PML-RARA Fusion Research Articles

Impact of Simultaneous Presence of Multiple PML-RARA Isoforms on Phenotype in Patients with Acute Promyelocytic Leukaemia.

To determine the coexistence of multiple PML-RARA transcripts in adult APL (acute promyelotic leukaemia) patients, and its impact on the patients' laboratory parameters, treatment responses, and prognoses. Cross-sectional study. Place and Duration of the Study: Department of Medical Genetics, Medical Faculty of Necmettin Erbakan University, Konya, Turkiye, from January 2015 to March 2023. The study group consisted of individuals diagnosed with APL. RNA isolation was performed by taking blood or bone marrow samples and the presence of breakpoints in PML-RARA bcr1, bcr2, and bcr3 was detected using the real-time PCR. However, the quantification of PML-RARA fusion transcripts cannot be provided using the utilised kit. Twelve women and eight men were examined with a mean age of 38 years (range: 19-80), and 46.5 years (range: 22-60) were examined, respectively. When evaluating patients based on isoforms, it was found that 40% had multiple isoforms. Nineteen (95%) patients achieved haematologic remission after the treatment. Only one patient who had three different isoforms did not achieve remission. The estimated median survival for patients with a single isoform and those with multiple isoforms was 78.1 months (95% CI: 37.8-117.6) and 71.7 months (46.2-97.2), respectively. Two of the patients with multiple isoforms were lost in the early stage, whereas no early-stage mortality was recorded among patients with a single isoform. Identifying PML-RARA isoform subtypes is important for predicting prognosis and informing clinical follow-up. Acute promyelocytic leukaemia, Breakpoint cluster region, Isoform, PML-RARA.

Impact of Simultaneous Presence of Multiple PML-RARA Isoforms on Phenotype in Patients with Acute Promyelocytic Leukaemia.

To determine the coexistence of multiple PML-RARA transcripts in adult APL (acute promyelotic leukaemia) patients, and its impact on the patients' laboratory parameters, treatment responses, and prognoses. Cross-sectional study. Place and Duration of the Study: Department of Medical Genetics, Medical Faculty of Necmettin Erbakan University, Konya, Turkiye, from January 2015 to March 2023. The study group consisted of individuals diagnosed with APL. RNA isolation was performed by taking blood or bone marrow samples and the presence of breakpoints in PML-RARA bcr1, bcr2, and bcr3 was detected using the real-time PCR. However, the quantification of PML-RARA fusion transcripts cannot be provided using the utilised kit. Twelve women and eight men were examined with a mean age of 38 years (range: 19-80), and 46.5 years (range: 22-60) were examined, respectively. When evaluating patients based on isoforms, it was found that 40% had multiple isoforms. Nineteen (95%) patients achieved haematologic remission after the treatment. Only one patient who had three different isoforms did not achieve remission. The estimated median survival for patients with a single isoform and those with multiple isoforms was 78.1 months (95% CI: 37.8-117.6) and 71.7 months (46.2-97.2), respectively. Two of the patients with multiple isoforms were lost in the early stage, whereas no early-stage mortality was recorded among patients with a single isoform. Identifying PML-RARA isoform subtypes is important for predicting prognosis and informing clinical follow-up. Acute promyelocytic leukaemia, Breakpoint cluster region, Isoform, PML-RARA.

Clinical Significance of Genetic and Molecular Changes in Primary Myeloid Sarcoma

