Positive Acute Promyelocytic Leukemia Research Articles

Trinary Fusion with Ligand Binding Domain Castration Is Crucial in Rarg-Driven APL and Render Unresponsive to ATRA Via Allosteric Disability Mechanism

Introduction 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 that manifest as APL, and they often carry FGs involved in RARA, RARB, or RARG. X::RARG positive APL ( RARG-APL) cases have been increasingly reported in recent years, thanks to the utilization of transcriptome sequencing (RNA-seq) and whole genome sequencing (WGS). Almost all evaluable RARG-APL cases were resistant to ATRA, and no mechanism has been elucidated. Intriguingly, cells transformed by artificial X::RARG were extremely sensitive to ATRA (PMID: 25510432), indicating intricate mechanisms unrevealed. This study first and systematically unveils the distinctive features and pivotal molecular mechanism of RARG-driven APL. Methods A total of 22 RARG-APL cases with RNA-seq and WGS data from 11 centers were enrolled (Fig. 1A). We used Arriba for routine FG calling and manually investigated the non-coding fusion sequence. Reverse transcription PCR (RT-PCR) and Sanger sequencing were used to validate the FGs and to determine whether the RARG 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 15 males and 7 females, aged 0.9-69 (median 38) years. Morphological and immunophenotype analyses all showed features of APL. Routine FGs calling reported RARG fusion to a 5′ partner in each case, with variable RARG splicing sites (Fig. 1A). There were 7 RARG 5' partner genes, with CPSF6, HNRNPC, and NUP98 observed in multiple cases. In the 3 index cases, we identified that they all had RARG 3‘ fusion events besides the 5‘ one (Fig. 1A). RT-PCR confirmed that the 5′ and 3′ RARG fusion events were in the same cistron in each case. Such as, the fusion transcript is the insertion of RARG-e4_9 (e refers to exon) between NPM1-e4 and e11 in case #G1, rather than two separate transcripts of NPM1-e4:: RARG-e4 and RARG-9:: NPM1-e11. We named this novel form of tandem splicingtrinary fusion. Analysis of the 19 validation cases further confirmed that all RARG-APL cases had RARG 3′ fusion events (Fig. 1A). We also validated that the 5′ and 3′ RARG fusion events were in the same cistron in 3 more cases, of which archived cDNA were available. Remarkably, the 3' splicing sits of all cases were consistently at the end of RARG-e9, leading to RARG-e10 truncation. The RARG 3' partner gene was the same as its 5' partner in 11 cases. But in the other 11 cases, the RARG 3' partner was a transposon element (TE) sequence, the most common being a LINE-L2a (8 cases). Sequences analysis indicated that the committed locus of the involved LINE-L2a was at 4.2 Kbp upstream of the RARG gene. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARG 3' fusions confer a poly_A signal sequence to the fusion transcript, which was essential for a mRNA. In-depth analysis indicated that TEs participate in the formation of RARG-FGs through a transposition mechanism rather than a translocation mechanism. RARG-e10 encodes helix 11_12 (H11_12) of its ligand binding domain (LBD), which plays a pivotal role in allosteric response to ATRA (PMID: 31178221). The trinary fusion protein with RARG LBD-H11_12 castration will lose responsiveness to ATRA via an allosteric disability mechanism (Fig. 1B, 1C). Experiments confirmed that cells transfected with X::RARG fusion with intact LBD responded well to ATRA, while fusion with LBD-H11_12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1D, 1E). Conclusions This study clearly revealed that trinary fusion and LBD castration are crucial molecular etiologies in RARG-APL. The trinary fusion orchestrates the aberrant activation of RARG (5‘ splicing) and the complete allosteric disability in responding to ATRA (3‘ splicing). Thus, this study elegantly explains the mechanism of leukemogenesis and the extensive ATRA resistance of RARG-APL. The formation of trinary fusion requires two steps of genome splicing or transposition events, which explains the rarity of RARG-APL. This also clarifies that RARG-APL has a significant disparity in molecular mechanism from PML::RARA-positive APL, and we suggest that it should be considered as a separate entity of acute myeloid leukemia.

Donor cell leukemia after allogeneic hematopoietic stem cell transplantation: a case report

A 48-year-old man was diagnosed Acute myeloid leukemia with FLT3 ITD positive and PML/RAR?- negative. Remission was achieved after induction and consolidation chemotherapy. Allogeneic hematopoietic stem cell transplantation was performed from his full matched sibling donor. He relapsed after 5 years and his bone marrow examination revealed PML/RAR?- positive Acute promyelocytic leukemia. t(15:17) was positive and FLT-3 ITD was negative. Cytogenetic and molecular analysis confirmed donor cell origin. Donor didn’t develop Acute promyelocytic leukemia.

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.

