PML RARA Research Articles

Expression pattern and prognostic implication of zinc homeostasis-related genes in acute myeloid leukemia.

Zinc homeostasis is regulated by the SLC39A/ZIP, SLC30A/ZnT, and metallothionein (MT) protein families. The association of zinc homeostasis with acute myeloid leukemia (AML) is unclear. We previously demonstrated that zinc depletion by TPEN triggers apoptosis in NB4 AML cells with the degradation of PML-RARα oncoprotein, suggesting that zinc homeostasis may be associated with AML. The primary aim of this study was to explore the expression pattern and prognostic roles of zinc homeostasis-related genes in AML. Bioinformatics analyses were performed using integrated datasets from the TCGA and GTEx projects. The GEPIA tool was used to analyze the differential expression of zinc homeostasis-related genes. Correlations between zinc homeostasis-related genes were assessed with Spearman's correlation coefficient. OncoLnc was used to evaluate the prognostic roles of zinc homeostasis-related genes with Kaplan-Meier and Cox regression models. In both NB4 and U937 cells, the transcriptional regulation of zinc homeostasis-related genes by zinc depletion was detected through qPCR. We found that multiple ZIPs, ZnTs, and MTs were differentially expressed and correlated in AML tumors. In AML patients, higher expression of ZIP4 and lower expression of ZnT5 and ZnT7 predicted poorer survival. We further found that zinc depletion by TPEN upregulated ZIP7, ZIP9, ZIP10, ZIP13, and ZnT7 and downregulated ZIP14, ZnT1, ZnT6, and most of the positively expressed MTs in both NB4 and U937 AML cells. Our findings suggest high expression of ZIP4 and low expression of ZnT5 and ZnT7 as potential risk factors for the prognosis of AML. Zinc homeostasis may be a potential therapeutic target for AML, deserving further exploration.

Targeting Interferon Pathway in TBLR1-RARa-Driven Promyelocytic Leukemia in the Context of ATRA and ATO Unresponsiveness

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia (AML) mostly driven by the t(15;17) translocation that yields the PML-RARα (PR) fusion protein. Apart from PML, other partner genes have been uncovered to fuse with RARα forming rare X-RARα fusion genes. Among them, TBLR1-RARα (TR) is a novel oncogene to induce APL identified in our previous studies, which elicited differed response and worse prognosis in contrast to the canonical PR fusion gene. Despite the textbook rationale of all-trans retinoid acid (ATRA) and arsenic trioxide (ATO) turning most APL patients with PR fusion gene from highly fatal to highly curable, TR-driven APL did not yield long-term remission both in murine models and patients, highlighting the necessity to dissect the mechanism of drug unresponsiveness and explore novel targeted therapies. In this study, we first established two doxycycline-inducible leukemia cell lines to directly initiate the expression of TR and PR fusion protein in U937 cells, respectively. Transcriptome sequencing analysis showed that interferon (IFN) pathways were significantly suppressed in TR rather than PR-induced leukemia cells. Then we administered IFNs as well as the standard regimen of ATRA and ATO in TR- and PR-induced APL, comparing therapy responses and biological phenotypes with in vitro and in vivo studies. Main results are summarized as follows concerning TR- in comparison to PR-driven APL. Firstly, ATO was unresponsive in TR-induced leukemia cells, which failed to elicit oncoprotein degradation, apoptosis and loss of self-renewal. Secondly, lower doses of ATRA were inadequate. Although a lower dose of ATRA was sufficient to trigger differentiation, increasing doses of ATRA further promoted the degradation of oncoprotein and reduced the colony formation capacity of TR leukemic cells, which ultimately conferred survival benefits in TR mice. Thirdly, type I IFNs was promising, which induced apoptosis, cooperated with ATRA to boost differentiation, and exhibited potential to reduce self-renewal, reflecting anti-leukemia efficacy. Finally, type I IFNs combining with ATRA displayed survival advantage to TR mice, and murine STING agonist DMXAA, which acted upstream to produce type I IFNs, significantly endowed TR mice with extended survival time. Based on our results, we further propose mechanistic hypotheses of TR-APL in comparison with the classic PR-APL. In TR-driven APL, despite the unresponsiveness of ATO, we may utilize STING-IFN pathway agonist to upregulate PML expression, which may obliterate self-renewal independent of ATO response triggered by initial PML nuclear body disruption observed in PR-driven APL, providing therapeutic implications for APL driven by rare RARα fusion genes. Our study aims to gain further understanding of TR-driven APL and integrate insights into principles underlying leukemogenesis mediated by rare RARα fusion genes, which may benefit such a therapy-resistant population and enable APL to be a bona fide curable leukemia.

Complications during Induction Chemotherapy in Acute Promyelocytic Leukemia: An Institutional Experience.

Stalin Chowdary BalaIntroduction Acute promyelocytic leukemia (APL) has transformed from a highly fatal disease to a highly curable one. Induction deaths continue to represent one of the major impediments in modern therapy of APL. Sepsis, hemorrhage, and differentiation syndrome are the major complications during induction therapy in APL. The present study reports the incidence and prognostic factors of major complications during induction chemotherapy in patients with newly diagnosed APL. Materials and Methods The present study was a single institutional, observational, retrospective study. All cases of APL diagnosed by morphology and confirmed by RT PCR (PML RARα) were included in this study. Data were analyzed using Statistical Package for the Social Sciences (SPSS) version 25. Results A total of 73 patients were analyzed. The median age at presentation was 30 years (range, 3-60 years) with a female to male ratio of 1.02:1. The most common symptom at presentation was fever (80%), followed by fatigue (56%) and gum bleeding (37%). The majority of the patients at presentation were high risk (42.4%), followed by intermediate risk (38.4%) and low risk (19.2%). Fifty-seven (78%) patients achieved complete hematological remission and 16 (22%) succumbed during induction chemotherapy. Infection was the most common cause of induction death (50%), followed by hemorrhage (37.5%) and differentiation syndrome (12.5%). On univariate analysis of prognostic factors, bcr3 variant, grade 3/4 bleeding during induction, and low levels of albumin at presentation were significant for induction mortality ( p = 0.034, 0.041, and 0.008 respectively). On multivariate analysis, only serum albumin < 3.5 g/dL was an independent predictor for induction mortality ( p = 0.043). Conclusion The majority of patients were high risk at presentation. Sepsis was the most common complication during induction and also the leading cause of induction death. Identifying induction complications at the earliest and providing aggressive supportive measures can further improve outcomes in APL.