To investigate the clinical significance of genetic and molecular changes in primary myeloid sarcoma (MS). Fourteen patients with primary MS were selected in Jiading District Central Hospital Affiliated Shanghai University of Medicine & Health Sciences, The First People's Hospital of Lianyungang from September 2010 to December 2021. AML1-ETO fusion, PML-RARα fusion and CBFβ breakage were detected by fluorescence in situ hybridization (FISH), and the mutations of NPM1, CEBPA, FLT3, RUNX1, ASXL1, KIT and TP53 genes were detected by new generation sequencing (NGS). Among 14 patients, the MS occurred in bone, breast, epididymis, lung, chest wall, cervix, small intestine, ovary, lymph nodes and central nervous system. The tumor cells expressed MPO (13 cases), CD34 (7 cases), CD43 (8 cases), CD68 (7 cases), CD99 (8 cases) and CD117 (6 cases). Cytogenetic abnormalities were observed in 4 cases, including 3 cases of AML1-ETO fusion and 1 case of CBFβ breakage, while no PML-RARα fusion was detected. There were no significant differences in overall survival (OS) and leukemia-free survival (LFS) between patients with and without AML1-ETO fusion/CBFβ breakage (both P >0.05). Among the 14 patients, the number of NPM1, CEBPA, FLT3-ITD, RUNX1, ASXL1, KIT and TP53 gene mutations was 5, 3, 5, 3, 2, 2, 1, respectively, of which 7 cases had at least one mutation in FLT3-ITD, RUNX1, ASXL1 and TP53 gene. The OS and LFS of patients with FLT3-ITD, RUNX1, ASXL1 or TP53 mutation were shorter than those without mutations (both P <0.01). The genetic and molecular abnormalities of primary MS can be detected by FISH and NGS techniques. FLT3-ITD, RUNX1, ASXL1 or TP53 mutation indicates a worse prognosis, but further clinical studies are needed to confirm it.

Acute promyelocytic leukaemia with PML - RAR a fusion presenting as pancytopenia and atypical morphology: A case report

Acute myeloid leukaemia (AML) is characterised by uncontrolled proliferation of malignant marrow stem cells and is associated with infection, anaemia and bleeding. An improved understanding of pathophysiology has led to revamping the diagnostic, prognostic and therapeutic landscape of AML. AML is classified based on the defined genetic abnormalities and based on the differentiation. AML with predominance of abnormal promyelocytes shows characteristic t(15;17)(q24.1;q21.2) leading to promyelocytic leukaemia - retinoic acid receptor Alpha (PML - RARA) fusion oncoprotein. A 75-year-old male with features of acute promyelocytic leukaemia - hypogranular variant with atypical morphology and PML-RARA fusion is presented. The bilobed buttock shaped nuclei is an atypical presentation and is important to diagnose this morphology.

Function of PML-RARA in Acute Promyelocytic Leukemia.

The transformation of acute promyelocytic leukemia (APL) from the most fatal to the most curable subtype of acute myeloid leukemia (AML), with long-term survival exceeding 90%, has represented one of the most exciting successes in hematology and in oncology. APL is a paradigm for oncoprotein-targeted cure.APL is caused by a 15/17 chromosomal translocation which generates the PML-RARA fusion protein and can be cured by the chemotherapy-free approach based on the combination of two therapies targeting PML-RARA: retinoic acid (RA) and arsenic. PML-RARA is the key driver of APL and acts by deregulating transcriptional control, particularly RAR targets involved in self-renewal or myeloid differentiation, also disrupting PML nuclear bodies. PML-RARA mainly acts as a modulator of the expression of specific target genes: genes whose regulatory elements recruit PML-RARA are not uniformly repressed but also may be upregulated or remain unchanged. RA and arsenic trioxide directly target PML-RARA-mediated transcriptional deregulation and protein stability, removing the differentiation block at promyelocytic stage and inducing clinical remission of APL patients.

Establishment of a PML-RARa Copy Number Estimator for Prototype Xpert ® PML-RARa Assay

Statement of the Problem: Acute Promyelocytic Leukemia (APL) represents 10-15% of Acute Myeloid Leukemia (AML). The PML-RARa fusion transcript is expressed in more than 95% of APL patients. Three PML-RARa isoforms (bcr1, bcr2, and bcr3) are identified in 90-95% of PML-RARa positive cases. Prototype Xpert ® PML-RARa, an automated cartridge-based assay for measuring PML-RARa fusion transcript levels (bcr1, bcr2, and bcr3), is standardized to quantify the amount of PML-RARA relative to ABL1 control gene based on delta Ct in peripheral blood. Since PML-RARa level is crucial for diagnosis and ongoing therapeutic monitoring in APL, it can be useful to obtain the PML-RARa copy number (CN). The aim of this work is to develop PML-RARa CN estimator and to compare %PML-RARa/ABL1 reporting between delta Ct-based and CN-based methods. Methodology &amp; Theoretical Orientation: Four levels of PML-RARa (bcr1, bcr2, and bcr3) and ABL1 IVT-RNA panels as well as two lots of prototype Xpert ® PML-RARa assay were used to generate standard curves for CN and %CN reporting. The samples with spiked-in bcr1 IVT-RNA and APL clinical samples containing PML-RARa fusion transcript were examined to evaluate the CN and %CN between two lots of the prototype Xpert ® PML-RARa assay and to compare the delta Ct-based and CN-based methods for reporting %PML-RARa/ABL1. Findings: Linearity was demonstrated in Ct vs CN input for PML-RARa (R 2&amp;gt;0.98) and ABL1 (R 2&amp;gt;0.97). Less than 2-fold difference was exhibited for CN and %CN across two different lots. Less than 2-fold difference was observed in %PML-RARa/ABL1 reporting between delta Ct-based and CN-based approaches. Conclusion &amp; Significance: A PML-RARa copy number estimator for prototype Xpert® PML-RARa assay was established, which will provide helpful information for diagnosis and prognosis of APL. (Product in development. Not for use in diagnostic procedures. Not reviewed by any regulatory body.)

