PML-RARA Transcript Research Articles

Cytoreductive chemotherapy in induction therapy plays a key role in the prognosis of patients with low-risk acute promyelocytic leukaemia.

In order to explore the risk factors of relapse and potential optimized therapeutic regimen of low-risk acute promyelocytic leukaemia (APL), here we retrospectively analysed 282 patients who were diagnosed between February 2014 and September 2021. The median follow-up was 59 (9-102) months. The 5-year overall survival and cumulative relapse incidence were 97.9% and 5.9%, respectively. In terms of different cytoreductive 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 the induction therapy. The hydroxycarbamide treatment group did not decrease the relapse rate compared to the no cytoreduction group (11.4% vs. 5.9%, p = 0.289). Compared with the hydroxycarbamide group, the anthracyclines/cytarabine treatment group showed improved 5-year RFS (88.145% vs. 98.113%, p = 0.008). Multivariate Cox regression analysis revealed that myeloblasts in bone marrow at diagnosis, and PML-RARA transcript level of 6.5% or more after induction therapy were associated with a subsequent risk of relapse. The only factor positively reducing the relapse rate was anthracyclines/cytarabine cytoreductive treatment. In conclusion, cytoreductive chemotherapy in induction therapy plays a potential key role in the prognosis of low-risk APL.

B-242 Fast and Cost-effective RNA Extraction in Onco-hematology Laboratories

Abstract Background Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of a BCR-ABL1 rearrangement; its detection and quantification by real-time quantitative PCR (RQ-PCR) plays a central role in CML diagnosis, therapy monitoring, and sequencing to identify resistance mutations in case of therapy failure. To evaluate the molecular response of CML, BCR-ABL1 quantification must be calculated on the international scale (IS%) for which RQ-PCR has the necessary sensitivity (up to 10−5). Currently, BCR-ABL1 quantifications are ubiquitous in onco-hematology laboratories (OHLs; ∼70% of tests performed in our OHL), whereby quality, cost, and turn-around-time are of critical importance. Today, the majority of OHLs in Brazil rely on laborious manual RNA-extraction methods (e.g., Trizol) that usually require 8 mL of peripheral blood (PB) to monitor CML. Here, we report a strategy that requires only 2 mL of PB in combination with the automated magnetic DNA/RNA-extraction kit Extracta-MPTA (Loccus do Brasil) for CML diagnosis and monitoring. Methods: BCR:ABL1 was quantified and its IS% determined using our developed kit, which was calibrated using ERM-AD263 and UK-NEQAS (external quality assessment). Total RNA was extracted from 40 PB samples, after erythrocyte lysis using two different methods: 1) Leukocytes from 8 mL of PB were extracted manually using Trizol; 2) leukocytes from 2 mL of PB were automatically extracted using Extracta-MPTA (Loccus do Brasil). BCR-ABL1 quantification, BCR-ABL1 IS%, and BCR quantification (internal control) were compared statistically (paired t-test). The reproducibility was evaluated by analyzing 20 replicates; the low limit of the leucocyte number to BCR:ABL1 quantification using Extracta-MPTA were also evaluated. Median and moving means of 135 unpaired samples were used to monitor BCR-ABL1 IS% between Trizol, Extracta-MPTA, while EQA from UK-NEQAS was also evaluated using both methods. In parallel, RNA was extracted by both methods from 20 samples for the diagnosis of acute lymphoblastic leukemia (ALL) (BCR-ABL p190 transcript) and acute promyelocytic leukemia (APL) (PML-RARA transcript). Results A comparison of BRC-ABL1, BCR, and %IS in 40 paired samples showed that the results obtained from Trizol or Extracta-MPTA are comparable (paired t-test: P = 0.4222, P = 0.9263, and P = 0.071, respectively). The reproducibility was evaluated by BCR quantification (24%). A comparison of the medians from 135 unpaired samples extracted using Trizol and Extracta-MPTA revealed that BCR-ABL1 IS% distribution among our patients is stable over time (P = 0.7704; Mann Whitney: r2 = 0.0012). EQA was extracted using both methods and the results were identical. A comparison between Trizol and EXTRACTA_MPTA for ALL and APL was 100% identical (Fischer; P = 1.000). Conclusion Both RNA extraction methods are suitable for OHLs for PML-RARA and BCR-ABL (p190 and p210) detection and CML diagnosis/monitoring by BCR-ABL1 detection/quantification. By using Extracta-MPTA the amount of PB used to obtain RNA can be reduced by 75%, test results can be obtained within 4 h, and direct reagent costs are reduced by 25% with a minimum of hands-on time.