22. Reducing diagnostic FISH turn-around-time: A rapid pipeline for BCR::ABL1 and PML::RARA suspected leukemias

Successful long-term outcome of patients with BCR::ABL1 positive chronic myeloid leukemia (CML) and PML::RARA positive acute promyelocytic leukemia (APL) is critically dependent on rapid cytogenetic and molecular studies. Fluorescence in situ hybridization (FISH) serves as a standard-of-care diagnostic assay in the workup of suspected BCR::ABL1 and PML::RARA positive leukemias. Nonetheless, turn-around-time for FISH results can be upwards of days, delaying highly-effective personalized treatments. To reduce FISH turn-around-time for patients with a suspected BCR::ABL1 or PML::RARA positive leukemia, our cytogenetics laboratory developed a rapid specimen processing algorithm to consistently return diagnostic BCR::ABL1 and PML::RARA FISH results within 24-hours from sample receipt. Our algorithm, a time-dependent decision matrix, leverages modified sample culturing, harvesting, and FISH hybridization protocols to return diagnostic BCR::ABL1 and PML::RARA FISH results as quickly as 6 hours from sample receipt. This pipeline enables accelerated treatment for patients with BCR::ABL1 and PML::RARA positive leukemias.

Metachronous occurrence of gastrointestinal stromal tumor and acute promyelocytic leukemia in a patient with ankylosing spondylitis: A case report

Gastrointestinal stromal tumors are the most common mesenchymal tumor of the alimentary canal. Acute promyelocytic leukemia (APL) is a unique subtype of acute myeloid leukemia. A 57-year-old male patient with a previous record with history of gastrointestinal stromal tumors (GIST) and ankylosing spondylitis, was hospitalized with right ankle and foot swelling and pancytopenia. A diagnosis of APL was made by bone marrow aspiration and cytogenetic study. After a combined treatment with all-trans retinoic acid and arsenic trioxide, the patient achieved cytogenetic and molecular remission. According to past studies, the synchronous or metachronous coexistence of GISTs with other malignancies, such as lymphoma, liver cancer and pancreatic tumors has been widely reported. This study describes a patient with the metachronous occurrence of a CD34 positive GIST and APL. We suggest, the minor alterations in hematological parameters must be taken into consideration for such cases.

Arsenic trioxide induces proteasome dependent TBLR1-RARα degradation to improve leukemia eradication through cell differentiation enhancement.

Background: Acute promyelocytic leukemia (APL) mainly harbors PML-RARα fusion gene, which is sensitive to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) treatment. However, APL harboring other RARα fusion genes exhibit different drug sensitivity. Here, we investigated the role and mechanism of TBLR1-RARα, a rare RARα fusion gene, on ATO treatment in leukemia cells.Methods: By constructing two cell models of leukemia cell line HL-60 and U937 with overexpressed TBLR1-RARα, we detected the cell differentiation in the two cell models after ATO treatment by flow cytometry and Wright staining. Meanwhile, cell viability, colony formation and apoptosis were also determined after ATO treatment.Results: We found that TBLR1-RARα enhanced ATO-induced apoptosis and cell proliferation inhibition. Besides, TBLR1-RARα also promoted ATO-induced cell differentiation. Furthermore, we found that the mitochondrial caspase pathway was involved in the apoptosis induced by ATO treatment in TBLR1-RARα positive leukemia cells. Moreover, ATO mediated TBLR1-RARα protein degradation via proteasome pathway, which accounts for the transcriptional activation of RARα target gene and is further involved in cell differentiation of TBLR1-RARα positive leukemia cells.Conclusions: Our study provides evidence that TBLR1-RARα positive APL patients may benefit from ATO treatment, thereby improving the appropriate management in TBLR1-RARα positive APL.

Outcome and complications of pediatric acute promyelocytic leukemia in Bangladesh

Pediatric acute promyelocytic leukemia (APL) is one of the most curable subtypes of acute myeloid leukemia of childhood. But it may have many early complications, especially in developing countries. This study aims to describe the outcome and complications of pediatric APL patients in Bangladesh. This prospective observational study was conducted in the pediatric hematology and oncology department of Bangabandhu Sheikh Mujib Medical University, Dhaka from September 2017 to March 2019. In this study, PML:RAR-α (Promyelocytic leukemia-retinoic acid receptor-α) positive APL cases were included and observed while being treated with risk-directed ATRA (All-trans-retinoic acid) based chemotherapy. Among twenty PML:RAR-α positive APL cases, 13 children were in the high risk group and hemorrhagic manifestations were present in 95% of patients. Post-induction remission was achieved in 85% of the patients. 3-year overall survival was 70% (45-85% with 95% confidence interval). There was no refractory disease or relapses. Neutropenic sepsis was the most common complication and also the most common cause of mortality. In Bangladesh, the 3-year overall survival of pediatric APL is 70% (45-85% with 95% CI). Post-chemotherapy neutropenic sepsis is the most common complication and also the most common cause of mortality in this potentially curable malignancy in Bangladesh.

A Modified AIDA Protocol With Anthracycline-Based Consolidation Results in High Antileukemic Efficacy and Reduced Toxicity in Newly Diagnosed PML/RAR∝-Positive Acute Promyelocytic Leukemia: Presented in part at the 40th meeting of the American Society of Hematology, Miami Beach, FL, December 4-8, 1998.