PB1847: ACUTE MYELOID LEUKEMIA – IS IT MORE BESIDES GENETIC ABNORMALITIES?

Background: Acute myeloid leukaemia (AML) still carries a poor prognosis, despite novel aproaches. Age and previous myelodysplastic syndrome (MDS) are important risk factors. Environmental factors: professional benzene exposure, residential background and exposure to chemotherapy (CT)/radiotherapy (RT) have also been implicated. Aims: To evaluate environmental factors of AML patients, its genetic characteristics and overall survival (OS), in a tertiary centre. Methods: We retrospectively revised newly diagnosed AML in our centre between 2018 - 2020. Clinical and laboratory data were obtained by clinical files review Results: We included 235 patients, 47.2% (n=111) male. Median age 69 (19-97) years. (67 [28.5%]≤60 years old. – YOUNG and 168 [71.5%] with >60 years old - OLD). The most common WHO 2016 category was AML with myelodysplasia-related changes (MRC) (37.0%; n=87), followed by AML NOS (21.3%; n=50); 15 (6.4%) patients had acute promyelocytic leukaemia with PML―RARA, 21 (8.9%) therapy-related myeloid neoplasm and 17 (7.2%) post-myeloproliferative neoplasm (post-NMP) AML. The main category within YOUNG patients was AML with mutated NPM1 (19.4%) and in the OLD group was AML-MRC (45.2%). Genomic classification (GC) (Papaemmanuil, 2016) was available for 117 cases (49.8%): AML with no detected driver mutations (n=28; 23.9%); AML with mutated chromatin, RNA-splicing genes, or both (n=26; 22.2%). Considering YOUNG vs OLD patients, the main category was AML with mutated NPM1 (25%) vs AML with no detected driver mutations (32.3%). Regarding ELN risk stratification: 20.0% were low-risk, 27.1% intermediate risk and 52.9% high-risk category. OS for total cohort was 4.2 months, with 177 events (75.3% of included patients). The YOUNG patients had a median OS of 18,7 months while the OLD had median OS of 2,6 months, with P<0,001 (HR 2,31). Ninety nine (42.1%) patients lived in industrial regions. We found no correlation between living place and dysplasia, even after adjusting for age, ELN risk or GC. We found an apparent association of WHO subtype with living place (p=0.013), as post-MPN AML was more common in non-industrial (10.3 vs 3.0%) and AML with mutated NPM1 in industrial areas (11.1 vs 7.3%), but they were lost after adjusting for age. There was no OS difference between industrial and non-industrial places even after adjusting for age. Regarding professional exposure (available for 85 patients), 7.1% were exposed to benzene, 2.4% to agricultural activities and 31.8% to other chemicals, 58.8% with no significant exposure, with no differences in WHO classification, GC nor age. Smoking appeared associated with longer OS (6.6 vs 34.9%; p=0.026; HR 0.53), but this effect is lost after adjusting for age, as smoking was more common in YOUNG (65.7 vs 25.0%). There was no correlation with AML subtype or risk. Concerning other risk factors like alcohol consumption, obesity, family history of cancer, previous solid neoplasm, previous chemotherapy or previous radiotherapy none of them had impact in AML subtype, ELN risk or OS (p=NS). Summary/Conclusion: In our cohort, the majority of patients were over 60 years old and presented with AML-MRC, as described in the literature. Concerning GC, the dominant category was AML with no detected driver mutations. Looking at environmental factors, in our cohort only residential background correlated with AML subtype, despite non-significance impact in prognosis.

Effects of interferon regulatory factor 9 on the biological phenotypes in PML-RARα-induced promyelocytic leukemia

目的研究急性早幼粒细胞白血病（APL）中干扰素调节因子9（IRF9）的表达特征、预后意义和生物学功能，探索IRF9作为潜在治疗靶点的临床转化意义。方法挖掘TCGA公共数据分析IRF9在APL中的表达水平及其表达高低对患者生存的影响；利用Dox诱导的慢病毒载体系统构建可诱导表达PML-RARα（PR）融合基因的U937细胞系，诱导PR融合基因早期表达并加入全反式维甲酸（ATRA）检测IRF9表达变化；构建可诱导表达IRF9的NB4细胞系，进行细胞分化和集落形成实验，初步探讨IRF9诱导表达对NB4白血病细胞生物学功能的影响。结果①TCGA数据库分析显示，在各种类型的AML中，IRF9在APL中表达水平最低且与预后不良相关。②成功构建可诱导表达PR融合基因的U937细胞系；PR表达引起IRF9蛋白水平下调，在NB4细胞系中IRF9表达缺失，ATRA处理后可表达上调。③成功构建可诱导表达IRF9的NB4细胞系，IRF9诱导表达促进NB4细胞的分化，且与较低剂量ATRA有协同促分化作用；IRF9诱导表达显著抑制了NB4细胞的集落形成能力。结论IRF9在APL中低表达且与预后不良相关，提示存在特异性调控机制；上调IRF9表达可发挥抗白血病效应，为其成为临床潜在治疗靶点奠定生物学基础。