Fatal intracranial haemorrhage in acute promyelocytic leukemia patients with short isoform of PML-RARα: Review of molecular and radiological data

Three major PML-RARα fusion gene transcripts (long [bcr1], variant [bcr2], and short [bcr3]) are currently used in clinical laboratories for the diagnosis and treatment monitoring of APL patients. Despite highly improved outcome, relapse and intracranial haemorrhage that may lead to early death is still an unsolved complication in APL. We reviewed APL patients confirmed by qPCR for the presence of PML-RARα transcripts (n = 27) and studied their outcome in relation to the isoform expression at diagnosis and follow-up in King Fahad Medical City. Eight in twenty-seven patients showed bcr3 and nineteen patients with bcr1 as major isoforms at diagnosis. Half of the bcr3 patients (n = 4/8) showed early mortality, prolonged qPCR positivity, 4-fold higher neutrophil/lymphocyte ratio, higher creatinine levels, and significantly reduced relapse free and overall survival time compared with bcr1 patients. Radiological findings in bcr3 patients revealed CNS involvement in the form of intracranial haemorrhage and periventricular microangiopathy and no CNS involvement in bcr1 patients. In conclusion, PML-RARα isoform expression at diagnosis in selective patients influences disease course over time and may even lead to early mortality due to haemorrhage. Thus, timely reporting of the specific PML-RARα isoform by clinical laboratories and CNS assessment by radiology can prevent complications leading to death in some APL patients.

Treatment Outcomes Of Patients With Newly Diagnosed Acute Promyelocytic Leukemia; Experience From A Developing Country.

Acute promyelocytic leukaemia (APL) characterized by t (15;17) leading to formation of fusion protein PML-RARA is an acute leukaemia with highest mortality. A remarkable improvement in the outcomes has been witnessed due to evolution of highly effective targeted therapies replacing the traditional chemotherapy is most patients. However limited data is available regarding treatment outcomes of APL using various novel regimens from developing countries like Pakistan. This was a retrospective descriptive study which included APL patients treated at AFBMTC Rawalpindi from 2005 to 2020. It included a total of 51 eligible patients with a diagnosis of de novo APL confirmed by the presence of PML-RARA transcript or presence of t (15;17) by cytogenetics or FISH analysis. The protocols used for treatment included the UKAML MRC 12, the LPA-99/LPA-2005 PETHEMA, the APML4 and non-chemotherapy based ATO-ATRA protocol. The study included 51 patients in which 31 (60.78%) were male and 20 (39.2%) were female. The median age at diagnosis was 30 years (range 5-70). The commonest symptom was fever seen in 43 (84.3%) patients and bruising was the commonest physical finding present in 44 (86.3%) patients. High-risk patients were 23 (46.1%), 18 (35.3%) were intermediate risk and 10 (19.6%) were low risk. The LPA99/LPA2005 was most frequently employed protocol being used in 36 (72%) patients. There were 2 deaths during induction and 44 (86.3%) achieved CR post induction. The median follow up time was 32 months (range 1 to 190 months) with an overall survival (OS) of 76.5% and a relapse free survival (RFS) of 66.7. Our study shows APL is a highly curable malignancy and outcomes have improved with newer non chemotherapy based therapies. It can also be concluded that outcomes of APL gradually improved over the past 2 decades due to improvement in supportive care, provision of blood products and use of newer protocols. The prognosis remains less favourable in high risk patients.

Acute Promyelocytic Leukemia Masquerading as Sero-negative Polyarthritis: Case Report

AbstractMusculoskeletal manifestations as the sole presentation in acute leukemia is rare in adults. Acute promyelocytic leukemia (APML) is a subtype of acute myeloid leukemia (AML) with reported incidence of 10 to 15% of total AML cases. APML presenting as polyarticular arthritis has never been reported in the literature. We present an interesting case of 20-year-old male patient who manifested with polyarticular arthritis mainly of small joints as the initial presentation, followed by pancytopenia and eventually was diagnosed as a case of APML on bone marrow morphology and molecular analysis for PML-RARα transcript. He was successfully treated with all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO). Arthritis also resolved with complete remission of APML. Arthritis in a case with pancytopenia should promptly be evaluated prior to treatment with steroids and anti-metabolites. Arthritis can be a presenting manifestation of APML and responds to prompt management of leukemia as in other cases of leukemic arthritis.