AbstractThe Spanish PETHEMA group designed a protocol for newly diagnosed PML/RAR∝-positive acute promyelocytic leukemia (APL) in which induction and consolidation followed the original AIDA regimen, except for the omission of cytarabine and etoposide from consolidation. Induction consisted of 45 mg/m2 all-trans retinoic acid (ATRA) daily until complete remission (CR) and 12 mg/m2 idarubicin on days 2, 4, 6, and 8. Patients in CR received 3 monthly chemotherapy courses: idarubicin 5 mg/m2/d × 4 (course no. 1), mitoxantrone 10 mg/m2/d × 5 (course no. 2), and idarubicin 12 mg/m2/d × 1 (course no. 3). Maintenance therapy consisted of 90 mg/m2/d mercaptopurine orally, 15 mg/m2/wk methotrexate intramuscularly, and, intermittently, 45 mg/m2/d ATRA for 15 days every 3 months. Between November 1996 and December 1998, 123 patients with newly diagnosed PML/RAR∝-positive APL from 39 centers were enrolled. A total of 109 patients achieved CR (89%; 95% confidence interval [CI], 83 to 95), 12 died of early complications, and the remaining 2 were resistant. Consolidation treatment was associated with very low toxicity and no deaths in remission were recorded. Molecular assessment of response by reverse transcriptase-polymerase chain reaction (RT-PCR) showed conversion to PCR-negative in 48 of 99 (51%) and 82 of 88 patients (93%) after induction and consolidation, respectively. The 2-year Kaplan-Meier estimates of overall survival and event-free survival were 82% ± 4% and 79% ± 4%, respectively. For patients who achieved CR, the 2-year disease-free survival (DFS) was 92% ± 3%. These data indicate that a significant reduction in toxicity might be obtained in APL using a less intensive consolidation without apparently compromising the antileukemic effect. These results also suggest a minor role for cytarabine and etoposide in the treatment of newly diagnosed PML/RAR∝-positive APL patients.

Deep Learning for Distinguishing Morphological Features of Acute Promyelocytic Leukemia

Acute Promyelocytic Leukemia (APL) is a subtype of Acute Myeloid Leukemia (AML), classified by a translocation between chromosomes 15 and 17 [t(15;17)], that is notably distinguished clinically by a rapidly progressive and fatal course. Due to the acute nature of its presentation, prompt and accurate diagnosis is required to initiate appropriate therapy that can be curative. However, the gold standard genetic tests can take days to confirm a diagnosis and thus therapy is often initiated on high clinical suspicion based on both clinical presentation as well as direct visualization of the peripheral smear. While there are described cellular morphological features that distinguish APL, there is still considerable difficulty in diagnosing APL from direct visualization of a peripheral smear by a hematopathologist. We hypothesized that deep learning pattern recognition would have greater discriminatory power and consistency compared to humans to distinguish t(15;17) translocation positive APL from t(15;17) translocation negative AML. To best tackle the problem of diagnosing APL rapidly from a peripheral smear, study patients with APL and AML were identified via retrospective chart review from a list of confirmed FISH t(15;17)-positive (n = 34) and -negative (n = 72) patients presenting at The Johns Hopkins Hospital (JHH). Additional inclusion criteria included new disease diagnosis, no prior treatment, and availability of peripheral blood smear image uploaded to CellaVision. Patients were separated into a discovery cohort presenting prior to 1/2019 (APL, n = 22; AML, n=60) and a validation cohort presenting on or after 1/2019 (APL, n = 12; AML, n = 12). A multiple-instance deep learning model employing convolutional layers at the per-cell level (Figure 1A) was trained on the discovery cohort and then tested on the independent prospective validation cohort to assess generalizability of the model. When compared to 10 academic clinicians (denoted with red +) who consisted of leukemia-treating hematologists, oncologists, and hematopathologists, the deep learning model was equivalent or outperformed 9/10 readers (Figure 1B) with an AUC of 0.861. We further looked at the performance of using proportion of promyelocytes (per CellaVision classification) as a biomarker of APL which had an AUC of 0.611. Finally, we applied integrated gradients, a method by which to extract per-pixel importance to the classification probability to identify and understand the morphological features the model was learning and using to distinguish APL (Figure 1C). We noted that the appearance of the chromatin in the non-APL leukemias was more dispersed and focused at the edge of the cell whereas in APL, the chromatin was more condensed and focused at the center of the cell. These morphological features, taught to us by the model, have not been previously reported in the literature as being useful for distinguishing APL from non-APL. Our work presents a deep learning model capable of rapid and accurate diagnosis of APL from universally available peripheral smears. In addition, explainable artificial intelligence is provided for biological insights to facilitate clinical management and reveal morphological concepts previously unappreciated in APL. The deep learning framework we have delineated is applicable to any diagnostic pipeline that can leverage a peripheral blood smear, potentially allowing for efficient diagnosis and early treatment of disease. Figure 1. Disclosures Streiff: Bayer: Consultancy, Speakers Bureau; Dispersol: Consultancy; BristolMyersSquibb: Consultancy; Janssen: Consultancy, Research Funding; Pfizer: Consultancy, Speakers Bureau; Portola: Consultancy; Boehringer-Ingelheim: Research Funding; NHLBI: Research Funding; PCORI: Research Funding; NovoNordisk: Research Funding; Sanofi: Research Funding. Moliterno:Pharmessentia: Consultancy; MPNRF: Research Funding. DeZern:MEI: Consultancy; Abbvie: Consultancy; Astex: Research Funding; Celgene: Consultancy, Honoraria. Levis:Astellas: Honoraria, Research Funding; Menarini: Honoraria; Amgen: Honoraria; FujiFilm: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria.