Relapse Analysis and Resistance Mutations of PML-Rara Fusion Gene in Acute Promyelocytic Leukemia Patients Treated with All-Trans Retinoic Acid and Arsenic

The clinical manifestations, management, and prognosis of acute promyelocytic leukemia (APL) are distinctive in acute myeloid leukemia (AML). Administration of all-trans retinoic acid (ATRA) and arsenic agents have greatly improved the outcome of APL from the most lethal to the most curable subtype AML. Unfortunately, relapse is still observed. And in the case of combined medication with ATRA and arsenic, the resistance mutation spectrum of PML-RARA is far from being revealed. We retrospectively analyzed 40 APL patients that admitted to our hospital from Jan. 2013 to Jul. 2020. There were 21 males and 19 females, aged 6-65 years (median age 31.5), and follow-up time was 3-210 months (median time 58.85 months).There were 3 cases that developed secondary tumors, although APL remission has been achieved. One developed refractory AML with KMT2A-MLLT3 fusion during the consolidation treatment one year after the diagnosis of APL. One case relapsed with APL 5 years after the initial diagnosis, and she developed refractory AML with RUNX1-MECOM fusion 3.5 years more after enduring multiple relapses and treatment courses. Another case developed AML-M2 3.5 years after the initial diagnosis of APL, and achieved sustained remission again through allogeneic hematopoietic stem cell transplantation (allo-HSCT).A total of 8 cases came to our hospital at the initial onset of APL, and 1 came during remission. All these cases got sustained remission through combined triple therapy (ATRA, arsenic, and chemotherapy), with the follow-up time of 3 to 37 months (median 20.5 months). No PML-RARA resistant mutation was detected in these 9 cases.A total of 28 cases came to our hospital after APL relapse. The time from APL onset to the first relapse was 9-189 months (median 34 months). PML-RARA resistance mutations were detected in 9 cases, including 8 cases that carry single RARA mutations each, and 1 case that relapsed with multiple extramedullary infiltrations carry quadruple mutations (PML S214L-A216T and RARA R276W-H298N) (Figure 1a and b). A total of 4 cases underwent allo-HSCT after APL relapse. One of them relapsed 6 months after allo-HSCT, and the other 3 got sustained remission with the follow-up time of 47, 39, and 52 months, respectively.There were 2 cases relapsed after maintaining sustained remission for more than 5 years. One was a 58-year-old male who achieved sustained remission after 2.5 years of combined triple therapy. However, APL recurred 16 years after the primary diagnosis. He achieved remission again and now underwent the consolidation course of combined triple therapy. Another was a 23-year-old male who achieved sustained remission after combined arsenic and chemotherapy, but APL relapsed 75 months after the primary diagnosis. He was resistant to combined triple therapy and achieved remission through allo-HSCT. We failed to retrieve archived specimens at the first onset of APL for genomic variation comparison with the second onset. Therefore, we could not determine whether these two cases were relapsed from the original APL or developed another APL.Although we have achieved a high remission rate and long-term survival for APL through combined triple therapy, relapse and secondary tumors still occur in some cases. Especially for cases that endure heavy treatment, genomic toxicity may promote the oncogenesis of secondary tumors. Also, APL may recur over 5 years after the primary diagnosis and sustained remission. Whether there were inherited predisposing factors in these cases is worthy of further investigation.The incidence of PML and RARA resistant mutations in cases resistance for ATRA and arsenic is not very high. This may be related to the combination use of chemotherapeutic drugs and suggests underlying resistance mechanisms. Although both ATRA and arsenic were used in most cases, the incidence of ATRA resistance mutations is significantly higher than that of arsenic, which might attribute to the sparse interaction sites of arsenic and PML.A better combination strategy of arsenic, ATRA, with or without chemotherapy is worthy of further investigation to reduce the incidence of secondary tumors and relapse. Adhering to a systematically planed treatment course is essential for acquiring rapid remission and reduction of relapse. Treatment compliance should be paid attention to guaranteed, especially during the oral administration courses outside the hospital. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Myeloid Sarcoma Type of Acute Promyelocytic Leukemia With a Cryptic Insertion of RARA Into FIP1L1: The Clinical Utility of NGS and Bioinformatic Analyses

BackgroundAcute promyelocytic leukemia (APL) is characterized by the presence of coagulopathy at onset and translocation t (15; 17) (q22; 21), meanwhile, other translocation variants of APL have also been reported. The FIP1L1–RARA fusion gene has recently been reported as a novel RARA-associated fusion gene.ObjectivesWe report a case of de novo myeloid sarcoma (MS) type of APL with FIP1L1–RARA found by next-generation sequencing (NGS) that was not detected by conventional analyze analysis for RARA translocations.MethodsWe performed typical morphological, magnetic resonance imaging (MRI), conventional tests for PML–RARA dual-fusion translocation probe, high-through sequencing and NGS. Meanwhile, bioinformatics analyses were done by using public repositories, including ONCOMINE, COSMIC, and GeneMANIA analysis.ResultsA 28-month-old girl with a complex karyotype that includes 46,XX,t(4;17)(q12;q22)[9]/46,idem,del(16)(q22)[3]/45,idem,-x,-4,-9,-15,del(16)(q22),+marl,+mar2,+mar3[7]/46,xx[3], c.38G>A (p.Gly13Asp) in the KRAS gene, and a cryptic insertion of RARA gene into the FIP1L1 gene was diagnosed with APL complicated by the de novo MS.ConclusionWe report a FIP1L1–RARA fusion in a child with APL who presented with an extramedullary tumor in the skull without the classic karyotype using NGS, whom we treated with good results. NGS analysis should be considered for APL variant cases. Further experimental studies to the association between the mutation in KRAS gene and FIP1L1–RARA fusion on the clinical phenotype and progression of APL are needed to identify more effective therapeutic targets for APL.