PB1819: CHARACTERISTICS AND OUTCOME OF PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIA IN SOUTHERN TUNISIA

Background: All-trans retinoic acid (ATRA) is the major advance in the treatment of acute promyelocytic leukemia (APL). Its introduction in the treatment of APL has revolutionized the outcome of this disease. Aims: The current study reports the clinical features and treatment outcome of patients with acute promyelocytic leukemia (APL) treated at the hematology department of Sfax from Tunisia. Methods: Our study was retrospective. It concerned all patients with the diagnosis of APL and followed in the hematology department of Hedi Chaker Hospital Sfax, from January 2000 until December 2019. The diagnosis of APL is based on morphological, cytogenetic and molecular study. The specific treatment is similar to that of the French protocol APL93 until 2013, and then similar to that of Spanish protocol LPA2005 since January 2014. We evaluate the rates of remission, death and overall survival (Kaplan-Meier method). Results: We collected 79 patients. The sex ratio was 1.3 with a median age of 34 years (extreme: 2-75 years). Hyperleukocytosis (GB> 10000 / mm3) was noted in 29 patients (37%). The average white blood cell count was 21900 / mm3. The disseminated intravascular coagulation (DIC) was found in the majority of cases (72% of cases). The t(15,17) was found in 89% of cases. The PML-RARA transcript was positive in 100% of cases. Eight patients (10%) died before treatment with hemorrhage. Thirty three patients were treated by the APL 93 (group1) and thirty eight patients were treated by the protocol LPA2005 (group 2). Fifteen patients (22%) died early during the induction course before evaluation. Causes of death during treatment were multiple: respiratory disease due to the differentiation syndrome, a severe haemorrhagic syndrome and or septic shock. Complete remission was obtained in 55 patients (98% of the evaluable patients and 77% of the treated patients) and one patient failure (2%). No deaths were reported during consolidation or maintenance treatment. A late bone marrow relapse was observed in 8 patients (14,5%). At five years of follow up, overall survival and event-free survival are 66% and 64%, respectively, and relapse-free survival is 77%. Summary/Conclusion: In our series, the DIC rates are correlated with that of the literature, as well as early death rate. The complete remission and overall survival remain lower than that of the literature. This is explained by a high rate of early mortality (during induction) by hemorrhage, of the higher frequency of hyper-leukocyte forms and the delay in diagnosis and management for our patients.

Has Hematopoietic Stem Cell Transplantation a Role in the Treatment of Children and Adolescents with Acute Promyelocytic Leukemia?

The past three decades have brought major therapeutic advances in treating acute promyelocytic leukemia (APL) both in adults and children. The current state-of-the-art treatment with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) in combination or not with chemotherapy results in long-lasting remission and cure in more than 90% of newly diagnosed patients. These treatments have made relapse a rare event. The detection of PML-RARA transcript by polymerase chain reaction (PCR) during treatment and follow-up can predict a hematological relapse. All studies have suggested a survival benefit in patients with molecular relapse given pre-emptive therapy compared with those treated at the time of overt hematological relapse. ATO-based regimens seem to be effective for achieving a second molecular complete remission (CR). Patients in second molecular CR are generally considered candidates for autologous hematopoietic stem cell transplant (HSCT), while for those with a persistent molecular disease, allogeneic HSCT should be offered if a suitable donor is identified.Except for sporadic pediatric reports, most of the evidence for using HSCT to treat relapsed/refractory APL comes from adult literature. Therefore, we now provide a review of published pediatric data that evaluated the role of HSCT in children with refractory/recurrent APL disease.

AML-214: Case Report: A Rare Case Presentation and Detection of Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is an uncommon subtype of acute myeloid leukemia (AML) characterized by the translocation t (15;17) (q22; q21) with a favorable prognosis upon immediate recognition and treatment. It is very rare when the insertion into PML could not be demonstrated by initial FISH and demonstrated by molecular analysis. We present a case of a 44-year-old female who presented in October 2020 to the Emergency Room with severe fatigue and blood tests showing Hb=6 g/dL, thrombocytopenia, and leukopenia (WBC 2.5 x10⁹/L). The patient was admitted to the hospital, where bone marrow biopsy and flow cytometry showed a negative result. Initial diagnosis was mainly related to a viral infection, when the patient had blood transfusion. On December 15, 2020, the patient presented with leukocytosis (55.31 x10⁹/L), anemia (Hb 7.6 g/dL), thrombocytopenia PLT (67 x10⁹/L), and swelling in her right knee. Repeated flow cytometry showed signs of dysplasia with abnormal myeloid lineage. Total body scans showed findings mostly related to underlying myeloproliferative or lymphoproliferative disease with hepatosplenomegaly, as well as retroperitoneal soft tissue thickening encasing the abdominal aorta, with aortic wall mostly related to associated para-neuroplastic aortitis. Stills disease was diagnosed. Initiation of steroids 1 mg/kg was given, leading to amelioration of the patient and less of blood transfusion. After 1 week, a repeated cytogenetics and molecular analysis showed PML-RARA transcripts and t (15;17), negative for acute leukemia, hypercellular aspirate showing left-shifted myeloid hyperplasia, and <1% blasts. She was diagnosed with promyelocytic leukemia and was started on treatment with ATRA + arsenic per guidelines and is now in complete remission.

