Promyelocytic Leukemia-retinoic Acid Receptor Alpha Research Articles

Phase separation of PML/RARα and BRD4 coassembled microspeckles governs transcriptional dysregulation in acute promyelocytic leukemia

In acute promyelocytic leukemia (APL), the promyelocytic leukemia-retinoic acid receptor alpha (PML/RARα) fusion protein destroys PML nuclear bodies (NBs), leading to the formation of microspeckles. However, our understanding, largely learned from morphological observations, lacks insight into the mechanisms behind PML/RARα-mediated microspeckle formation and its role in APL leukemogenesis. This study presents evidence uncovering liquid-liquid phase separation (LLPS) as a key mechanism in the formation of PML/RARα-mediated microspeckles. This process is facilitated by the intrinsically disordered region containing a large portion of PML and a smaller segment of RARα. We demonstrate the coassembly of bromodomain-containing protein 4 (BRD4) within PML/RARα-mediated condensates, differing from wild-type PML-formed NBs. In the absence of PML/RARα, PML NBs and BRD4 puncta exist as two independent phases, but the presence of PML/RARα disrupts PML NBs and redistributes PML and BRD4 into a distinct phase, forming PML/RARα-assembled microspeckles. Genome-wide profiling reveals a PML/RARα-induced BRD4 redistribution across the genome, with preferential binding to super-enhancers and broad-promoters (SEBPs). Mechanistically, BRD4 is recruited by PML/RARα into nuclear condensates, facilitating BRD4 chromatin binding to exert transcriptional activation essential for APL survival. Perturbing LLPS through chemical inhibition (1, 6-hexanediol) significantly reduces chromatin co-occupancy of PML/RARα and BRD4, attenuating their target gene activation. Finally, a series of experimental validations in primary APL patient samples confirm that PML/RARα forms microspeckles through condensates, recruits BRD4 to coassemble condensates, and co-occupies SEBP regions. Our findings elucidate the biophysical, pathological, and transcriptional dynamics of PML/RARα-assembled microspeckles, underscoring the importance of BRD4 in mediating transcriptional activation that enables PML/RARα to initiate APL.

Outcomes of Acute Promyelocytic Leukaemia in Paediatric Patients: Insights from a Low-Middle-Income Country.

To describe the prognostic variables and course of paediatric acute promyelocytic leukaemia (APL) in Pakistan. Cohort study. Place and Duration of the Study: Department of Paediatric Oncology, Combined Military Hospital, Rawalpindi, Pakistan, from January 2012 to December 2022. Patients aged 1-15 years, clinically confirmed APL with promyelocytic leukaemia- retinoic acid receptor alpha (PML-RARA) were enrolled. Initial admission included a thorough examination, recording demographic and clinical data, reporting time, prior treatment, and socioeconomic status. Statistical analysis used SPSS 25.0, with significance at p <0.05. This study included 50 cases of APL. Out of which, 32 (64%) were males and 18 (34%) were females. The mean age at diagnosis was 7.02 ± 3.86 years. Pallor (96%) and fever (88%) were common presentations. The average white blood cell count was 28.70 ± 35.39 x109/L. Treatment protocols include 48% International Consortium for Childhood (ICC)-APL, and 52% arsenic trioxide (ATO). High-risk cases were 54%. Neutropenic fever and differentiation syndrome were common induction complications. Delays over one month increased induction deaths (6.7 to 35%, p = 0.011), reducing disease-free survival (DFS), (76.7 to 35%, p = 0.001), and overall survival (OS), (80 to 45%, p = 0.007). After 40.90 ± 45.19 months' follow-up, 10-year OS and DFS were 66.0% and 60.0%, respectively. The best OS and DFS, at 80%, were observed in standard-risk cases treated with ATO. Neutropenic fever and bleeding were the primary causes of mortality in paediatric APL induction. Treatment delay was a key prognostic factor. ATO-based therapy offered safer, improved DFS, and OS suitable for primary healthcare settings. Acute promyelocytic leukaemia, Chemotherapy, Neutropenic fever.

Mutations at proximal cysteine residues in PML impair ATO binding by destabilizing the RBCC domain.