High Throughput Microfluidics Platform to Assess Synthetic Lethality and Novel Therapeutic Drug Combinations

Molecularly targeted therapies have reshaped cancer treatment in recent decades; however, not all patient cancers are eligible for or effectively treated by such approaches. In addition, resistance mechanisms (both pre-existing and acquired) limit clinical benefit. To these ends, combination therapies involving targeted agents are increasingly being developed for their potential to induce potent synthetic lethalities, bypass drug resistance, promote durable responses, limit adverse side effects via reduced dosing, and expand treatment options. In the context of acute myeloid leukemia (AML) specifically, several efficacious combination therapies have been approved for specific patient subsets, such as all-trans retinoic acid (ATRA) plus arsenic trioxide in the PML-RARA fusion acute promyelocytic subtype and Midostaurin plus Cytarabine and Daunorubicin in FLT3-mutant AML. Despite the availability of these and other therapies, there remains an unmet need for treatments against AML subtypes lacking actionable driver alterations or for which current therapies are insufficient. A major hurdle limiting novel combination therapy discovery is the sheer combinatorics associated with mixing two or more agents (e.g., screening all possible pairs of 100 drugs in a single cell line requires nearly 5,000 combination experiments). Thus, there is a practical need for methodologies that prioritize test compounds among broad candidate sets and high throughput technologies for rapid and efficient screening. We have developed a combination drug screening workflow that integrates machine learning-based predictions of potentially synergistic compound pairs with a droplet microfluidics-based screening platform. Our physical platform, termed the FlowMatrix, contains a series of nano wells arrayed in a 96 x 96 grid (for 9,216 total wells) in which cells are incubated and compounds delivered via emulsified droplets. To facilitate drug combination screening, the FlowMatrix is loaded column-wise with a 96-element library and subsequently loaded a second time along its rows with either the same or an independent drug library. In our current setup, one FlowMatrix can capture complete data for up to 100 unique drug pairs, which includes control, single-agent, and 4x4 dose combination measurements for each pair. Following multi-day incubation, viability for each well is assessed by the number of live cells imaged with fluorescence microscopy. Combinations with high scoring synergy based on Bliss independence criteria are then pushed to validation and further characterization pipelines. We are currently focused on identifying novel combination therapies for AML. Using small molecule libraries of diverse compound classes and machine learning to prioritize initial screening candidates, we have to-date profiled &amp;gt;8,500 AML cell line-drug combinations in our system. This encompasses &amp;gt;2,900 unique drug combinations profiled in 7 distinct AML cell lines. Among ~270 hits that emerged from these screens are established clinical and preclinical AML combination therapies, in addition to candidates that, to the best of our knowledge, are novel in the context of AML. Strong established hits in our screen include ATR inhibition plus Gemcitabine treatment as well as several combinations involving the BCL-2 inhibitor Venetoclax with chemotherapies (Decitabine and Daunorubicin), Quizartinib, Idasanutlin, and mTOR inhibitors. Thus, we have developed an efficient and cost-effective high throughput drug combinations profiling system that has uncovered candidate therapies that may expand treatment options for patients afflicted by AML.