Acute promyelocytic leukemia derived extracellular vesicles conserve PML-RARα transcript from storage-inflicted degradation: a stable diagnosis tool in APL patients.

The early death, which is more common in acute promyelocytic leukemia (APL) patients rather than other types of acute myelocytic leukemia (AML) highlights the importance of appropriate diagnostic method for early detection of this disease. The low sensitivity of the conventional methods, low tumor burden in some patients, and the need for bone marrow sampling are some of the diagnostic challenges on the way of proper detection of APL. Given these, we aimed to compare the efficacy of extracellular vesicles (EVs), as a diagnostic tool, with the existing methods. RT-PCR, qPCR, and flow cytometry were applied on EVs and their corresponding associated cellular component collected from 18 APL new cases, 23 patients with minimal residual disease (MRD), and NB4 cell line. RT-PCR results were positive in both cellular and vesicular components of all new cases, NB4 cells, and EVs in contrary to MRD cases. Normalized copy numbers (NCN) of PML-RARα were 5100 and 3950 for cell and EVs, respectively (p < 0.05). There was a significant difference in the NCN of PML-RARα between cells and EVs in BM samples. Investigating the effect of storage at room temperature revealed that PML-RARα level was retained near to the baseline level in EVs, but there was a significant reduction in its copy number in the cellular component during 7days. Taken together, given to the acceptable stability, EVs could be introduced as a non-invasive liquid biopsy that alongside existing methods could remarkably change the paradigm of APL diagnostic approaches.

PML-RARa of the Short but Not the Long/Variant Isoform Initiate from CD34+ TIM-3+ LSCs with Hierarchical Leukemic Organization

Acute promyelocytic leukemia (APL) is successfully treated with PML-RARa-targeted therapies. APL is divided into two groups with distinct PML-RARa subtypes: Long/variant (the L/V type) and short (the S type) isoforms. The L/V type PML-RARa is observed ~70% of APL patients, whereas the S type isoform, which lacks nucleus localization sequence located in exon6 of PML gene, is involved in ~30% of APL cases. PML-RARa contributes to leukemogenesis presumably by repressing transcriptional activities via its DNA binding properties in nucleus. However, recent studies have shown that PML-RARa mainly localizes in nucleus in the L/V type APL, whereas it dominantly distributes in cytoplasms in the S type APL. This study was planned to clarify difference in the role of PML-RARa in leukemogenesis in APL patients with the L/V or the S type.We performed the transcriptome analysis of APL blast cells from 4 cases each of the L/V type and the S type. Surprisingly, APL cells of the L/V and the S type showed significantly different gene expression pattern by gene set enrichment analysis. For example, pathways for apoptosis involving BCL-2, prostaglandin and T lymphocyte maturation were enriched only in the S type APL, whereas cytokine pathways were mainly enriched in the L/V type APL. More interestingly, the gene sets associated with functional leukemic stem cells (LSCs) (K.Eppert, et al. Nat Med, 2011) were enriched in the S type APL (NES 1.72, FDR q-value 0.083).In most AML of FAB types except for M3 (APL), self-renewing LSCs are concentrated in the CD34+CD38- population, and transplantation of CD34+CD38- LSCs of AML patients resulted in reconstitution of human AML in xenograft models. Based on this data, it is widely accepted that AML is hierarchically organized and is maintained by LSCs, like normal hematopoiesis is initiated from normal HSCs. However, it has been reported that reconstitution of human APL is usually difficult in xenograft models (D.Thomas and R.Majeti. Blood, 2017), although human APL was reconstituted in rare cases by a recent report (S.Patel, et al. Leukemia, 2012). We have reported that self-renewing AML LSCs express TIM-3, a signaling molecule activating the β-catenin pathway (Y.Kikushige, et al. Cell Stem Cell, 2015), but TIM-3 expression was negative in leukemic cells in most cases of APL. However, because the S type but not the L/V type APL has LSC signatures by transcriptome analysis, we revisited to test the LSC activity of the S type APL.By FACS analysis, CD34+CD38- cells were rarely found in the L/V type APL patients, and they largely lacked TIM-3 expression and did not have PML-RARa transcript (n=10), suggesting that these CD34+CD38-TIM-3- cells may be residual normal hematopoietic stem cells (HSCs). In contrast, CD34+CD38- cells from the S type APL strongly expressed TIM-3 and PML-RARa transcript (n=12), suggesting that they may be functional LSCs. We then tested whether CD34+ cells of the S type APL can reconstitute human APL in a xenograft model. Purified 0.4-3.0 x106 CD34+ APL cells were transplanted into sublethally irradiated NSG mice. Strikingly in the S type APL, CD34+ but not CD34- cells reconstituted human PML-RARa+ APL in NSG mice in 3 out of 5 patient tested, whereas in the L/V type APL, CD34+ cells were incapable of reconstitution in all 3 cases tested. The hCD45+CD34+TIM-3+ cells harvested from the primary NSG recipients again reconstituted the S type APL in serial xenotransplantion experiments. These results collectively suggest that the S type APL have LSCs capable of self-renewal within the CD34+ leukemic population, and may use TIM-3 signaling for self-renewal as in other FAB types of AML.Thus, APL with the S type, but not the L/V type PML-RARa have expression signature related to LSCs, and have functional LSCs with TIM-3 expression. Therefore, the leukemic hematopoiesis of APL with the S type is hierarchically organized as in case of AML of other FAB types. The mechanisms by which these 2 types of PML-RARa exert different functions to the LSC property should be critical to understand pathogenesis of APL. DisclosuresNo relevant conflicts of interest to declare.