Acute promyelocytic leukemia (APL) is characterized by the fusion gene promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) and is conventionally treated with arsenic trioxide (ATO). ATO binds directly to the RING finger, B-box, coiled-coil (RBCC) domain of PML and initiates degradation of the fusion oncoprotein PML-RARA. However, the mutational hotspot at C212-S220 disrupts ATO binding, leading to drug resistance in APL. Therefore, structural consequences of these point mutations in PML that remain uncertain require comprehensive analysis. In this study, we investigated the structure-based ensemble properties of the promyelocytic leukemia-RING-B-box-coiled-coil (PML-RBCC) domains and ATO-resistant mutations. Oligomeric studies reveal that PML-RBCC wild-type and mutants C212R, S214L, A216T, L217F, and S220G predominantly form tetramers, whereas mutants C213R, A216V, L218P, and D219H tend to form dimers. The stability of the dimeric mutants was lower, exhibiting a melting temperature (Tm) reduction of 30 °C compared with the tetrameric mutants and wild-type PML protein. Furthermore, the exposed surface of the C213R mutation rendered it more prone to protease digestion than that of the C212R mutation. The spectroscopic analysis highlighted ATO-induced structural alterations in S214L, A216V, and D219H mutants, in contrast to C213R, L217F, and L218P mutations. Moreover, the computational analysis revealed that the ATO-resistant mutations C213R, A216V, L217F, and L218P caused changes in the size, shape, and flexibility of the PML-RBCC wild-type protein. The mutations C213R, A216V, L217F, and L218P destabilize the wild-type protein structure due to the adaptation of distinct conformational changes. In addition, these mutations disrupt several hydrogen bonds, including interactions involving C212, C213, and C215, which are essential for ATO binding. The local and global structural features induced by these mutations provide mechanistic insight into ATO resistance and APL pathogenesis.

Cytoreductive Chemotherapy in Induction Therapy Plays a Key Role in the Prognosis of Patients with Low-Risk Acute Promyelocytic Leukemia

Introduction: Despite the success for low-risk acute promyelocytic leukemia (APL) in the all-trans retinoic acid (ATRA) plus arsenicals era, several important clinical issues continue to account for treatment failure including early death (ED) and disease relapse. The present study was conducted to explore the potential role of cytoreduction during induction therapy on prognosis. Methods: All consecutive low-risk APL patients who received ATRA plus arsenic for induction and consolidation therapy at the Peking University People's Hospital, Peking University Institute of Hematology, between February 2014 and September 2021, were analyzed. Kaplan-Meier was used to estimate relapse rates and OS. Exploration of factors associated with relapse was based on Cox proportional hazards regression model. Results: Here 282 patients were diagnosed with low-risk APL and received ATRA plus arsenic as an induction and consolidation therapy. During induction therapy, six patients died early. Nine patients were withdrawn after induction therapy. Of the 282 patients evaluable for OS, the 5-year OS was 97.9%. Of the 267 patients evaluable for relapse, 14 patients (5.2%) relapsed throughout follow-up, with the 5-year cumulative relapse incidence of 5.9%. The median time to relapse, including hematological, molecular and extramedullary relapse, was 18.5 (IQR 12.25-24.5, range 6.2-52.0) months after hematological CR. After induction therapy, all 276 evaluable patients (100%) achieved hematological CR. Cytoreduction therapy, including hydroxycarbamide, anthracyclines and cytarabine during induction therapy was administrated based on the 2018 Chinese APL guidelines. Given the unavailability and inconveniences of intravenous chemotherapy at some stage in the COVID-19 pandemic, our center explored the routine of oral etoposide as cytoreductive therapy from January 2020. Etoposide was administered to patients with a WBC count of 3.2-9.8×10 9/L at diagnosis. The cumulative mean dose of etoposide was 706.5 mg (range, 150-1700 mg) during the induction therapy. In terms of different cytoreduction therapies, 86 patients were administered with hydroxycarbamide (30.5%), 113 with anthracyclines or cytarabine (40.1%), 31 with etoposide (11.0%), and 52 with no cytoreductive therapy (18.4%) during induction therapy. We previously explored that promyelocytic leukemia retinoic acid receptor alpha (PML-RARA) transcript level of 6.5% or more after induction therapy was associated with a subsequent risk of relapse. Compared to patients with no cytoreduction therapy or only hydroxycarbamide during induction therapy, the incidence of PML-RARA transcript levels of ≥6.5% was significantly decreased at the end of induction therapy in those who received anthracyclines/cytarabine and etoposide (27.5% vs. 26.2% vs. 15.5% vs. 0%, P = 0.003, Table 1). Notably, 2-year cumulative relapse rates were significantly lower in the anthracyclines/cytarabine treatment group and the etoposide treatment group (3.962% vs. 8.861% vs. 1.887% vs. 0%, P = 0.040, Table 1). The hydroxycarbamide treatment group did not decrease the relapse rate compared to the no cytoreduction group (11.4% vs. 5.9%, P = 0.289). To estimate RFS, the hydroxycarbamide treatment group and the no cytoreduction treatment group were merged to create a single group (the H/N group, 5-year RFS, 90.391%). Compared with the H/N group, the anthracyclines/cytarabine treatment group and the etoposide treatment group showed improved 2-year RFS (93.064% vs. 98.113% vs. 100.0%, P = 0.046, Figure 1). Multivariate Cox regression analysis revealed that myeloblasts in bone marrow at diagnosis, Leukocytosis during induction therapy, PML-RARA of ≥6.5%, and anthracyclines/cytarabine cytoreductive treatment were associated with a subsequent risk of relapse. Conclusions: In summary, the present study indicated that cytoreductive chemotherapy had a dual effect of both cytoreduction and prevention of relapse in low-risk APL. Oral etoposide as cytoreduction therapy combined with oral ATRA plus RIF is expected to be a regimen with low relapse, good tolerance and great convenience. A prospective, randomized, controlled, interventional study on oral etoposide versus intravenous daunorubicin for cytoreductive chemotherapy in induction therapy in patients with low-risk APL is now going on (NCT05832320).

