the RARα gene might predispose to myeloid and lymphoid disorders.

Identification of complex t(15;17) in APL by FISH

Fluorescence in situ hybridization (FISH) provides a sensitive and effective approach in identifying the RAR-α/PML fusion event in acute promyelocytic leukemia (APL) with the t(15;17). In the present study we describe the use of this assay for the identification of the RAR-α/PML fusion in bone marrow (BM) cells from three APL patients with complex t(15;17) translocations.