Oral Arsenic and Retinoic Acid for Children with Non High-Risk Acute Promyelocytic Leukemia

ObjectiveAcute promyelocytic leukemia (APL) has now become a highly curable disease with the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) as first-line treatment. Adult non high-risk APL patients (WBC< 10×109/L) can be cured using only ATRA and ATO without chemotherapy. We also provided similar results using a totally oral (oral arsenic and ATRA), chemo-free outpatient protocol (Beijing protocol) for adult patients with non high-risk APL. However, experiences in children with APL are scarce. These prompted us to try to extend the Beijing protocol to children patients with non high-risk APL.MethodsWe conducted a single-centre pilot study to evaluate the efficacy of oral arsenic and ATRA for children patients with non high-risk APL from April 2014 through October 2016. Nine patients with the median age of 16 years old (range 13-18) were enrolled and received oral arsenic realgar-indigo naturalis formula (RIF, 60 mg/kg) and ATRA (25 mg/m2) as induction therapy until hematologic complete remission (CR). Hydroxyurea and/or cytarabine (without anthracyclines) were used to diminish the increased WBC during induction treatment. The post-remission therapy included RIF in a 4-week on and 4-week off , and ATRA in a 2-week on and 2-week off schedule for 7 months. The primary endpoint was the complete molecular response (CMR) post-consolidation, defined as absence of detectable PML-RARA transcripts by quantitative PCR. Secondary endpoints included CR, safety, hospital stay. Median follow-up was 15 months (range 6-36 months) by April 2017.ResultsAll 9 patients achieved hematologic CR after a median time of 30 days. CMR rate was 100% at 3 and 6 months. Grade1,2,3, and 4 liver adverse events was 2,3,1,0, respectively. Differentiation syndrome occurred in 3 patients during induction (Table1). No hematological relapse or molecular relapse occurred at the last follow-up. Both the 2-year estimated EFS and OS were 100%.The total hospital time was 17 days (4-37 days) during the treatment phase. Of note, 100% (9/9) of patients completed the post-remission therapy on an outpatient basis without hospitalization. Patients resumed their usual lifestyle during post-remission therapy and their QoL was nearly normal during post-remission therapy.ConclusionsOur study, which employed a largely home-based treatment protocol with complete oral regimen, chemo-free during induction in children patients with non high-risk APL, suggested to be effective, convenient. However, lager sample studies and long-term outcome are warranted. DisclosuresNo relevant conflicts of interest to declare.

All-trans Retinoic Acid, Arsenic Trioxide, and Anthracycline-based Chemotherapy Improves Outcome in Newly Diagnosed Acute Promyelocytic Leukemia Regardless of FLT3-ITD Mutation Status.