Acute Promyelocytic Leukemia Treatment Masking Hepatic Tuberculosis: A Management Dilemma

Acute promyelocytic leukemia is a form of acute myeloid leukemia (AML) that is characterized by presence of a promyelocytic leukemia-retinoic acid receptor alpha fusion. In most patients, this fusion is detected on conventional karyotype as the t(15;17)(q24.1;q21.2) translocation, but some patients have cryptic translocations with a normal karyotype. Historically, AML is associated with a poor prognosis. Treatment with all-trans retinoic acid and arsenic trioxide assures long-term survival in the majority of patients. This treatment is generally well-tolerated but may cause hepatotoxicity. This is usually identified by transaminitis but resolves after temporary cessation of treatment. Our patient's hepatotoxicity did not resolve following all-trans retinoic acid and arsenic trioxide cessation which posed a diagnostic dilemma. This prompted exploration of other possible causes of hepatotoxicity. An eventual liver biopsy identified acid-fast bacilli, confirming a diagnosis of hepatic tuberculosis. A broad differential diagnosis is imperative when investigating abnormalities in liver function, especially in chemotherapy patients when treatment cessation may cause cancer progression.

Case report: A rare case of TBL1XR1-RARB positive acute promyelocytic leukemia in child and review of the literature.

Some forms of acute myelogenous leukemia (AML) share typical morphological and immunophenotypic features of acute promyelocytic leukemia (APL) but are negative for promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) fusion. These forms of AML are known as variant APL. Some variants of APL present with retinoic acid receptor beta (RARB) fused or rearranged with partner genes. RARB-positive APL is very rare, resistant to all-trans retinoic acid (ATRA), and associated with poor prognosis. Here, we reported one case with TBL1XR1-RARB positive APL, featured by early onset and no apparent bleeding tendency or coagulation dysfunction. This patient was resistant to ATRA and arsenic trioxide (ATO), but was good responsive to conventional chemotherapy for AML. The case report was followed by a literature review.

Metabolic adaptation drives arsenic trioxide resistance in acute promyelocytic leukemia

AbstractAcquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain most disease recurrence seen in the clinic. We have generated stable ATO-resistant promyelocytic cell lines that are less sensitive to all-trans retinoic acid (ATRA) and the combination of ATO and ATRA compared with the sensitive cell line. Characterization of these resistant cell lines that were generated in-house showed significant differences in immunophenotype, drug transporter expression, anti-apoptotic protein dependence, and promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) mutation. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in these ATO-resistant APL cell lines. Glycolytic inhibition by 2-deoxyglucose (2-DG) was sufficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell line. 2-DG was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO-resistant cell lines. In contrast, the resistant cell lines were significantly affected by compounds targeting mitochondrial respiration when combined with ATO, irrespective of the ATO resistance-conferring genetic mutations or the pattern of their anti-apoptotic protein dependency. Our data demonstrate that combining mitocans with ATO can overcome ATO resistance. We also show that this combination has potential for treating non-M3 acute myeloid leukemia (AML) and relapsed APL. The translation of this approach in the clinic needs to be explored further.