Outcomes of Paediatric Acute Promyelocytic Leukaemia in the UK

Acute Promyelocytic Leukaemia (APL) is characterized by unique genetic and molecular abnormalities, clinical features, and treatment strategies. It accounts for 5-10% of paediatric acute myeloid leukaemia (AML) cases. APL is defined by the t(15;17)(q24;q21) chromosomal translocation, leading to the formation of the PML-RARA fusion protein, which plays a crucial role in leukemogenesis. All-trans retinoic acid (ATRA) has revolutionized APL management by targeting the PML-RARA fusion protein and inducing complete remission in the majority of patients. Arsenic trioxide (ATO) has emerged as an effective therapeutic agent, particularly in cases with ATRA resistance or relapse. ATO is now widely recommended as part upfront treatment. However, access to ATO has been limited by funding sources in certain countries. This retrospective national study presents the treatment strategies, complications, and survival outcomes of 50 paediatric patients, aged 1 to 18 years, diagnosed with APL in the UK over an 8-year period, between 1 st November 2014 and 31 st October 2021. There were 21 (42%) high risk (HR) patience and 29 (58%) non-HR patients in the cohort. The majority of patients received a chemotherapy-free regimen comprising ATRA and ATO, with additional agents used selectively. Chemotherapy was usually given to patients in the event of lack of access to ATO or clinical urgency. Availability to ATO varied between countries within the UK. Event-free survival (EFS) and overall survival (OS) were evaluated, with a minimum 2 years follow up from diagnosis and median follow-up of 8 years. Overall, the cohort demonstrated a high OS of 94%, with no significant difference observed between HR and non-HR patients. However, HR patients experienced significantly lower EFS, with all relapses occurring in this group. Relapsed patients received varied treatment regimens, including ATRA, ATO, and chemotherapy, with favourable outcomes. This study contributes valuable insights into the management and outcomes of paediatric APL in the UK, emphasizing the importance of early diagnosis and risk stratification for optimal treatment decisions. Addressing disparities in ATO availability and promoting consistent funding across NHS Trusts will be crucial in further improving patient outcomes and reducing relapse rates in APL. Future research should focus on refining treatment protocols and exploring novel therapeutic strategies to enhance the long-term survival of paediatric APL patients. The study highlights the challenges associated with ATO availability across NHS Trusts, impacting treatment decisions and outcomes for APL patients.

AML-503 An Unusual Presentation of Acute Promyelocytic Leukemia (APML): Complex Cytogenetic Isochromosome 17 and WT1 Gene Mutation in the Absence of PML-RARA Fusion Protein

AML-503 An Unusual Presentation of Acute Promyelocytic Leukemia (APML): Complex Cytogenetic Isochromosome 17 and WT1 Gene Mutation in the Absence of PML-RARA Fusion Protein

Impact of Next-Generation Sequencing in Diagnosis, Prognosis and Therapeutic Management of Acute Myeloid Leukemia/Myelodysplastic Neoplasms

For decades, the diagnosis, prognosis and thus, the treatment of acute myeloblastic leukemias and myelodysplastic neoplasms has been mainly based on morphological aspects, as evidenced by the French-American-British classification. The morphological aspects correspond quite well, in a certain number of particular cases, to particular evolutionary properties, such as acute myelomonoblastic leukemias with eosinophils or acute promyelocytic leukemias. Advances in biology, particularly “classical” cytogenetics (karyotype) and molecular cytogenetics (in situ hybridization), have made it possible to associate certain morphological features with particular molecular abnormalities, such as the pericentric inversion of chromosome 16 and translocation t(15;17) in the two preceding examples. Polymerase chain reaction techniques have made it possible to go further in these analyses by associating these karyotype abnormalities with their molecular causes, CBFbeta fusion with MYH11 and PML-RAR fusion in the previous cases. In these two examples, the molecular abnormality allows us to better define the pathophysiology of leukemia, to adapt certain treatments (all-transretinoic acid, for example), and to follow up the residual disease of strong prognostic value beyond the simple threshold of less than 5% of marrow blasts, signaling the complete remission. However, the new sequencing techniques of the next generation open up broader perspectives by being able to analyze several dozens of molecular abnormalities, improving all levels of management, from diagnosis to prognosis and treatment, even if it means that morphological aspects are increasingly relegated to the background.