All-trans retinoic acid (ATRA) and pre-upfront arsenic trioxide (ATO) have revolutionized the therapy of acute promyelocytic leukemia (APL). However, internal tandem duplication of FMS-like tyrosine kinase 3 (FLT3-ITD) mutations is associated with increased risk of relapse. The aim of this study was to analyze the prognostic impact of FLT3-ITD on APL patients who received remission induction with ATRA, idarubicin (IDA) and/or ATO, followed by ATRA plus ATO along with anthracycline, as consolidation therapy. A total of 72 patients newly diagnosed with APL were included in this study. 83.3% of the patients achieved complete remission (CR) after induction therapy. FLT3-ITD mutations were detected in 16 (22.2%) patients and closely related to bcr-3 PML-RARa transcript (P<0.001). The 5-year overall survival (OS) rate was 100% in both FLT3-ITDpositive and FLT3-ITDnegative groups, and there was no significant difference in 5-year event-free survival (EFS) between the two groups (78.3% vs. 83.3%, P=0.85). ATRA plus ATO and anthracycline-based chemotherapy achieved great outcome in newly diagnosed APL regardless of the FLT3-ITD mutation status.

Characterization of Cryptic Rearrangements and Variant Translocations in Acute Promyelocytic Leukemia

AbstractAcute promyelocytic leukemia (APL) is typified by the reciprocal translocation, t(15; 17)(q22; q21), leading to the formation of PML-RARα and RARα-PML fusion genes. We have characterized 7 cases of morphologic APL found to lack the t(15; 17) on conventional cytogenetic assessment. In 6 of 7 cases, cryptic PML-RARα rearrangements were identified by reverse transcriptase-polymerase chain reaction and fluorescent in situ hybridization (FISH); whereas, in the remaining patient, APL was associated with the variant translocation, t(11; 17)(q23; q12-21), leading to the formation of PLZF-RARα and RARα-PLZF fusion genes. In each of the cases with cryptic PML-RARα rearrangements, PML-RARα transcripts were detected in the absence of RARα-PML, consistent with the concept that PML-RARα is the critical oncogenic fusion protein. In 4 of these cases with evaluable metaphase spreads, the occurrence of a nonreciprocal translocation was confirmed by FISH with sole formation of the PML-RARα fusion gene; in 3 cases with morphologically normal chromosomes 15 and 17, RARα was inserted into PML on 15q, whereas in the remaining patient the PML-RARα fusion arose due to insertion of 15q-derived material including PML into RARα on 17q. Immunofluorescence studies were performed using antibodies raised against PML and PIC 1, a ubiquitin-homology domain protein previously identified as an interaction partner of PML. In acute myeloid leukemia (AML) of subtypes other than M3, PIC 1 was localized to the nuclear membrane and colocalized with PML within discrete nuclear bodies. In APL cases with cryptic PML-RARα rearrangements, the characteristic microparticulate pattern of PML staining was detected with partial colocalization with PIC 1, indicative of disruption of the nuclear bodies; whereas in t(11; 17)-associated APL, PML and PIC 1 remained colocalized within discrete nuclear bodies, as observed in non-APL cases. Although deregulation of the putative growth suppressor PML and delocalization of other nuclear body constituents have been advocated to play a key role in the development of t(15; 17)-associated APL, the present study shows that disruption of PML nuclear bodies per se is not a prerequisite for the pathogenesis of APL.

No prognostic significance of normalized copy number of PML-RARA transcript at diagnosis in patients with acute promyelocytic leukemia

BackgroundAcute promyelocytic leukemia is a peculiar disease with few studies that have investigated the prognostic significance of PML/RARA transcript level at diagnosis. Patients and methodsThis retrospective study included all cases diagnosed with acute promyelocytic leukemia over the period from June 2015 to March 2019. The normalized copy number (NCN) was tested by real-time polymerase chain reaction at diagnosis, and at the end of induction regimen. ResultsOur study included 83 de novo APL patients, 53 (63.9%) were adults and 30 (36.1%) were children. The median (range) age of our patients was 28.0 (1.0–70.0) years. The pediatric group had a significantly higher prevalence in males (p = 0.02), higher incidence of disseminated intravascular coagulopathy (p = 0.014), and high-risk groups (p = 0.017). At diagnosis, the median NCN (%) of the entire group at 22.5 was set as the cut off value. There was no significant association between NCN at diagnosis and other prognostic variables except for bone marrow promyelocytes (p = 0.006). High-risk group APL patients as well as those presenting with hemorrhage had an inferior overall survival (OS) (p = 0.007; p < 0.001) respectively. PML-RARA NCN at diagnosis did not have an impact on the OS or increased risk of relapse of our patients (p = 0.434; p = 0.721). Conclusionthe initial PML/RARA tumor burden is not a prognostic factor for APL. The initial TLC at 10x109/L cut off is the most important predictive for OS. Early detection and close monitoring are required to decrease the high rate of early deaths in developing countries.