Cerebral Thrombosis As an Initial Presentation of Pediatric Acute Promyelocytic Leukemia

Disseminated intravascular coagulopathy and bleeding are common manifestations of acute promyelocytic leukemia(APL) which are both associated with significant mortality and morbidity. However APL related thrombosis is very rare and majority of the reported cases are adults in the literature. In the past 14 years, 14 cases with APL were found in our hospital database.There were 6 female and 8 male children with a mean age of 10.7±4.6 years (2-17y). Here, we report two pediatric APL case who presented with cerebral thrombosis at the initial presentation. 8/14(57%) had disseminated intravascular coagulation at the initial presentation and one patient had intracardiac and cerebral sinovenous thrombosis during the induction period. Case 1:A 13-year-old boy was admitted to our hospital with a complaint of headache for two days. His physical examination revealed a papilledema and cranial MRI did show sinovenous thrombosis. At the time of the evaluation his WBC count was found to be low and there were myeloid blasts on his peripheral smear,bone marrow aspiration was done and diagnosed as translocation t(15;17)positive APL. He remain in remission more than 4 years without any event however deceased after relapse. Case 2: A 8-year-old boy was admitted to our hospital with a complaint of right hemiparesis, there was a thrombotic occlusion of the left middle cerebral artery associated cerebral infarct on his MRI.His WBC was 5.9x109/l with 76% blasts and bone marrow aspiration and flow-cytometry were both compatible with APL. Patient died after 2 weeks of admission because of thrombosis and bleeding. The incidence of APL related thrombosis was 4.5 and 5.6% in two large adult cohorts. Both arterial and venous thrombosis may ocur in patients with APL. To the best of our knowledge there were limited number of pediatric APL case reports in the literature who presented with thrombosis. In our small group of pediatric APL patients 14% presented with thrombosis and the total number of thrombotic events were found to be 28% during the follow up. Disclosures No relevant conflicts of interest to declare.

Insulin-like Growth Factor Binding Protein 7 Activates the Retinoid Acid Differentiation Pathway in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired transcriptional differentiation programs. Despite important advances in AML therapy, the five-year overall survival rate of AML patients remains a disappointing 30-40%. This poor prognosis is mainly caused by survival of chemotherapy resistant leukemic cells, named leukemic stem cells (LSC), re-initiating relapse. However, for AML patients with PML-RARA positive acute promyelocytic leukemia (APL), treatment with all trans retinoic acid (ATRA) results in cure rates of >90%. Upon ATRA treatment, APL cells can restore transcription leading to granulocytic differentiation, and in combination with arsenic trioxide APL cells go into apoptosis. While the success of ATRA treatment has been demonstrated for APL patients, so far it has not proved effective for non-APL AML patients.Previously, we demonstrated that insulin-like growth factor binding protein 7 (IGFBP7), a negative regulator of IGF1 receptor (IGF1R) activity, induces apoptosis of AML cells and sensitizes AML cells to chemotherapy-induced cell death. Since it has been shown that IGF1R inhibitors can eliminate therapy-resistant cells by modifying their chromatin state, we hypothesized that IGFBP7 may also have the capacity to modify an epigenetic state and unlock the ATRA-driven differentiation response. To this end, we generated APL cell lines with down- or upregulated IGFBP7 levels and demonstrated that knockdown of IGFBP7 in NB4 cells blocked ATRA-induced differentiation, whereas overexpression of IGFBP7 led to an 8-fold increase in differentiation in the presence of low concentrations of ATRA, together suggesting a role for IGFBP7 in ATRA-induced differentiation in APL cells.Strategies to increase efficacy of ATRA-based therapy might also improve treatment outcomes for non-APL AML, and therefore we investigated the potential of IGFBP7 to induce susceptibility for ATRA-driven differentiation in this group. ATRA and IGFBP7 treatment of non-APL AML cell lines and primary AML cells derived from patients at diagnosis demonstrated an enhanced efficiency of the combination therapy to induce differentiation of myeloid CD45dimCD33+ AML cells (2.5-fold increase in CD11b-expression) and/or to reduce viability of AML CD45dim cells as compared to single treatments (from 30-39% to 70% reduction upon IGFBP7, ATRA or combination therapy, respectively), in 50% of tested primary AML samples, while this combination therapy did not influence normal hematopoietic cell survival. Remarkably, ATRA-IGFBP7 treatment diminished the in vivo engraftment potential of primary AML cells as compared to treatment with either drug alone in NSG mice (from 28% and 52% to 17% engraftment upon ATRA, IGFBP7 or combination therapy, respectively). Re-transplantation of human AML derived from first transplanted mice into secondary recipients demonstrated that the ATRA-IGFBP7 combination treatment also eliminated LSC more effectively (1.4-fold reduction). Together these data suggest that IGFBP7 enhances sensitivity of AML (stem) cells to ATRA, and is able to induce a transcriptional program sensitizing AML cells for ATRA-induced differentiation and cell death.To identify factors responsible for IGFBP7-induced ATRA sensitivity, we performed gene expression profiling of primary AML samples treated with IGFBP7, and identified growth factor independent protein 1 (GFI1) as one of the top down-regulated genes upon IGFBP7 stimulation. As overexpression of GFI1 in non-APL AML patient samples resulted in a >2.5-fold reduction in IGFBP7-induced susceptibility to ATRA-driven differentiation, low GFI1 expression is suggested to be associated with susceptibility to ATRA in AML cells.In conclusion, our results indicate that treatment of AML patient with a combination of ATRA and IGFBP7 might be successful in preventing relapse and improving AML patient survival, which has to be confirmed in further clinical studies. DisclosuresOssenkoppele:Karyopharm: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Genmab: Research Funding; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding.