Identification of variant APL translocations PRKAR1A-RARα and ZBTB16-RARα (PLZF-RARα) through the MI-ONCOSEQ platform

The cornerstone of management in patients with acute promyelocytic leukemia (APL) is early diagnosis and prompt initiation of treatment with an all-trans retinoic acid (ATRA)-based regimen. Identification of the t(15;17)(PML-RARA) chromosomal translocation through conventional cytogenetics fluorescence in-situ hybridization (FISH) or detection of the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) fusion through RT-PCR represent the current standard of care for diagnosing APL. However, about 1–2% of patients with APL have a variant translocation involving other fusion partners with RARα besides PML. These patients present a unique diagnostic and clinical challenge in that conventional cytogenetics in addition to FISH and/or RT-PCR for PML-RARα may fail to identify these clinically relevant genetic lesions leading to an inappropriate diagnosis and treatment. We present two cases of patients who had APL with variant translocations whose bone marrow specimens were sent to the University of Michigan for enrollment in the MI-ONCOSEQ study (HUM00067928) after standard testing failed to identify PML-RARα or t(15;17) despite a phenotypic concern for this diagnosis. In these two patients, whole exome and transcriptome profiling via the MI-ONCOSEQ platform identified a PRKAR1A-RARα fusion in one patient and ZBTB16-RARα fusion in another patient. These cases illustrate the utility of whole exome and transcriptome profiling in diagnosing variant translocations in patients in whom there is a high clinical suspicion for APL based on hematopathology review.

Arsenic trioxide inhibits anaplastic lymphoma kinase (ALK)‐positive diffuse large B‐cell lymphoma through targeting ALK‐fusion oncoprotein

Arsenic trioxide inhibits anaplastic lymphoma kinase (ALK)‐positive diffuse large B‐cell lymphoma through targeting ALK‐fusion oncoprotein

CUDC-101 overcomes arsenic trioxide resistance via caspase-dependent promyelocytic leukemia-retinoic acid receptor alpha degradation in acute promyelocytic leukemia.

Although arsenic trioxide (ATO) treatment has transformed acute promyelocytic leukemia (APL) from the most fatal to the most curable hematological cancer, many high-risk APL patients who fail to achieve a complete molecular remission or relapse become resistant to ATO. Herein, we report that 7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide (CUDC-101) exhibits specific anticancer effects on APL and ATO-resistant APL in vitro and in vivo, while showing negligible cytotoxic effect on the noncancerous cells including normal CD34 cells and bone marrow mesenchymal stem cells from APL patients. Further mechanistic studies show that CUDC-101 triggers caspase-dependent degradation of the promyelocytic leukemia-retinoic acid receptor alpha fusion protein. As a result, APL and ATO-resistant APL cells undergo apoptosis upon CUDC-101 treatment and this apoptosis-inducing effect is even stronger than that of ATO. Finally, using a xenograft mouse model, we demonstrated that CUDC-101 significantly represses leukemia development in vivo. In conclusion, these results suggested that CUDC-101 can serve as a potential candidate drug for APL, particularly for ATO-resistant APL.

Potentiation of ATRA Activity in HL-60 Cells by Targeting Methylation Enzymes

All Trans Retinoic Acid (ATRA) is a Differentiation Inducer (DI) of Acute Promyelocytic Leukemia (APL) with proven clinical utility. Its benefits for other types of Acute Myelocytic Leukemia (AML) have been limited. In APL, ATRA targets the PML-RARA (promyelocytic leukemia/retinoic acid receptor-alpha)/DNA methyltransferase (DNMT)/Histone Deacetylase (HDAC) complex, facilitating its

Overall survival of adult acute myeloid leukemia based on cytogenetic and molecular abnormalities during 5 years in a single center study.

Objectives:To determined the 5-year overall survival (OS) rates for adult patients with acute myeloid leukemia (AML) patients at King Abdulaziz Medical City, Jeddah, Saudi Arabia, based on cytogenetic and molecular abnormalities.Methods:A retrospective cohort study reviewing adult AML patient files between 2011 and 2018. Sixty-three patients were excluded due to pediatric age and secondary AML. The remaining 87 adult patients with de novo AML were enrolled in this study.Results:The most frequent cytogenetic abnormalities were t(15;17) (17.2%), followed by complex cytogenetic (13.8%) and t(8;21) (5.7%). The most frequent molecular abnormalities were promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) (16.1%) and Nucleophosmin 1 (NPM1) (11.5%). The highest OS rate was associated with t(15;17), PML-RARA, and NPM. However, complex cytogenetic was associated with the lowest OS rate; fms-like tyrosine kinase 3 (FLT3)-internal tandem duplication was independently correlated with low OS rate.Conclusion:The study describes cytogenetic and molecular abnormalities observed in adult AML patients and gives an overview of prognostic factors and determine the OS, with comparable results with recent published data by the WHO.