Acute promyelocytic leukemia in a child with reticulin fibrosis

Reactive myelofibrosis (MF) may be associated with infectious, inflammatory, or neoplastic conditions. Extensive myelofibrosis and indolent course are frequent in acute megakaryoblastic leukemia, but these findings are rarely reported in acute promyelocytic leukemia (APL). Patients with neoplastic disorders associated with MF are reported to have an inferior outcome. However, the effect of this association is not apparent in APL patients. This report presents a case of APL in a pediatric patient in association with significant reticulin fibrosis and osteosclerosis. The case highlights that fibrosis in the marrow can make it more challenging to diagnose acute leukemia. The PML-RARA transcripts can be present with subtle clinical or hematological findings, emphasizing the importance of molecular studies in unexplained cytopenias. Furthermore, in the case of APL, as opposed to other myeloid neoplasms, fibrosis does not appear to be associated with a worse prognosis and can resolve after treatment.

MiR-638 in circulating leukaemia cells as a non-invasive biomarker in diagnosis, treatment response and MRD surveillance of acute promyelocytic leukaemia.

MicroRNA (miRNA) expression has been implicated in leukaemia. In recent years, miRNAs have been under investigation for their potential as non-invasive biomarkers in acute promyelocytic leukaemia (APL). We investigated whether miR-638 in circulating leukaemia cells is a non-invasive biomarker in diagnosis, assessment of the treatment response and minimal residual disease (MRD) surveillance of APL. Sixty cases of acute myeloid leukaemia (AML), including 30 cases of APL and 30 cases of non-APL AML, were selected. Thirty healthy controls were also selected. Bone marrow (BM) and peripheral blood (PB) samples were collected from APL patients at diagnosis and post-induction. Microarray analysis and quantitative real-time PCR (qRT-PCR) were performed for miRNA profiling and miR-638 expression analysis, respectively. For statistical analysis, Mann-Whitney U test, Wilcoxon Signed Rank test, receiver operating characteristic (ROC) curve analysis and Spearman's rho correlation test were used. Both microarray and qRT-PCR data showed that miR-638 was significantly upregulated in BM after APL patients received induction therapy. Moreover, miR-638, which is specifically downregulated in APL cell lines, was upregulated after all-trans retinoic acid (ATRA)-induced myeloid differentiation. Receiver operating characteristic (ROC) curve analyses revealed that miR-638 could serve as a valuable biomarker for differentiating APL from controls or non-APL AML. Furthermore, miR-638 expression was sharply increased after induction therapy and complete remission (CR). An inverse correlation was observed between miR-638 and PML-RARα transcripts levels in BM samples, while a positive correlation was revealed between PB miR-638 and BM miR-638 levels in APL patients after induction therapy. Our study suggested that miR-638 may serve as a potential APL biomarker for diagnosis and assessment of the response to targeted therapy, and PB miR-638 could be used for non-invasive MRD surveillance in APL.

Clinical significance of increased PML-RARa transcripts after induction therapy for acute promyelocytic leukaemia

Objective: To analyze the clinical and biological significance of the acute promyelocytic leukemia (APL) whose PML-RARa transcripts increased after induction therapy.Methods: We analyzed 9 cases of APL whose PML-RARa transcripts increased after induction treatment and compare them with APL whose PML-RARa transcripts decreased.Results: The only factor affecting increased PML-RARa transcripts was the induction protocol. The cases of increased PML-RARa transcripts received induction treatment mainly based on ATRA and ATO. The evaluation of bone marrow aspirate cytology showed that the cell percentage from myelocyte to segmented neutrophil of the patients with increased PML-RARa transcripts was significantly higher than that of the patients with decreased PML-RARa transcripts. In the follow-up, MRD in 9 cases was consistently negative.Conclusions: Our studies showed the increased PML-RARa transcripts after induction treatment had different clinical significance from the decreased PML-RARa transcripts.