Multicenter randomized trial of arsenic trioxide and Realgar-Indigo naturalis formula in pediatric patients with acute promyelocytic leukemia: Interim results of the SCCLG-APL clinical study.

Intravenous arsenic trioxide (ATO) has been adopted as the first‐line treatment for acute promyelocytic leukemia (APL). Another arsenic compound named the Realgar‐Indigo naturalis formula (RIF), an oral traditional Chinese medicine containing As4S4, has been shown to be highly effective in treating adult APL. In the treatment of pediatric APL, the safety and efficacy of RIF remains to be confirmed. This randomized, multicenter, and noninferiority trial was conducted to determine whether intravenous ATO can be substituted by oral RIF in the treatment of pediatric APL. From September 2011 to January 2017, among 92 patients who were 16 years old or younger with newly diagnosed PML‐RARa positive APL, 82 met eligible criteria and were randomly assigned to ATO (n = 42) or RIF (n = 40) group. The remaining 10 patients did not fulfilled eligible criteria because five did not accept randomization, four died and one had hemiplegia prior to arsenic randomization due to intracranial hemorrhage or cerebral thrombosis. Induction and consolidation treatment contained ATO or RIF, all‐trans‐retinoic acid and low intensity chemotherapy. End points included event‐free survival (EFS), adverse events and hospital days. After a median 3‐year follow‐up, the estimated 5‐year EFS was 100% in both groups, and adverse events were mild. However, patients in the RIF group had significantly less hospital stay than those in the ATO group. This interim analysis shows that oral RIF is as effective and safe as intravenous ATO for the treatment of pediatric APL, with the advantage of reducing hospital stay. Final trial analysis will reveal mature outcome data.

HLA‐DR Positive Acute Promyelocytic Leukemia (APL)

BACKGROUNDAcute promyelocytic leukemia (APL) with t(15;17)(q22;q21)/PML‐RARα is a subtype of acute myeloid leukemia(AML) with distinct morphologic and immunophenotypic characteristics. It is a highly aggressive disease that requiresrapid diagnosis and early intervention. Compared with other types of AML, APL typically displaysa triad of absent or weak CD34, absent HLA‐DR, and positive CD117. HLA‐DR positive APL is extremely rareand its clinical and pathological features have not been reported. A total of 45 cases of APL with t(15,17)/PMLRARα were diagnosed at Harbor‐UCLA Medical Center from year 2006 to 2015. Among them, only two cases were positive for HLA‐DR by flow cytometry immunophenotyping. Here we describe the clinical, morphologic, immunophenotypic, and cytogenetic features of these two cases.RESULTS AND DISCUSSIONThe patients in both cases presented with anemia, thrombocytopenia, and decreased WBC count. In case A, the peripheralblood smear showed many myeloblasts and only occasional abnormalpromyelocytes. Although a thin Auer rod was found in rare blasts, neitherthe thick Auer rods that typically seen in APL nor faggot cells were identified. While case B showed characteristic morphologic features of hypergranular variant of APL in the bone marrow, no blasts orabnormal promyelocytes were identified in the peripheral bloodsmear. Therefore, APL should not be excluded even when abnormalpromyelocytes are not present in the peripheral blood smears. In addition, MPO cytochemical stain performed on the bone marrow aspiratesmear of case A did not show uniform strong staining pattern that is commonly observed in APL. Besides HLA‐DR, CD34 was also expressed on the blasts of case A. These atypical morphologic and immunophenotypic features may be attributed to the additional cytogenetic abnormalities found in case A.CONCLUSIONHLA‐DR‐positive APL is very rare and represents about 4% of all APL cases in our study. Due to its rare occurrence, continuous reporting of HLA‐DR positive APL will bring to the awareness of the broad clinical and pathological spectrum of APL, provide better clinicopathologic correlation, and facilitate rapid diagnosis and thus promptinitiation of appropriate management of this aggressive malignant tumor.Support or Funding InformationWe declare no benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

Therapy-related myelodysplastic syndrome after successful treatment of acute promyelocytic leukemia: case report and literature review