AML-142: Implementation of Combined DNA and RNA Sequencing in Myeloid Neoplasms

Context Identifying targetable and driver mutation and fusions in myeloid neoplasms through NGS platform. Objective The aim of this study was to optimize and validate Oncomine Myeloid Research Assay (comprising of 40 DNA target genes, 29 driver genes, and a broad fusion panel) for establishing assay sensitivity, specificity, and precision using Ion torrent PGM and implementation into clinics. Patients or other participants Archived DNA samples (n=17) with known mutations in (FLT3-ITD, CEBPA, NPM1, PTPN11, cMPL1, IDH1/2, TET, EZH2, U2AF1, SETBP1, ASLX1, TP53) and RNA samples (n=11) with FISH/RT-PCR-confirmed fusions (NUP 214, RUNX1, PML RARA BCR 1, PML RARA BCR 3, MLLT3, B14A3, B19A2) were retrieved as positive controls and negative controls for the validation. The sequences were analyzed using variant calling software (Ion Reporter Annotate variants version 5.4, 5.6, and 5.10) caller to identify variants relevant to the clinical indication. After thorough optimization and validation, this panel was used for prospective analysis in patients with suspected myeloid disorders. [AML (n=36), MDS (n=12), MPN (n=9)] AUL (n=1), Cytopenia/anaemia under evaluation (n=8) and CMML (n=3)]. Intervention FLT3-ITD by fragment analysis was performed in all cases after validation. Main outcome measures After implementation of NGS platform in myeloid neoplasms FLT3 and TET2 genes were the most common pathogenic variant in DNA sequencing and KMT2A rearrangement on RNA sequencing. Results In the four validation runs (318 chip - 4 DNA/RNA) comprising 28 known variants, all mutations/rearrangements were concordant except two cases (FLT3-ITD > 200bp and SETBP1, G870S 3.2%). For the prospective myeloid disorders (n=69), mutations were observed in 31 genes, maximum cases having FLT3 (n=8) and TET2 (n=8). The other frequently mutated genes were SRSF2 (n=7), NRAS (n=6), RUNX1 (n=6), ASXL1 (n=6), CEBPA (n=5), U2AF1 (n=4), SH2B3 (n=4), BCOR (n=4). All FLT3-ITD-positive and -negative cases were confirmed by Fragment Length Analysis. In eleven cases, fusions were detected; KMT2A (n=4), BCR-ABL (n=3), PML-RARA (bcr3) (n=1), and RUNX1T1 (n=2) and ETV6-ABL fusions Conclusions We demonstrate the validation and implementation of a combined targeted DNA and RNA sequencing for routine clinical use in myeloid malignancies.

Gata2 Expression and Function in PML-RARA-driven Preleukemia and APL

Self-renewal is a key feature of the cells that initiate acute myeloid leukemia. To identify the mechanisms involved in PML-RARA (PR)-driven self-renewal, we made use of Ctsg-PR mice, which have PR knocked into the UTR of the first exon of Ctsg. Ctsg-PR drives the expression of PR in myeloid progenitor cells and gives these cells the ability to serially replate in methylcellulose-based colony assays. Most Ctsg-PR mice develop acute promyelocytic leukemia (APL) with an average latency of ~300 days. To identify target genes regulated by Ctsg-PR, we performed single cell RNA-seq (scRNA-seq) on whole bone marrow from young, preleukemic Ctsg-PR mice or age-matched littermates. We identified 959 differentially expressed genes (DEGs) within myeloid progenitors (546 upregulated by PR, and 413 downregulated). Gata2 was identified as a DEG in this analysis, and we confirmed this phenotype with bulk RNA-seq of purified promyelocytes from young, preleukemic WT vs. Ctsg-PR mice. All APLs derived from Ctsg-PR mice also expressed high levels of Gata2. To identify the immediate-early target genes of PR, we transduced human CD34+ cord blood cells with MSCV-IRES-GFP retroviruses containing PR, a mutant PR with a RARA DNA binding mutation (C88A, known to abolish PR replating), or an empty vector. ScRNA-seq analysis of these cells after 7 days of ex vivo culture identified 1815 DEGs (1301 upregulated and 514 downregulated by PR) in GFP+ cells expressing PR, compared to GFP+ cells transduced with PRC88A or empty vector. Among the DEGs, Gata2 was upregulated 5-fold in the PR GFP+ cells. Identical short-term retroviral overexpression studies with mouse marrow revealed that PR expression caused an expansion of hematopoietic progenitor cells that overexpressed Gata2. Finally, although normal human promyelocytes do not express GATA2, virtually all primary human APL samples do. Combined, these studies strongly suggest that GATA2 is a target gene of PR, and may therefore play a role in the development of APL. Based on our expression data, we hypothesized that Gata2 inactivation would reduce PR-driven self-renewal. To test this hypothesis, we bred Ctsg-PR mice to Rosa26-Cas9 mice, which ubiquitously express Cas9. Marrow from the resulting Ctsg-PR x Cas9 mice was electroporated with guide RNAs (gRNAs) targeting the zinc finger 1 (ZF1) domain or exon 2 of Gata2, or control loci (Actb intron 5 or Rosa26 intron 1) and serially replated in methylcellulose with SCF, IL-3, and IL-6. CRISPR-Cas9 efficiently induced a wide array of indel mutations at all gRNA target sites. Two days after electroporation, the frequency of Gata2 alleles with indels at the target site ranged from 64% to 93% (n=4); hundreds of different Gata2 indels were generated in each experiment. To our surprise, the Gata2 targeted Ctsg-PR cells replated with dramatically higher efficiency than control locus targeted cells (Figure 1). The enhanced replating efficiency was dependent upon PR, since Gata2 targeted Rosa26-Cas9 bone marrow did not serially replate. Further, cells with Gata2 indels were positively selected for over time. For example, in one experiment, the frequency of a 12 bp deletion in Gata2 (that caused an in-frame deletion in ZF1) rose from an initial variant allele frequency (VAF) of 3% to 70% after 8 weeks of replating. A larger Gata2 deletion was co-selected at a similar frequency, and resulted in the deletion of Gata2 exon 4, which encodes 90% of ZF1. Virtually all of the positively selected cells contained Gata2 indels, demonstrating the competitive advantage of Gata2 loss-of-function mutations in this setting. Control gRNAs did not lead to significant changes in plating efficiency, nor were any indels selected for. Additionally, Ctsg-PR x Cas9 cells with Gata2 indels shifted from a neutrophilic phenotype (Gr1+ CD11b+) to a monocytic one (CD11b+ Gr1-) at late passages. To further investigate the role of Gata2 in Ctsg-PR induced APL, we sequenced the genomes of 16 mouse APLs and found that 2 samples had spontaneous mutations in Gata2 (R330L and N363fs, with VAFs of 74% and 39% respectively). In summary, these data provide evidence that PR positively regulates Gata2, and that Gata2 in turn promotes neutrophilic differentiation of committed, "reprogrammed" myelomonocytic progenitors. Surprisingly, Gata2 appears to contribute to the lineage fate and proliferative capacity of PR-expressing hematopoietic progenitor cells. Disclosures No relevant conflicts of interest to declare.