Laboratory Assays in Patients with Acute Promyelocytic Leukemia and FLT3-TKD Mutations

Acute Promyelocytic Leukemia (APL) is a well-characterized subtype of Acute Myeloid Leukemia (AML). The majority of patients with acute promyelocytic leukemia harbor the reciprocal translocation of t(15;17)(PML-RARα). Recent studies have shown that FMS-Like tyrosine kinase receptor-3 (FLT-3) mutations occur in approximately onethird of AML patients. Two major categories of clinically significant FLT3 activating mutations have been identified: Internal tandem duplication (ITD) mutations within the juxtamembrane domain of FLT-3 gene and missense point mutations in the tyrosine kinase domai. Several studies have demonstrated a poor overall survival with FLT-3 ITD mutation which is found in approximately 20-40% of AML patients. In contrast, the prognostic significance of FLT-3 TKD alone is still unclear, but some studies indicate that it also confers a poor prognosis in AML patients. The current study was performed on 66 [Male: 27(41%); Female: 39(59%)] APL patients to analyze and compare hematological parameters between APL patients with wild-type FLT-3(FLT3-WT) [87% (n=58)] and APL patients with mutant FLT3- TKD [12% (n=8)]. Quantification of PML-RARα transcripts was performed by Real-time quantitative PCR. FLT3-TKD mutation was detected by Polyacrylamide Gel Electrophoresis and CBC values were measured with a SYSMEX automated hematology analyzer. The participants of both cohorts were stratified into high-risk and low-risk groups based on their hematological parameters. The findings indicated that APL patients with FLT-3 TKD mutation had laboratory features with more favorable outcome compared with patients with wild-type FLT-3.

Detection of Exosomal PML-RARA Fusion Gene Expression Level by Droplet Digital PCR

To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR). By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique. The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected. A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.

PS1045 INDIRECT NUMBER NEEDED TO TREAT: COMPARATIVE EFFECTIVENESS OF GLASDEGIB + LOW-DOSE CYTARABINE VS AZACITIDINE OR DECITABINE IN ACUTE MYELOID LEUKEMIA PATIENTS INELIGIBLE FOR INTENSIVE CHEMOTHERAPY

Background: Acute promyelocytic leukemia (APL) has a characteristic peculiar morphologic, genetic features and more favorable outcome. Aims: is to study the differential effect of BCR-1 and BCR-3 isoforms of PML-RARA together with FLT3-ITD mutational status on outcome of APL patients. Methods: All patients referred to the outpatient medical and pediatric oncology clinic, National cancer institute (NCI), Cairo University, Egypt in the period from May 2012 to January 2018. The diagnosis of APL was based on morphologic examination of the bone marrow and peripheral blood smears and was confirmed by the detection of the PML-RARA transcript by FISH and RT-PCR. Results: the study included 118 patients with newly diagnosed de novo APL (71 (60%) adults and 47 (39.8%) children). Based on the initial TLC, pediatric APL patients have higher (48.9%) risk than adults (30%) (p = 0.044). The CR rate was 91% (77/84), after excluding early induction related deaths (18) and patients who did not receive treatment at NCI (no beds available) (16). All pediatric patients (both BCR-1 and BCR-3) and all adult patients with BCR-3 achieved CR while 22/28 (78.5%) of patients with BCR-1 achieved CR (p = 0.04). Adult patients with FLT3 mutation (5/8 = 62.5%) received more high-risk regimen than patients with wild FLT3-ITD (10/38 = 26.3%)(p = 0.047). FLT3-ITD mutation was associated with more hemorrhage and DIC (p = 0.038), and lower 2-year OS (96% v 44.6%)(p = 0.001) in pediatric patients. Also DIC and Hemorrhage are associated with lower 2-year OS in pediatric patients (91.4% v 48.6%)(p < 0.001). The 1-year and 2-year OS for pediatric patients were better than adults (56.2% v 83% and 80.7 % v 49.8% respectively) with a median of 39.4 month versus 12month respectively (p = 0.005). On multivariate analysis, Age was an independent prognostic variable affecting survival, HR 2.6 (95% CI 1.3–5.3, P = 0.007)(adults had hazards ratio of death 2.6 times higher than pediatrics). DIC & FLT3-ITD were independent prognostic variables affecting survival in the pediatric APL with HR 12.3 (95% CI 1.46–104.61, P = 0.021) and 5.2 (95% CI 1.01–26.95, P = 0.048) respectively.Summary/Conclusion: BCR-1 isoform of PML-RARA is associated with decreased response to ATRA-based regimens in adult APL patients. DIC & FLT3-ITD are adverse prognostic factors in pediatric APL patients. Age is an independent prognostic factor in APL patients (whole group). Despite higher high risk groups in pediatric patients, the outcome is better than adult patients.