Abstract In the 2016 revision of the World Health Organization classification the term therapy-related myeloid neoplasia (t-MN) defines a subgroup of acute myeloid leukemia (AML) comprising patients who develop myelodysplastic syndrome (MDS-t) or acute myeloid leukemia (AML-t) after treatment with cytotoxic and/or radiation therapy for various malignancies or autoimmune disorders. We report the case of a 36 year old patient with t-MN (t-MDS) after achieving complete remission (CR) of a PML-RARA positive acute promyelocytic leukemia (APL) at 32 months after diagnosis. Initially classified as low risk APL and treated according to the AIDA protocol - induction and 3 consolidation cycles - the patient achieved a complete molecular response in September 2013 and started maintenance therapy. On follow-up PML-RARA transcript remained negative. In January 2016 leukopenia and thrombocytopenia developed and a peripheral blood smear revealed hypogranular and agranular neutrophils. Immunophenotyping in the bone marrow aspirate identified undifferentiated blast cells that did not express cytoplasmic myeloperoxidase. The cytogenetic study showed normal karyotype. The molecular biology tests not identified PMLRARA transcript. A diagnosis of t-MDS (AREB-2 - WHO 2008) was established. Treatment of AML was started with 2 “3+7” regimens and 1 MEC cycle. Two months from diagnosis, while in CR, an allogeneic HSCT from an unrelated HLA compatible donor was performed after myeloablative regimen. An unfavorable clinical evolution was followed by death on day 9 after transplantation. The occurrence of t-MNs during CR of APL represents a particular problem in terms of follow-up and differential diagnosis of relapse and constitutes a dramatic complication for a disease with a favorable prognosis. This work was supported by the grants PN 41-087 /PN2-099 from the Romanian Ministry of Research and Technology

HLA-DR antigen-positive acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) with t(15;17)(q22;q21)/PML-RARα is a subtype of acute myeloid leukemia (AML) with distinct morphologic and immunophenotypic characteristics. It is a highly aggressive disease that requires rapid diagnosis and early intervention. In addition to morphologic evaluation, flow cytometry has been widely used to facilitate prompt diagnosis of this disease. Compared with other types of AML, APL typically displays a triad of absent or weak CD34, absent HLA-DR, and positive CD117. HLA-DR positive APL is extremely rare and its clinical and pathological features have not been reported. A total of 45 cases of APL with t(15,17)/PML-RARα were diagnosed at Harbor-UCLA Medical Center from year 2006 to 2015. Among them, only two cases were positive for HLA-DR by flow cytometry immunophenotyping. Here we describe the clinical, morphologic, immunophenotypic, and cytogenetic features of these two cases.

Coagulopathy in Acute Promyelocytic Leukemia: Strategies to Improve Assessment of Hemostatic Risk

In patients with acute promyelocytic leukemia (APL), in spite of recent advances, early treatment related mortality (TRM) still remains a challenge and is largely related to the associated coagulopathy. It is well recognized that conventional coagulation parameters used in the clinic do not accurately predict the risk of adverse outcomes like major bleeding or thrombosis. There is limited data on the clinical utility of whole blood viscoelastic assays and extended coagulation parameters for evaluation of coagulopathy in patients with APL.Consecutive patients with PML-RARA positive APL were enrolled. Blood samples were collected prior to infusion of blood products on day 1 and again on day 4 for thromboelastometry done on whole blood and activated with recombinant tissue factor at a final dilution of 1:50,000 (ROTEM analyzer, Tem Innovations GmbH, Basel) and an extended panel of coagulation parameters done by immunoassays (TAT complex, protein C, ATIII, PIC complex, tPAIC, d-dimer and thrombomodulin). Platelet count and conventional coagulation parameters, which were prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and thrombin time (TT), were measured daily for the first 14 days (more frequently as clinically indicated). Patients received platelet transfusions to target a platelet count of 30x109/L (in the absence of bleeding). Fresh frozen plasma and cryoprecipitate transfusions were done to target a normal PT and aPTT and maintain a fibrinogen level above 140 mg%. Major bleeding, thrombosis and death during induction therapy were the outcome variables.Of the 61 patients enrolled, 11 were excluded (3 did not continue treatment at our hospital, 2 died within 48 hours of admission, and 6 took discharge against medical advice before completing induction). Of the remaining 50 cases that were evaluated, 40 were newly diagnosed and 10 were relapsed. The median age was 34.5 years (range 9-68). 25 were males (50 %). The WBC count was above 10 x 109/L in 20 patients (40%). The median platelet count was 28 x 109/L (range 4-339). There were 13 (26%) deaths in induction. Six patients had a major bleeding event. Five patients had a thrombotic event. Of the evaluated conventional coagulation parameters, extended coagulation parameters, platelet counts and ROTEM parameters at diagnosis (ROTEM data not available at diagnosis in 2 cases) only the ROTEM parameter of maximum clot firmness (MCF) as a continuous variable was significantly associated with death (p=0.012). On univariate cox regression analysis with death as the outcome, MCF≤ 30mm was a poor prognostic marker (hazard ratio of 13.24, 95%CI 1.71-102.19; p =0.013). On multivariate cox regression analysis including high WBC count (>10 x 109/L), low platelet count (< 40 x 109/L) and history of relapse, MCF ≤30mm was an independent poor prognostic variable (HR: 11.89; 95% CI 1.43 - 98.75; p 0.022). Four of the 6 major bleeding events, four of five thrombotic events and all deaths related to coagulopathy occurred in patients with MCF ≤30mm. The descriptive statistics of patients with MCF >30mm (n=21) vs those with MCF ≤ 30mm (n=27) are shown in table 1. Figure 1 illustrates the type of discordance one can see between conventional coagulation parameters and ROTEM values.This study suggests that the MCF value from a ROTEM assay at diagnosis is probably a better measure of the hemostatic defect at diagnosis in APL. Patients with APL with low MCF on ROTEM at diagnosis are at an increased risk of death during induction therapy despite an adequate blood product replacement strategy which was based on conventional coagulation parameters. In these patients, there is a need to develop novel and possibly a more intensive replacement strategy.Table 1Comparison of baseline characteristics and clinical outcomes.VariablesMCF ≤30 (n = 27)N (%) /Median (range) / Mean±SDMCF > 30 (n=21)N (%) /Median (range) / Mean±SDp valueAge (years)40(12-68)27(9-48)0.006WBC >10 x x109/L17(62.96)2(9.52)0.003Platelet count (x109/L)17(4-89)45(10-339)0.001PT in seconds14.5(11-87.5)11.8(10.1-14.8)0.004aPTT in seconds28(23.2-77.9)30.2(22-53.6)0.104Fibrinogen in mg%134.8(24.7-393)172.2(14.7-549.5)0.066Major bleeding4(14.8)1(4.8)0.368Thrombosis4(14.8)1(4.8)0.369Death12(44.4)1(4.8)0.003 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD2 positive acute promyelocytic leukemia is high risk of relapse and associated with expression of CD34 and FLT3-ITD mutation