Local Field Radiotherapy Induced Therapy Related Myeloid Neoplasms and Bone Marrow Suppression

Introduction: Different cytotoxic agents have been implicated in development of therapy related myeloid neoplasm (t-MN). These include alkylating agents, topoisomerase II inhibitors, antimetabolites, and ionizing field radiation given to large field including active bone marrow (BM) (Swerdlow, 2016). This report highlight the impact of local field radiotherapy on BM through discussing few patients diagnosed in our center who showed profound BM hypocellularity or developed t-MN following exposure to local field radiotherapy without chemotherapy. Local field Radiotherapy as a causative agent for t-MN: A result of retrospective study performed in our center through (2012-2018), a total of 20 patients of t-MN out of 232 total AML patients (8.6%) were detected. Two patients out of 20 (10%) in our cohort had developed t-MN following exposure to local field radiotherapy. Patient (1): 50-year old Asian male diagnosed as Vocal cord tumor, received local field radiotherapy for 2 months with no chemotherapy. 15.8 months later, he presented to with two days history of gum bleeding associated with pancytopenia (WBCx10^3/µl:2.3, Hb g/dl:6.7, Platelets x10^3/µl:100). Peripheral blood smear showed infiltration with many leukaemic promyelocytes and diagnosed as Acute promyelocytic leukemia (APL). The diagnosis was confirmed by BM examination and detection of PML RARA classic rearrangement fusion indicating PML/RARA, t(15;17) in 88.5% cells. Reticulin stain shows increased reticulin fibrosis (1-2/ 3). The patient was stratified as intermediate risk and started on ATRA followed by PETHEMA protocol with complete remission. Patient (2): A 62-year old Egyptian lady with background of hepatitis C and previous stroke, diagnosed as Endometrioid Endometrial Cancer FIGO grade I. The patient received Brachytherapy 21 Gy dose to vaginal vault, in 3 fractions (7 Gy each) without complications. 22 months later, she developed progressive neutropenia and diagnosed as high grade myelodysplastic syndrome (MDS-EB-2) with complex karyotype and P53 somatic mutation, managed by palliative therapy. Discussion: Generally laryngeal cancers radiotherapy is directed to confined area and secondary cancers are more common in the area of close proximity to radiation field. Brachytherapy is a limited source of radiation placed into a cylinder and inserted within the prostate or the vagina for treatment of cancer prostate or endometrial carcinoma respectively. Compared to external radiotherapy (RT), the limited field radiotherapy (including external beam RT and brachytherapy), the doses received by BM is very low and hence the risk of secondary leukaemia is expected to be significantly lower. Although local field RT appears to be associated with a small increase in the incidence of solid cancers, only a few studies have evaluated the risk of developing myeloid malignancies after limited field radiotherapy. Numerous cases of (t-APL) have been reported in the literature mostly following TOPO isomerase II inhibitors. However, up to our knowledge no previous reports of t-MN with recurrent genetic translocations (including t-APL) following local field radiotherapy. Local field radiotherapy induced bone marrow suppression: Two male patients with previous history of cancer prostate and limited field radiotherapy. BM examination for both patients revealed profound hypocellularity (&lt;5-5%) with suppressed trilineage hematopoiesis. Patient (3): A 66 year-old male diagnosed as cancer prostate, subjected to radical prostatectomy, followed by salvage radiotherapy. Two months later, the patient was referred for persistent cytopenia (WBC 2.9 x10^3/uL, Hemoglobin 12.8 gm/dL and platelets (92 x10^3/uL). Follow-up revealed persistent thrombocytopenia. No follow-up BM was performed. Patient (4): A 73-year old male patient with previous history of marginal zone lymphoma and cancer prostate treated by brachytherapy. He underwent BM examination for lymphoma staging. Discussion: For patients who have received adjuvant, salvage or radical RT, a significant portion of the pelvic BM receives a substantial dose of RT. To our knowledge, there are no significant published data assessing the impact of limited field radiotherapy on BM particularly radiation induced BM suppression. Larger studies are required in order to accurately assess the actual effect of local field radiotherapy on hematopoiesis and its genetic profiling. Disclosures No relevant conflicts of interest to declare.