Improved Outcomes of All-trans-retinoic Acid and Arsenic Trioxide Plus Idarubicin as a Frontline Treatment in Adult Patients With Acute Promyelocytic Leukemia

IntroductionThe purpose of this study was to explore the outcomes of all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) plus idarubicin (IDA) as a frontline treatment in adult patients with acute promyelocytic leukemia (APL). Patients and MethodsWe analyzed the outcomes of ATRA and intravenous ATO plus IDA as a frontline induction therapy in 118 patients with APL with high-risk (HR) and standard-risk (SR) disease from January 2008 to December 2017. The medical records of 118 patients with APL (HR, n = 45; SR, n = 73) who received the frontline triple combination regimen at Henan Provincial People’s Hospital and Tongji Hospital were retrospectively reviewed. Consolidation therapy comprised 6 cycles of ATO and ATRA plus IDA, and IDA was administered in 1 to 4 cycles of consolidation therapy based on the comparable clinical efficacy compared with 6 cycles and fewer side effects to myocardium without subsequent maintenance therapy. ResultsOf 118 patients, there were 3 (2.5%) early deaths and 115 (97.5%) hematologic complete remissions; 102 (88.7%) of 115 patients achieved molecular complete remission following induction therapy, and all patients were polymerase chain reaction-negative for promyelocytic leukemia-retinoic acid receptor alpha after the first cycle of consolidation therapy. The 5-year overall survival (OS) and event-free survival (EFS) were 93.0% ± 2.9% and 92.4% ± 3.0%, respectively. Early death, hematologic complete remissions, molecular complete remissions, and toxicities were comparable between the HR and SR groups. The cumulative incidence of relapse in the HR group was 4.7% (n = 2), and the cumulative incidence of relapse in the SR group was 0. The OS and EFS of the SR and HR groups were comparable (92.3% ± 4.5% vs. 92.8% ± 4.0%; X2 = 0.263; P = .608; 92.3% ± 4.5% vs. 91.1% ± 4.2%, X2 = 0.917; P = .338). The total dosage of IDA was approximately 181 to 258 mg, and no patient experienced cardiotoxicity. OS and EFS were not influenced by fms-related tyrosine kinase 3 internal tandem duplication mutation status (P = .405 and P = .528, respectively). ConclusionThe triple combination of ATRA and ATO plus IDA as both an induction and consolidation therapy for the HR and SR groups attained excellent outcomes, and this regimen was effective, safe, and easy, without maintenance therapy. The triple combination treatment might be a preferred frontline therapy for patients with APL, especially for those with HR or the APL fms-related tyrosine kinase 3 internal tandem duplication mutation.

Prognostic Significance of bcr-1 and bcr-3 Isoforms of PML-RARA and FLT3-ITD in Patients With Acute Promyelocytic Leukemia

BackgroundAcute promyelocytic leukemia (APL) has a characteristic peculiar morphologic and genetic features as well as a more favorable outcome. We studied the differential effect of bcr-1 and bcr-3 isoforms of the promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) transcript together with FLT3 internal tandem duplication (FLT3-ITD) mutation status on the outcome of newly diagnosed de novo APL patients. Patients and MethodsThis cohort study included all patients diagnosed with APL at outpatient medical and pediatric oncology clinics of the National Cancer Institute, Cairo University, Cairo, Egypt, from May 2012 to January 2018. ResultsThe study included 118 patients with APL, 71 adults (60.2%) and 47 children (39.8%). Median (range) age was 25 (1.5-70) years. Children had significantly higher total leukocyte count (≥10 × 109/L), disseminated intravascular coagulation (DIC), and thrombocytopenia (< 40 × 109/L) than adults (P = .04, .03, and .04, respectively), while the latter group had significantly higher hemorrhage than children (P = .04). FLT3-ITD mutation was detected in the whole group, children, and adults in 23.7%, 30.6%, and 24.6%, respectively. FLT3-ITD mutation was significantly associated with leukocytosis in the whole group (P = .039). bcr-3 was significantly associated with FLT3-ITD mutation in the whole APL cohort and in adults (P = .011, P = .022). All children (both bcr-1 and bcr-3) and all adult patients with bcr-3 experienced CR, while 22 (78.5%) of 28 patients with bcr-1 experienced CR (P = .04). APL patients with DIC and hemorrhage had significantly lower overall survival (P = .002 and < .001, respectively). Overall survival for APL in children was significantly better than in adults (P = .005). Multivariate analysis indicated that age was an independent prognostic variable affecting survival (hazard ratio = 2.6; 95% confidence interval, 1.3-5.3; P = .007) (adults had hazard ratio of death 2.6 times higher than children). DIC and FLT3-ITD were independent prognostic variables affecting survival in children with APL (hazard ratio = 12.3; 95% confidence interval, 1.46-104.61; P = .021; and hazard ratio = 5.2; 95% confidence interval, 1.01-26.95; P = .048, respectively). ConclusionAge is an independent prognostic factor for APL. bcr-3 is significantly associated with FLT3-ITD in adults with APL. DIC and FLT3-ITD are adverse prognostic factors in children with APL. Despite children being at higher risk, outcome is better than in adults.