CD2 positive acute promyelocytic leukemia is high risk of relapse and associated with expression of CD34 and FLT3-ITD mutation

Development and validation of a 3-Plex RT-qPCR assay for the simultaneous detection and quantitation of the three PML-RARa fusion transcripts in acute promyelocytic leukemia.

Rapid diagnosis of acute promyelocytic leukemia (APL) with promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa) contributes to a highly effective therapy with all-trans retinoic acid (ATRA). Real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) is a valuable tool to diagnose APL with PML-RARa. However, a single RT-qPCR analysis, which is laborious and costly, has to be performed in three reactions to determine whether one of the three PML-RARa transcripts is present and to quantify the involved transcript. This paper describes a novel TaqMan MGB probe-based 3-plex RT-qPCR assay in a single reaction to detect simultaneously the three PML-RARa transcripts. Specific primers and probe were designed, and the results were further normalized to the Abelson gene. The detection results for the serially diluted plasmid indicate that the analytical sensitivity was 10 copies per reaction for PML-RARa bcr1, bcr2, and bcr3. A relatively high sensitivity of 10-4 was achieved with this assay when analyzing the bcr1 transcripts obtained from the NB4 cell line. The reproducibility was satisfactory because the coefficients of variation of cycle threshold values were less than 3% for both inter- and intra-assays. After testing 319 newly diagnosed patients with leukemia (including 61 APL cases), the results of the 3-plex RT-qPCR assay completely agreed with the traditional methods used for the detection of PML-RARa. The quantitative results of the 3-plex RT-qPCR were highly correlated with the single RT-qPCR and showed similar assay sensitivity for 60 PML-RARa positive APL samples at diagnosis and 199 samples from 57 patients during follow-up. Interestingly, one PML-RARa bcr2 case at diagnosis with breakpoint at 1579, which was not detected by the single RT-q-PCR, was detected by the 3-plex RT-qPCR assay. The 3-plex RT-qPCR assay is a specific, sensitive, stable, and cost-effective method that can be used for the rapid diagnosis and treatment monitoring of APL with PML-RARa.

Tumor necrosis factor inhibitors and acute leukemia in two cases.

e18010 Background: Inhibitors of tumor necrosis factor alpha (anti-TNF agents) are commonly used in Crohn’s disease and rheumatoid arthritis. Relationship between TNF inhibitors and development of malignancy, e.g., lymphoma and solid tumors, is defined recently. However, a few authors reported leukemia. Crohn’s disease and also azothioprine may also be associated with leukemia. Here, we are presenting two acute myeloid leukemia (AML) cases using azothioprine and anti-TNF inhibitors for Crohn’s disease. Methods: Observational case report. Results: Case 1: A 56-year-old male patient with active Crohn’s disease for 4 years was refractory to first line mesalazine, corticosteroid and azothioprine treatment. So, anti-TNF monoclonal antibody (moAb) (Adalimumab) had been given. After second dose of adalimumab treatment AML (AML-M2 as FAB) developed. Case 2: A 38-year-old male had been diagnosed with ankylosing spondylitis for 7 years and Crohn’s disease for 2 years. He had been treated with salazopyrin and indomethacin. Anti-TNF etanercept (50 mg/wk) was started 30 months ago due to severe active pulmonary disease. After 1 year, he developed active Crohn’s disease. So, the treatment changed to infliximab. Also, mesalazine and azothioprine were added to treatment. On the 18th month of the infliximab t(15;17) positive acute promyelocytic leukemia were developed in the patient. Conclusions: Blocking TNF with anti TNF agents may cause blocking apoptosis and inhibit natural killer activity. So, antitumoral activity of natural killer cells is blocked by these agents. TNF alpha gene mutation has been associated with CLL, lymphoma, myelodysplastic syndrome and secondary AML. Azothioprine may also increase leukemia, lymphoma and solid tumors risk at patient on anti TNF treatment. Following the diagnosis of Crohn’s disease without immunomodulator treatment a case had developed acute leukemia in the literature. In our patients, anti-TNF, azothioprine and inflammatory bowel disease may be cause acute leukemia cumulatively. Physicians should be careful and regularly check the patients using anti-TNF agents in considering of malignancy development. In conclusion, the pathogenesis of leukemia development after TNF blockage should be clarified.