Unexpected diagnosis in not so usual clinical scenarios: Acute promyelocytic leukemia – A tertiary care hospital experience

Background: Acute promyelocytic leukemia (APL) is a distinct subtype of Acute myeloid leukemia (AML) with arrest in maturation of cells of myeloid series in bone marrow. PML-RARA fusion resulting from t(15;17) translocation forms the genetic basis of this haematological malignancy. Introduction of specific differentiating targeted therapy i.e. All-trans retinoic acid (ATRA) have remarkably improved the outcome of this previously dreaded AML. Aims and Objective: The current study presents five cases of Acute promyelocytic leukemia with unusual presentations followed by effective treatment to revert their adverse outcome. Materials and Methods: In this retrospective evaluation of Acute Myeloid leukemia cases, five cases were included from duration of November 2019 to November 2021. The diagnosis of AMLs was made according to EGIL 1998/WHO 2016 guidelines. Peripheral blood (PB) and bone marrow aspirate (BMA) were stained with Giemsa and cytochemistry included myeloperoxidase (MPO) and periodic acid-schiff (PAS). Results: Five color flowcytometric analyses were performed on BMA and PB samples. Chemotherapy was administered in the patients and follow-up was done. 03/05 patients faced mortality due to rapid disease progression. Conclusion: The fast track clinical course from presentation to the outcome of APL is dreadful for the treating clinician and the heamatopathologist. Cytomorphology and Cytochemistry are as important in rapid recognition of APL as Immunophenotyping and Cytogenetics for establishing accurate diagnosis.

The Pattern of Chromosome Aberrations and Molecular Markers in the Population of Hematological Patients Diagnosed at the University Clinical Center Tuzla

Introduction: Besides cardiovascular, malignant diseases are one of the leading causes of death in Bosnia and Herzegovina. At the top of this list are hematological diseases. This research aimed to identify cytogenetic and molecular biomarkers in patients treated for different types of hematological neoplasms. Methods: The retrospective study included 1600 samples of patients with different hematological diseases in the period from January 2006 to May 2022. The Polymerase Chain Reaction (RT-PCR) method was used to determine the presence of genetic rearrangements and to confirm the findings of conventional cytogenetic analysis. Results: Chromosomal aberrations were found in 739 (46,18%) patients. Using the RT-PCR technique, positive cases were increased by 1,5%. The BCR-ABL fusion gene was present in e14-a2 transcript form in 73% of samples, e13-a2 isoform in 21%, e1-a2 in 2%, while e14-a2/e1-a2 transcript coexpression was present in a percentage of 4% of the samples. The PML-RARa fusion gene was found in the form of bcr 1 transcripts in 21%, bcr2 32% and bcr3 59% of the samples. In twelve cases A type of the CBFB-MYH11 fusion transcript was detected. The MLL-AF4 fusion was found in only one case. Conclusion: The obtained percentages of frequency of individual molecular gene isoforms are in accordance with the results of most other researchers. This refers to the Balkan population and the Caucasian ethnic group.

UBR5 Is a Hect E3 Ubiquitin Ligase That Regulates Chromatin Bound Nuclear Hormone Receptor Stability