B1 oligomerization regulates PML nuclear body biogenesis and leukemogenesis

ProMyelocyticLeukemia (PML) protein can polymerize into a mega-Dalton nuclear assembly of 0.1–2 μm in diameter. The mechanism of PML nuclear body biogenesis remains elusive. Here, PMLRBCC is successfully purified. The gel filtration and ultracentrifugation analysis suggest a previously unrecognized sequential oligomerization mechanism via PML monomer, dimer, tetramer and N-mer. Consistently, PML B1-box structure (2.0 Å) and SAXS characterization reveal an unexpected networking by W157-, F158- and SD1-interfaces. Structure-based perturbations in these B1 interfaces not only impair oligomerization in vitro but also abolish PML sumoylation and nuclear body biogenesis in HeLaPml-/- cell. More importantly, as demonstrated by in vivo study using transgenic mice, PML-RARα (PR) F158E precludes leukemogenesis. In addition, single cell RNA sequencing analysis shows that B1 oligomerization is an important regulator in PML-RARα-driven transactivation. Altogether, these results not only define a previously unrecognized B1-box oligomerization in PML, but also highlight oligomerization as an important factor in carcinogenesis.

PB1736 IMPACT OF BCR FUSION TRANSCRIPTS ON OUTCOMES IN ACUTE PROMYELOCYTIC LEUKEMIA (APL)

Background:Acute promyelocytic leukemia (APL) results from fusion of PML RARα gene and accounts for 10% of AML. The breakpoints on chromosome 17 are consistently located in the second intron of the RARα gene, but on chromosome 15, there are different breakpoint cluster regions, namely bcr1, bcr2, and bcr3 located in intron 6, exon 6, and intron 3, respectively. About 70% of patients with APL have bcr‐1 or long (L)‐type transcript, 20% of patients with APL have bcr‐3 or short (S)‐type transcript, and 10% of patients have bcr‐2 or variant (V)‐type transcriptAims:The primary objective of this analysis was to study patient characteristics and outcomes in relation to bcr fusion transcripts of APLMethods:Data of all patients diagnosed as APL by PCR for PML RARα with known bcr transcripts were retrospectively analysed. Deaths within 72 hours of admission were excluded from the study. Statistical analysis was done using SPSS software, version 25. Overall survival was plotted using the Kaplan‐Meier methodResults:A total of 54 patients were analysed. bcr 1, bcr 3, bcr 2 and bcr 2’3 transcripts were seen in 29(53.7%), 20(37%), 3(5.6%) and 2(3.7%) respectively. Ten out of 11 paediatric patients (91%) had bcr1 transcript at presentation. The baseline characteristics and chemotherapy regimens used are tabulated in Table 1. Differentiation syndrome during induction chemotherapy was seen in 6 (21%) and 3 (15%) patients in bcr1 and bcr 3 transcripts respectively. Five patients with bcr3 transcript, 2 patients with bcr1 transcript and one patient each in bcr2 and bcr2’3 transcripts succumbed during induction therapy. Sepsis (45%), followed by DIC (45%) were the most common causes of mortality. At a median follow up of 19 months, the overall survival rate was 93.1% and 74.3 % in bcr1 and bcr3 transcript groups respectively (p = 0.09).Summary/Conclusion:Most of the paediatric patients had bcr1 transcript at presentation. High risk at presentation was significantly higher in bcr3 group. Sepsis and DIC were the most common causes of induction mortality. Patients with bcr3 transcript had nonsignificant inferior survival compared to bcr1image

PML–RARα induces all-trans retinoic acid-dependent transcriptional activation through interaction with MED1

ABSTRACTTranscriptional activation by PML–RARα, an acute promyelocytic leukemia-related oncofusion protein, requires pharmacological concentrations of all-trans retinoic acid (ATRA). However, the mechanism by which the liganded PML–RARα complex leads to the formation of the preinitiation complex has been unidentified. Here we demonstrate that the Mediator subunit MED1 plays an important role in the ATRA-dependent activation of the PML–RARα-bound promoter. Luciferase reporter assays showed that PML–RARα induced significant transcription at pharmacological doses (1 μM) of ATRA; however, this was submaximal and equivalent to the level of transcription driven by intact RARα at physiological doses (1 nM) of ATRA. Transcription depended upon the interaction of PML–RARα with the two LxxLL nuclear receptor recognition motifs of MED1, and LxxLL→LxxAA mutations led to minimal transcription. Mechanistically, MED1 interacted ATRA-dependently with the RARα portion of PML–RARα through the two LxxLL motifs of MED1. These results suggest that PML–RARα initiates ATRA-induced transcription through its interaction with MED1.