Acute promyelocytic leukemia: evaluation of diagnostic tests from 2000 to 2018 in a public hospital

ABSTRACT Introduction: Acute promyelocytic leukemia (APL) is caused by t(15;17)(q24;q21) translocation, which product is the fusion oncoprotein PML-RARa (promyelocytic leukemia-retinoic acid receptor alpha). The morphology of leukemic promyelocytes is usually characteristic, with the presence of faggot cells and coarse cytoplasmic granulations; immunophenotype is characteristic in most cases. However, definitive laboratory diagnosis should be performed by detecting t(15;17) or by PML-RARa fusion protein. Objectives: To compare cytomorphology, flow cytometry, and classical cytogenetic of bone marrow samples from patients with APL, treated at the Complexo Hospital de Clinicas da Universidade Federal do Parana (CHC-UFPR), as well as describe the possible discrepancies between the methodologies. Method: Retrospective analysis of APL cases treated at the CHC-UFPR from January 2000 to July 2018. Results: Eighty-eight patients (42 man/ 46 woman; mean age: 34 years), 42.1% of them presented a high-risk prognosis. Flow cytometry was performed in 83 cases (94.3%); karyotype was performed in 79 cases (89.7%), but translocation t(15;17) was confirmed in only 53 cases (60.2%). From the 28 patients with a non-conclusive karyotype; fourteen (15.9%) of them presented the PML-RARa transcript in the molecular analysis. In total, 35 patients (39 8%) performed research of the PML-RARa gene by molecular biology. Only 45 patients (51.1%) presented concordant diagnosis among the three technical exams (morphology, flow cytometry and cytogenetics). Overall survival was 67% at 4.8 years, with 29 deaths. Conclusion: Genetic confirmation was observed in 76.1% of samples, 60.2% by conventional cytogenetics and 15.9% by molecular biology. There was a disagreement between the methodologies, and a low sensibility of the conventional cytogenetics, demonstrating the importance of performing molecular techniques for diagnostic confirmation.

PML/RARA inhibits expression of HSP90 and its target AKT.

Essential for cell survival, the 90kD Heat Shock Proteins (HSP90) are molecular chaperons required for conformational stabilization and trafficking of numerous client proteins. Functional HSP90 is required for the stability of AKT, a serine-threonine kinase phosphorylated in response to growth factor stimulation. AKT plays a crucial regulatory role in differentiation, cell cycle, transcription, translation, metabolism and apoptosis. Acute promyelocytic leukaemia (APL) is characterized by the presence of the promyelocytic leukaemia/retinoic acid receptor alpha (PML/RARA) fusion protein, which deregulates expression of several genes involved in differentiation and apoptosis. Here, we report inhibition of HSP90AA1 and HSP90AB1 isomer transcription in blasts isolated from patients with APL, associated with reduction of HSP90 protein expression and loss of control on AKT protein phosphorylation. We show that invitro treatment of PML/RARA expressing cells with all-trans retinoic acid (ATRA) up-regulates HSP90 expression and stabilizes AKT. Addition of the HSP90-inhibitor 17-(allylamino)-17-demethoxygeldanamycin in combination with ATRA, blocks upregulation of AKT protein, indicating that HSP90 is necessary for ATRA action on AKT. This is the first report proving that expression of HSP90 isomers are directly and differentially repressed by PML/RARA, with critical results on cellular homeostasis of target proteins, such as AKT, in APL blasts.