Acute promyelocytic leukemia (APML) is driven by the oncogenic PML-RARA fusion, which is unique in that it is degraded by two small molecules, all-trans retinoic acid (ATRA) and arsenic trioxide. ATRA-induced degradation of retinoic acid receptor (RARA) is necessary for sustained anti-tumor effect (Ablain et al, JEM, 2013). More generally, nuclear hormone receptors (NRs) are degraded through a ligand-dependent manner of an unknown mechanism. Here we demonstrate that the HECT E3 ubiquitin ligase UBR5 is a ligand-dependent regulator of RARA and other NRs, solve its structure, and show it binds to NRs on chromatin, regulating transcription by degradation. We performed CRISPR-Cas9 screens on RARA and its partner, retinoid X receptor alpha (RXRA), to identify the ubiquitin ligase responsible for ATRA-dependent degradation. We found that UBR5 sgRNAs rescued ATRA-induced degradation. Moreover, we found that RARA and RXRA bind to UBR5 only in the presence of ATRA using co-immunoprecipitation experiments. NRs undergo a stereotypic structural change in the ligand binding domain (LBD) following ligand binding, revealing a conserved hydrophobic cleft that allows for nuclear co-activator (NCOA) binding through LxxLL peptide motifs, which are also present in UBR5. We performed mutagenesis screens on the RARA LBD and identified key residues within the cleft that, when altered, prevented ATRA-induced degradation and UBR5-RARA binding. Mutation of UBR5 LxxLL motifs also led to deficient RARA binding. We solved a 1.7Å crystal structure of RARA bound to a UBR5 LxxLL peptide, confirming ATRA dependent UBR5 binding in the cleft. To understand UBR5/RARA interactions in the context of full-length UBR5, we solved its structure via cryo-electron microscopy and performed a detailed characterization of its functional domains and binding sites. We confirmed the HECT domain is needed for degradation through a UBR5 CRISPR tiling screen and showed specific loss of function of the HECT domain through targeted sgRNAs led to a rescue of ATRA induced RARA degradation. RARA and PML-RARA are tightly chromatin associated, especially in the context of APML. To examine whether UBR5 binds RARA on chromatin, we performed chromatin-immunoprecipitation and sequencing (ChIP-seq) with anti-RARA and anti-UBR5 antibodies in an APML line (NB4) with and without ATRA treatment. Consistent with ATRA-induced degradation, we found significant loss of RARA peaks after ATRA treatment. RARA binding co-localized with UBR5 binding, suggesting UBR5 associates with RARA on chromatin. RNA-seq analysis of NB4 cells with or without UBR5 KD revealed significant alterations in transcript expression, implying that UBR5 regulates RARA-dependent transcription by degradation and therefore potentially downstream cellular behavior. To test this, we knocked down UBR5 in NB4s, treated with ATRA, and found that UBR5 KD led to increased ATRA resistance. More broadly, we found that UBR5 KD rescues ligand-induced degradation of other NRs including the glucocorticoid (GR), estrogen, vitamin-D, progesterone receptors and liver-X-receptor alpha. Through similar experiments we showed that UBR5 binds to GR in the presence of dexamethasone and recognizes and competes with NCOAs for a similar degron. ChIP and RNA-seq in A549 cells following dexamethasone treatment revealed that UBR5 also co-localizes with GR and regulates its transcription, suggesting UBR5-mediated degradation may be a general regulator of multiple NRs and their transcriptional activities. Our findings reveal a novel molecular mechanism regulating nuclear hormone receptors: ligand dependent E3 ligase binding on chromatin, leading to degradation and transcriptional regulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Unusual Extramedullary Presentation of an NPM1-Mutated Acute Myeloid Leukemia (AML) in a Young Adult

Abstract Introduction/Objective Pleural effusion presentation of AML, particularly without leukemic disease, has been rarely described in the literature. This case serves to investigate the value of an identified NPM1 mutation. Methods/Case Report A 25-year-old male with Down syndrome presented with dyspnea. Computerized tomography scan identified a moderate pleural effusion and pelvic lytic bone lesions. A complete blood count was normal. Pleural fluid cytologic evaluation showed a spectrum of immature myeloid precursors, and flow cytometry identified a 23% CD117 positive myeloblast population expressing CD33, CD13, and dim CD45 but lacking CD34 and HLA-DR. Fluorescence in-situ hybridization testing was negative for a PML-RARA fusion. Given the phenotype, molecular testing was initiated and confirmed an NPM1 mutation. Testing revealed additional pathogenic variants in DNMT3A and IDH1 with no FLT3 mutations. Peripheral blood smear and bone marrow evaluations yielded no clear leukemic involvement, and cytogenetics from bone marrow showed 47,XY,+21c[20]. The significance of this myeloid infiltrate was initially not entirely clear with no established leukemic involvement or tumor-forming mass as required for the World Health Organization diagnosis of myeloid sarcoma. However, the finding of the NPM1 mutation lent high confidence for a diagnosis of an extramedullary presentation of acute myeloid leukemia, though the lytic bone lesions were not biopsied to possibly demonstrate a myeloid sarcoma. Results (if a Case Study enter NA) N/A. Conclusion The identified NPM1 mutation was instrumental in diagnosing an unusual presentation of AML where material was limited. Subsequent to this presentation the patient developed more overt leukemic disease at an outside institution, additionally supporting this diagnosis.