Application of Fluorescence in situ Hybridization in Detection of PML/RARA Translocation in Patients with Acute Promyelocytic Leukemia

Background: Acute promyelocytic leukaemia (APML) is a sub-type of acute myeloid leukaemia (AML). APML accounts for 10% of all AML diagnosis. Diagnosis is made by presence of abnormal promyelocytes in circulating blood and bone marrow smear and presence of PML RARA fusion transcript which is responsible for leukemogenesis. Fluorescence in situ Hybridisation (FISH) plays key role in confirming diagnosis by detecting PML/RARA (15;17) translocation which will help in deciding treatment and prognosis in patients diagnosed with APML. Aims and objectives: To estimate presence of PML/RARA translocation in patients diagnosed with APML using FISH. Materials and methods: Bone marrow samples of 20 patients diagnosed with APML was collected for standardized lymphocyte culture method followed by standard protocol for FISH using commercially available probes for PML /RARA gene. Ethical clearance was obtained from institutional ethical committee. Results: Out of 20 patients, 18 patients had shown positive result for PML/RARA translocation. Hence 90% of patients showed positive results for fusion gene. Conclusion: Detection of PML/ RARA translocation helps in rapid diagnosis as well as treatment of patient with APML. Further it will also help in prognosis and follow up to detect residual disease in these patients.

Outcomes in Adult Acute Promyelocytic Leukemia (APL): A Real World Scenario

Background:Acute promyelocytic leukemia (APL) accounts for approximately 10% of acute myeloid leukemia(AML) cases in adults. With the advent of targeted therapy like all- trans retinoic acid (ATRA) and arsenic trioxide, survival rates have improved leading to cure in majority of patients. The present study was designed to analyze the outcomes in newly diagnosed APL.Aims and objectives:The primary objective of this study was to analyze overall survival and relapse rate in APL.Secondary objective was to study the parameters impacting outcomes in APL.Materials and methods:All newly diagnosed patients with APL between January 2005 and December 2017 were retrospectively analyzed. Diagnosis of APL was confirmed by demonstration of PML-RARA translocation by polymerase chain reaction or fluorescence in situ hydridization. Risk stratification was done using Sanz risk. Event free survival was defined as the time from diagnosis till relapse, death or lost to follow up. Overall survival was defined as time from diagnosis till death or lost to follow up. Statistical analysis was done using SPSS software, v.25. Overall survival curves were plotted using the Kaplan-Meier method.INCLUSION CRITERIA:All cases of APL diagnosed by morphology and confirmed by RT PCR (PML RARα) were included in this study.EXCLUSION CRITERIA:Age ≤ 18yrsDeath within 72 hours of admission / not taken treatmentPrior chemotherapy or radiotherapy for the treatment of malignancy.Results:Data of 1396 AML patients between 2005 and 2017 was collected, of which 190(13.6%) patients had APL. Of 190 patients, 111 patients who met inclusion and exclusion criteria were analyzed. The median age at presentation was 33 years (range,19-60). The male female ratio was 1.01:1. The median duration of symptoms at presentation was 15 days (range, 3-180). According to Sanz risk grouping, high, intermediate and low risk were seen in 48 (43.1%), 46 (42.4%), 17(14.5%) patients respectively. The baseline characteristics are tabulated in Table 1.Of 111 patients, 96 (86.5%) patients received induction chemotherapy with ATRA and daunorubicin and 15(13.5%) patients received ATRA and arsenic trioxide. Induction mortality was 23(20.7%). Eighty-eight (79%) patients survived induction chemotherapy, of which 87(98.8%) were in complete molecular remission (CMR) at the end of consolidation. At a median follow up of 30 months, the event free survival rate, overall survival rate and relapse rate were 72%, 74.7% and 9% respectively. The overall survival rate in low-intermediate and high-risk groups were 84.1% and 62.5% respectively. Of eight patients who relapsed, five patients received second line chemotherapy and attained second CMR (100%). On univariate analysis, the strongest predictors for OS were high risk and bcr3 variant(p=0.007, p=<0.001 respectively). On multivariate analysis, only high risk was significant for overall survival (p=0.02).Conclusion:Majority of APL patients were high risk at presentation. High risk and bcr3 variant had significant impact on survival outcome in APL. As relapse rates were low and second CMR can be achieved with salvage chemotherapy in majority of patients, improving induction outcomes will further improve survivals in APL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RNA Sequencing Based Whole Transcriptome Analysis Detected Precisely All Fusion Transcripts in Leukemias

Introduction: Fusion transcript is a chimeric RNA encoded by a fusion gene or by two different genes by subsequent trans-splicing. Detection of fusion transcripts is an integral part of routine diagnostics of hematological malignancies. However, most of previous analytical methods couldn't detect all fusion transcripts in leukemia. In this study, we developed accurate fusion transcript detection methood using whole transcriptome sequencing, fusion gene detection software and expression analysis.Methods: RNA sequencing (RNA-seq) for whole transcriptome was performed in 11 patients with hematological malignancies (4 AML, 2 APL, 2 ALL, and 3 CML) having fusion transcripts detected by multiplex RT-PCR (HemaVision, DNA Diagnostic, Risskov, Denmark). Library were prepared with 1 ug of total RNA for each sample by TruSeq mRNA Sample Prep kit (Illumina, San Diego, USA). The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Santa Clara, USA). Indexed libraries were then sequenced using the HiSeq2500 platform (Illumina). The data obtained from the sequencing was analyzed using STAR-Fusion (v1.2.0). Novel fusion transcripts were confirmed by conventional sequencing.Results: Using STAR-Fusion, average number of fusion candidates per sample was 949.8 (range, 286-1752). To exclude false positive results and obtain true positive results, we developed the following filtering algorithm. First filtering criterion is to have more than 5 junction reads, the second is to detect more than one number of spanning reads, and the third criterion is to be in-frame fusion, which type of fusion can actually synthesize intact protein. Fusion candidates remaining after applying the above three filtering criteria were 1-3 per sample. All known fusion transcripts (PML–RARA, RUNX–RUNX1T1, CBFB–MYH11, KMT2A–MLLT3, BCR–ABL1, DEK–NUP214, ETV6–RUNX1) by multiplex RT-PCR were also detected in RNA-seq. In addition, 10 novel fusion transcripts (IGKV4-1–IGKC, IGLV1-47–IGLC2, HBA2–HBB, DEFA3–MBNL1, HBB–HBA2, MPO–HBA2, HBS1L–AHI1, HBB–HBA2, IGKV4-1–IGKC, SS18L1–ADRM1) were detected and among them, 6 fusions were confirmed by conventional sequencing.Conclusions: Whole transcriptome sequencing and optimized filtering algorithms successfully detected all known fusion transcripts and various novel fusions. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular Heterogeneity in Acute Promyelocytic Leukemia - a Single Center Experience from India.