Abstract 1506: Characterization of fibronectin type III domain containing 3B (FNDC3B) as a novel fusion partner of retinoic acid receptor alpha in granulocytic differentiation

Abstract Acute promyelocytic leukemia (APL) is characterized by the promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA) fusion. Recently, we identified fibronectin type III domain containing 3B (FNDC3B) as a novel RARA fusion partner resulting from the t(3;17)(q26;q21) translocation in a variant APL patient. Unlike other RARA fusion partners, FNDC3B appears as the only partner involved in granulocytic differentiation but the molecular mechanisms underlying its actions remain to be characterized. FNDC3B mRNA expression in normal and malignant hematopoietic cells was determined by quantitative RT-PCR. Transcriptional control of FNDC3B was studied by reporter gene assays, site-directed mutagenesis and chromatin immunoprecipitation (ChIP) assays. FNDC3B-regulated pathways were identified by transcriptome analyses. Using BloodSpot, we observed that FNDC3B expression was progressively up-regulated as human hematopoietic stem cells (HSCs) differentiate along the granulocytic lineage, with the highest level in terminally differentiated polymorphonuclear cells. Concordantly, we demonstrated lower expression of FNDC3B in human cord blood CD34+ HSCs than HL-60 promyelocytes. The specific involvement of FNDC3B in granulocytic differentiation was further suggested by higher FNDC3B expression and promoter activity in human promyelocytic than monocytic cell lines. Mutational analyses indicated that two putative CCAAT-enhancer binding protein epsilon (CEBPE) and one Sp1 binding sites were critical for FNDC3B promoter activity in HL-60 cells. The specific association of CEBPE and Sp1 with the FNDC3B promoter could be confirmed by ChIP assays. Knockdown of CEBPE in HL60 cells by small interfering RNA (siRNA) reduced FNDC3B expression and promoter activity. Furthermore, we observed a strong positive correlation between CEBPE and FNDC3B expression in the Cancer Genome Atlas (TCGA)-acute myeloid leukemia (AML) dataset (n=173, r=0.66, P&amp;lt;0.0001). Collectively, these findings indicate CEBPE, which is critical in terminal granulopoiesis, as an important factor controlling FNDC3B transcription in granulocytic cells. Using RNA-seq, we found that FNDC3B knockdown in NB4 promyelocytes was associated with preferential downregulation of genes encoding granule and endoplasmic reticulum (ER) structural/functional proteins. Concordantly, DAVID functional annotation of genes that are positively correlated with FNDC3B in the TCGA-AML cohort revealed enrichment of Gene Ontology terms related to ER functions. Together, these findings suggest that FNDC3B mediates its functions primarily by targeting the ER/granule pathway, which is important for granulocytic maturation. In summary, our data indicate FNDC3B as a novel CEBPE target in the myeloid transcriptional hierarchy and may provide new clues in abnormal granulopoiesis. Citation Format: Zhe Wand, Chi Keung Cheng, Terry Wong, Kin Mang Lau, Thomas S.k. Wan, Kam Tong Leung, Margaret Heung-Ling Ng. Characterization of fibronectin type III domain containing 3B (FNDC3B) as a novel fusion partner of retinoic acid receptor alpha in granulocytic differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1506.

Evaluation of multiprobe fluoresence in-situ hybridization panel in the detection of common chromosomal abnormalities of acute myeloid leukemia

Background Numerous recurrent chromosomal aberrations have been identified in acute myeloid leukemia (AML), and their detection has become essential for accurate diagnosis, classification, and prognosis of the disease. Fluorescence in-situ hybridization (FISH) provides a powerful technique complementary and even alternative to chromosome banding studies for the identification of selected chromosome aberrations. Aim Evaluation of the multiprobe FISH panel in the detection of common cytogenetic abnormalities in AML and to investigate their association with clinical diagnosis, chemotherapy, and prognosis. Patients and methods This study was conducted on 20 newly diagnosed AML patients. All patients were subjected to full history taking, clinical examination, laboratory investigations including complete blood count, bone marrow aspiration, immunophenotyping, and interphase FISH using cytocell multiprobe AML/myelodysplastic syndrome panel designed to detect AML/Eight-twenty-one (ETO), Promyelocytic leukaemia- Retinoic acid receptor alpha (PML-RARA), and core-binding factor beta/Myosin, heavy chain 11, smooth muscle (CBFβ/MYH11) translocations, mixed-lineage leukaemia (MLL) break apart, P53, 5q, 7q, and 20q deletions. Results Interphase FISH analysis showed 5q deletion in 8/20 (40%) cases, positive PML/RARA in 3/20 (15%) cases p53 deletion in 15/20 (75%) cases, positive AML1/ETO in 1/20 (5%) cases, no MLL break apart cases (0%), 7q deletion in 4/20 (20%) cases, positive CBFβ/MYH11 fusion gene in 4/20 (20%) cases, 20q deletion in 9/20 (45%) cases, and trisomy 8 in 7/20 (35%) cases. There was a statistically significant relationship between 5q deletion and prognosis (P=0.028). Conclusion Multiprobe FISH is more cost effective and time effective compared with traditional FISH. It is an efficient technique for the detection of cytogenetic aberrations AML, providing critical information for diagnosis and prognosis, and for monitoring the course of the disease.