MOLECULAR CYTOGENETIC AND NEXT-GENERATION SEQUENCING STUDIES OF AN ADOLESCENT WITH ACUTE MYELOID LEUKEMIA AND INV(16) ASSOCIATED WITH A KIT PATHOGENIC MUTATION

Inv(16)(p13q22)/CBFb-MYH11 in Acute Myeloid Leukemia (AML) is hard to characterize and comprises about 5%‒7% of all cases and 5%‒9% in children. Inv(16) is associated with a good prognosis both in adults and children. However, approximately 30%‒40% of affected patients experience relapse. In this context, additional type I and II mutations have been related to inv(16). KIT mutations associated with inv(16) have been related to a worse prognosis. Here we describe the clinical and molecular data of a pediatric patient with AML, presenting inv(16) associated with a KIT pathogenic mutation. A 12-year-old boy with bleeding was referred to Pedro Ernesto University Hospital (HUPE), Rio de Janeiro, Brazil. The patient presented with suspected hyperleukocyte acute leukemia. Laboratory tests revealed a WBC count of 80 × 10 3 , a platelet count of 9 × 10 3 , and a hemoglobin level of 5.1 g/dL. Flow cytometry results demonstrated an immunophenotypic profile characteristic of LMA-M4. We conducted G-banding, FISH, and NGS studies. Cytogenetic analysis was non-informative. Based on the clinical and immunophenotypic data, we conducted an experimental design using PML-RARA and CBFB-MYH11 FISH probes. The patient was negative for the PML-RARA fusion, but CBFb-MYH11 was confirmed. The NGS Assay revealed a pathogenic missense mutation in KIT. The results were negative for FLT3/ITD mutations. AML patients presenting inv(16)/CBFb-MYH11 usually have a good prognosis with better remission and overall survival rates compared to those with normal karyotypes. However, due to the high rates of disease relapse, the prognostic value of alterations associated with inv(16)/CBFb-MYH11 has been widely discussed in the literature. Our patient presented with excessive bleeding and heavy hemorrhage, which initially raised the suspicion of acute promyelocytic leukemia. Due to the heterogeneity of the CBFb-MYH11 fusion, featuring distinct clinical and genetic profiles depending on its molecular size molecular and the association of these profiles with specific genomic alterations, a precise genomic characterization is necessary. KIT mutations, such as the one observed in our patient, are present as alterations associated with inv(16) in approximately 9%‒53% of AML cases. Previous studies have demonstrated the potential prognostic impact of KIT mutations in the affected patients. In this context, missense mutations may lead KIT to an overactive state. This type of mutation has been shown to play a pivotal role in mediating clinical response and disease refractoriness in patients treated with targeted therapeutics. Therefore, the information provided through the NGS can be crucial for treatment adaptation. In this work, we contribute with the literature showing the clinical, molecular, and genomic data of an adolescent with AML and inv(16) associated with a KIT pathogenic mutation. Besides, we highlight the importance of NGS studies in characterizing the genomic profile of patients affected by this neoplasm. Although, the prognostic implications of these findings remain to be investigated, especially in the context of new generation tyrosine kinase inhibitors targeting KIT in future clinical trials for inv(16)/CBFb-MYH11 pediatric patients.