Atypical breakpoints and variant APL cases involving alternative chromosomal aberrations are seen in a small subset of acute promyelocytic leukemia (APL) patients. Over seven different partner genes for RARA have been described. Although rare, these variants prove to be a diagnostic challenge and require a combination of advanced cytogenetic and molecular techniques for accurate characterization. Heterogeneity occurs not only at the molecular level but also at clinico-pathological level influencing treatment response and outcome. In this case series, we describe the molecular heterogeneity of APL with a focus on seven variant APL cases from a single tertiary cancer center in India over a period of two and a half years.We discuss five cases with ZBTB16-RARA fusion and two novel PML-RARA variants, including a Bcr3 variant involving fusion of PML exon4 and RARA exon3 with an additional 40 nucleotides originating from RARA intron2, another involving exon 6 of PML and exon 3 of RARA with addition of 126 nucleotides, which mapped to the central portion of RARA intron 2. To the best of our knowledge, this is the first case series of this kind from India.

The short isoform of PML‐RARα activates the NRF2/HO‐1 pathway through a direct interaction with NRF2

The NF-E2 p45-related factor 2 (NRF2)-Kelch-like ECH-associated protein 1 signaling pathway plays an important role in cytoprotection. In acute promyelocytic leukemia, fusion of the promyelocytic leukemia protein (PML) with retinoic acid receptor alpha (RARα) results in an oncogene, PML-RARα (PR). Although previous studies have shown that both RARα and PML inhibit NRF2 activity, how PR regulates NRF2 has not been reported. Here, we discovered that PR-S, the short isoform of PR, potentiates NRF2 activity in a tert-butylhydroquinone (tBHQ) concentration-dependent manner. Furthermore, PR-S colocalized with NRF2 in HeLa and HEK293T cells. The association of PR-S and NRF2 is mediated by the DNA-binding domains of RARα and the Neh7 domain of NRF2. Our results define a novel function of PR-S as a NRF2-transcriptional co-activator.

Quantitative assessment of Wilms tumor 1 expression by real-time quantitative polymerase chain reaction in patients with acute myeloblastic leukemia.

Background:The Wilms tumor 1 (WT1) gene is originally defined as a tumor suppressor gene and a transcription factor that overexpressed in leukemic cells. It is highly expressed in more than 80% of acute myeloid leukemia (AML) patients, both in bone marrow (BM) and in peripheral blood (PB), and it is used as a powerful and independent marker of minimal residual disease (MRD); we have determined the expression levels of the WT1 by real-time quantitative polymerase chain reaction (RQ-PCR) in PB and BM in 126 newly diagnosed AML patients.Materials and Methods:This study was done in molecular pathology and cancer research center from April 2014 to June 2015, RQ-PCR method was used to determine the WT1 gene expression in BM and/or PB samples from 126 patients of AML, we cloned both WT1 and ABL genes for creating a standard curve, and we calculate copy number of WT1 genes in patients.Results:A total of 126 AML patients consist of 70 males (55.6%) and 56 females (44.4%), with a median age of 26 years; 104 (81%) patients out of 126 show overexpression of WT1 gene. We also concomitant monitoring of fusion transcripts (PML RARa, AML1-ETO, MLL-MLL, CBFb-MYH11, or DEK-CAN) in our patients, the AML1-ETO group showing remarkably low levels of WT1 compared with other fusion transcript and the CBFB-MYH11 showing high levels of WT1.Conclusion:We conclude that WT1 expression by RQ-PCR in AML patients may be employed as an independent tool to detect MRD in the majority of normal karyotype AML patients.

PML-RARα kinetics and impact of FLT3-ITD mutations in newly diagnosed acute promyelocytic leukaemia treated with ATRA and ATO or ATRA and chemotherapy.

The APL0406 study showed that arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) are not inferior to standard ATRA and chemotherapy (CHT) in newly diagnosed, low-intermediaterisk acute promyelocytic leukaemia (APL). We analysed the kinetics of promyelocytic leukaemia-retinoic acid receptor-α (PML-RARα) transcripts by real-time quantitative PCR (RQ-PCR) in bone marrow samples from 184 patients and assessed the prognostic impact of fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) in 159 patients enrolled in this trial in Italy. After induction therapy, the reduction of PML-RARα transcripts was significantly greater in patients receiving ATRA-CHT as compared with those treated with ATRA-ATO (3.4 vs 2.9 logs; P=0.0182). Conversely, at the end of consolidation, a greater log reduction of PML-RARα transcripts was detected in the ATRA-ATO as compared with the ATRA-CHT group (6.3 vs 5.3 logs; P=0.0024). FLT3-ITD mutations had no significant impact on either event-free survival (EFS) or cumulative incidence of relapse in patients receiving ATRA-ATO, whereas a trend for inferior EFS was observed in FLT3-ITD-positive patients receiving ATRA-CHT. Our study shows at the molecular level that ATRA-ATO exerts at least equal and probably superior antileukaemic efficacy compared with ATRA-CHT in low-intermediaterisk APL. The data also suggest that ATRA-ATO may abrogate the negative prognostic impact of FLT3-ITD.