A Phase III Study of New Synthetic Retinoid Tamibarotene(Am80) Compared with ATRA in Maintenance Therapy for Newly Diagnosed Acute Promyelocytic Leukemia (APL): Japan Adult Leukemia Study Group (JALSG) APL204 Study

AbstractAbstract 410 Background:ATRA with anthracycline-based chemotherapy markedly improved the prognosis of APL, leading to disease free survival rates 70–80%. But continuous relapse in post-remission period has been a major problem. The role of maintenance therapy for patients in molecular remission after consolidation therapy is uncertain and previous study showed ATRA with or without 6-MP/MTX for 1 to 2 years would be the choice. JALSG APL204 study was intended to decrease the relapse rate in post remission period and applied new synthetic retinoid Tamibarotene (Am80) for maintenance therapy. Am80 is an oral synthetic retinoid with benzoic acid and RARα specific agonist invented in Japan in 1984. High differentiation potential and low affinity to cytoplasmic retinoic acid binding protein may contribute to a superior anti-APL cell effect than ATRA. Early phase II study showed CR rate was 58% for relapsed APL previously treated with ATRA (Blood 90:967–973, 1997). Furthermore Am80 has lower incidence of muco-cutaneous side effects because of no binding affinity to RARγ. Methods:APL204 study was designed as prospective randomized phase III trial comparing Am80 with ATRA in maintenance therapy. Patients (pts) with untreated PML/RARA positive APL between the age of 15 to 69 are eligible. Induction therapy was stratified according to the initial WBC counts: WBC <3,000/μl (Group A: ATRA only), 3,000/μl ≦ WBC <10,000/μl (Group B: ATRA and IDA/Ara-C: 2+5), WBC <10,000/μl (Group C ATRA and IDA/Ara-C: 3+7) and pts who experienced leukocytosis in each group were added chemotherapy (group D). Consolidation chemotherapy consisted of 3 courses containing MIT/Ara-C, DNR/Ara-C and IDA/Ara-C. Patients who achieved molecular remission after consolidation chemotherapy were randomly assigned to 2 groups of maintenance therapy with Am80 at a daily dose of 6 mg/m2 divided in 2 doses for 14 days and ATRA at a daily dose of 45 mg/m2 divided in 3 doses for 14 days. Each cycle was repeated every 3 months for 2 years and MRD was monitored every 6 months for additional 2 years. Primary endpoint is hematological or molecular relapse-free survival (RFS) during maintenance and follow up period. Results:Between April 2004 to December 2010, 347 pts were enrolled and 345 pts were evaluable. Median age was 48 (range 16–68) years old, male : female = 183 : 162 and M3 : M3v = 322 : 23. Median WBC count at diagnosis was 700 (70–127,000)/μl and number of pts in each induction group were A=133, B=56, C=70, D=86 pts. In induction therapy, overall CR rate was 94.5% (326/345) and 95.5%(A), 92.9%(B), 88.6%(C), 98.8%(D) in each group. During consolidation therapy 11 pts (3.4%) were dead, including 9 pts (2.8%) for therapy related toxicity and 2 pts (0.6%) for resistant disease. Two hundred and seventy pts (85.7%) were evaluable for maintenance randomization, 134 pts in Am80 and 136 pts in ATRA. With median follow up of 51 (15–97) months, 5-year RFS was 90.9% (Am80) and 83.2% (ATRA) (P=0.1284), OS 92.4% and 98.1% (P=0.3543) in each group. In group C (WBC ≧10,000/μl), 5-year RFS was 87.7% (Am80) and 59.9% (ATRA) (P=0.0297), it was statistically significant, but in group A and B there was no significant differences. Overall relapse rate was 13.7% (37 pts) after randomization. The most frequent adverse effect of Am80 was a transient hyperglycemia. For all patients in APL204 study, 5-year EFS were 76.6%, RFS 82.9%, OS 87.5%. Conclusions:Maintenance therapy with Am80 has a moderate effect to decrease the relapse rate compared with ATRA but the difference was not statistically significant at 5 years. Both ATRA and Am80 maintenance seem to be effective compared to our previous APL97 study. Notably Am80 is significantly effective in high risk group as WBC ≧10,000/μl (Group C). These results may lead to new strategy for high risk APL. In the future study applying ATO in consolidation therapy and Am80 in maintenance therapy may be a promising strategy to improve the curability and decrease the toxicity of therapy for newly diagnosed APL. This trial was registered at University Hospital Medical Information Network (UMIN)-CTR ID C000000154 in Japan. Disclosures:No relevant conflicts of interest to declare.