Isolated Central Nervous System (CNS) Relapse in Paediatric Acute Promyelocytic Leukaemia: A Systematic Review.

Extramedullary disease, as a whole, is rare in Acute Promyelocytic Leukaemia (APML). If at all relapse occurs, following sites are involved: Central Nervous System (CNS), skin, testes, mediastinum, gingiva, and ear. Isolated CNS relapses after complete morphological and molecular remission is rarer particularly in children. To review the literature systematically to find out the incidence of isolated CNS relapse in paediatric APML cases. A systematic search of major databases (Medline, Pubmed and Google Scholar) was conducted. We included all types of studies that reported about incidence or prevalence of isolated CNS relapse in children upto 18 years of age with APML. A total of nine studies (with 10 cases of isolated CNS relapse) were included. Majority (70%) was high risk patients, and 60% were ≤six-year-old. Nearly, 50% were having the mean time to relapse <12 months and most (60%) of them were male. The children who died were having shorter time to CNS relapse (around 12 months), and were older (>6 to 18 years). In the present review, disease in the high-risk group, male sex, younger age (≤six-years-old), and Promyelocytic Leukaemia/Retinoic Acid Receptor Alpha (PML-RARA) detection was found to be associated with isolated CNS relapse in children with APML. Cerebrospinal fluid (CSF) examination along with immunophenotyping and Reverse Transcription polymerase Chain Reaction (RT-PCR) for PML-RARA is required for a definite diagnosis and early treatment of patients to improve overall survival.

A rare case of acute promyelocytic leukemia with focal bone marrow involvement presenting as a paraspinal myeloid sarcoma

Acute promyelocytic leukemia (APL) accounts for 5–8% of acute myeloid leukemias (AMLs). Myeloid sarcomas (MS) are rare extramedullary tumors consisting of immature myeloid cells. These tumors may develop de novo or concurrently with AML and may precede or coincide with the occurrence of AML. The occurrence of MS in an APL patient is a rare clinical event. In these cases, it usually occurs as a sign of relapse. Focal bone marrow involvement by APL is also rare; the majority of cases of APL demonstrate a diffusely hypercellular bone marrow throughout the axial skeleton due to expansion of neoplastic blasts/promyelocytes. We describe a 23-year-old female who presented with gait problems, including multiple falling episodes, and lower extremity weakness. An MRI demonstrated a paraspinal soft tissue mass. Histopathologic analysis demonstrated a myeloid sarcoma expressing CD45, myeloperoxidase, CD117, BCL2, focal weak CD34, and weak CD99 and negative for CD30, TdT, CD20, CD3, and CD10. The bone marrow aspirate demonstrated blasts/promyelocytes accounting for approximately 26% of the total bone marrow aspirate cellularity. The bone marrow biopsy and clot section demonstrated normocellularity with a few blast/promyelocyte foci on the clot section accounting for approximately 5% of total bone marrow cellularity. These foci expressed MPO and CD117 and were negative for CD34. Flow cytometry demonstrated blasts accounting for approximately 31% of all CD45+ events expressing MPO, CD33, CD13, CD117, CD64, CD2, CD38, and CD71 and negative for CD34, HLA-DR, CD11b, CD14, CD15, CD16, CD19, CD22, CD10, CD56, CD61, and surface and cytoplasmic CD3. Fluorescence in situ hybridization (FISH) analysis was positive for the t(15;17)(q22;q12) promyelocytic leukemia/retinoic acid receptor alpha (PML/RARA) translocation. The patient was induced with arsenic trioxide and subsequently underwent consolidation chemotherapy with daunorubicin and all-trans-retinoic acid. Her post-chemotherapy bone marrow biopsy was negative for residual disease. Flow cytometry and FISH analysis were also negative. This is a rare case of APL involving both the initial presentation as myeloid sarcoma and focal bone marrow involvement.