EPIDEMIOLOGICAL AND MOLECULAR PROFILE OF PATIENTS WITH ACUTE MIELOID LEUKEMIA IN CEARÁ

Acute myeloid leukemia (AML) is the second most common leukemia in adults and older patients represent most cases. The states of the Northeast of the country have stood out in the frequency of leukemia cases, and the frequencies are higher than the rate of 35% compared to other neoplasms, especially in the metropolitan regions of Fortaleza. In the State of Ceará, the number of cases of leukemia expected for the year 2020 is 6.17 cases/100,000 inhabitants in men and 4.29 cases/100,000 inhabitants in women. In view of this information, the aim of this study was to identify the molecular and epidemiological profile of patients with AML in the state of Ceará. Sample collection was performed in patients with AML treated at the General Hospital of Fortaleza, considered the main and largest hematology outpatient clinic in the metropolitan region of Fortaleza, as well as the State of Ceará. All patients participating in this study read and signed the informed consent form. Patients with other types of leukemias or other hematological diseases were excluded. This study was approved by the Research Ethics Committee of the Federal University of Ceará under protocol no. 38680520.9.0000.5054. From July 2021 to July 2022, 31 patients with AML were treated, of which 8 are women and 23 are men. The mean age observed was 50.4 years. 54.8% of patients live in the capital, while the other 45.2% of patients live in rural areas. Through immunophenotyping it was possible to verify that the main markers presented by LMA patients were CD33, CD13, CD117, CD45, MPO, CD34, CD64, HLA-DR, CD11c, CD11b e CD38. Thirteen patients (42%) had abnormal and complex karyotypes, of which six died. Five patients (16,1%) had the t(15;17)(q24;q21.3) corresponding to the PML-RARA fusion. Six patients (19,3%) had the FLT3 mutation that confers a poorer prognosis. During the clinical follow up, a total of 16 patients died due to septic shock, COVID-19, or refractoriness to treatment. The incidence of cases of adults over 50 years of age with AML was 58%, as it corroborates the expected incidence of cases regarding the age described in the literature. Studies indicate that immunophenotyping is critical to the differential diagnosis among AML subtypes. AML is defined by the mainly expression of the markers MPO, CD117, CD13, CD33, HLA-DR and CD38, corroborating what was observed in the results. In addition, depending on the alteration observed in the karyotype, it may confer poor or good prognosis to the patient. The PML-RARA merger, for example, is known as M3 AML or acute promyelocytic leukemia (APL), a subtype of AML with a better prognosis for patients. The FLT3 mutation is present in 1 in 3 patients with AML. The presence of this altered gene may mean a worse prognosis and a greater possibility of recurrence but allows a specific and differentiated treatment. Epidemiological data from this preliminary study indicate that in the state of Ceará AML is more common in male patients aged 50-80 years. In addition, the use of targeted therapies for patients with abnormal karyotypes as well as the adoption of stricter measures to prevent hospital infections may increase patient survival.

AML-317 Long-Term Follow-Up of Acute Promyelocytic Leukemia: Experience From Clinical Hematology Department of Casablanca

Acute promyelocytic leukemia (APL) accounts for about 5-10% of all AML cases, its incidence is about 0.1/100,000. The present study was designed to analyze the clinicopathologic features and outcomes in APL. This is a retrospective descriptive study based on medical records and follow-up of APL patients from 2003 to 2021. It includes all patients diagnosed with APL at the Casablanca Hematology Department. The diagnosis was genetically confirmed by detecting the PML-RAR fusion gene or demonstrating the t(15;17). A total of 94 patients with APL were seen between November 2003 and July 2021 in our department. Median age at presentation was 34 years (range, 1-69 years). The gender ratio was 0.7 (40 men and 54 women). Patients from the northwest region of Morocco, which is dominated by Casablanca, accounted for 47 percent of all patients, followed by 15% from the north and 11% from the east. The median duration of symptoms before presentation was 32 days (range, 2-240 days). At the time of presentation, 51 (60%) of the patients had disseminated intravascular coagulation (DIC). The performance status was 1 in 72% of the cases. The hemorrhagic grade was 36% of stage 2. High-risk, intermediate-risk, and low-risk were seen in 32%, 55%, and 13% of patients, respectively. PML-RAR data was available for 34 patients (36%) and karyotyping data for 85 patients (91%). Except for 15 patients (16%) who died at diagnosis, all 79 patients who were followed up on received chemotherapy: 63 patients (78.8%) received APL protocol and 18 patients (19.8%) Pethema. At the end of induction chemotherapy, 68 patients (86%) achieved complete hematologic remission, while 11 patients (14%) died. 3 patients relapsed after maintenance treatment including 2 cases of neurological relapse and one case of bone marrow relapse, all died. At 4 years, the event-free survival and overall survival rates were 62% and 62%, respectively. Early death remains the major reason for treatment failure. Retrospective studies have reported an early death rate of 7.2-21.1%. In our population, it represents 16%. APL has progressed from a fatal disease to one that is highly curable in the last decade.

The role of adjuvant chemotherapy in the management of acute promyelocytic leukemia differentiation syndrome.

Acute Promyelocytic Leukemia (APL) is characterized by the t(15;17) chromosomal translocation resulting in a PML-RARA fusion protein. The all-trans-retinoic acid (ATRA) and Arsenic Trioxide (ATO) only regimens have demonstrated success in treating low- and intermediate-risk patients. However, induction with ATRA/ATO only regimens have been showing increased incidence of differentiation syndrome (DS), a potentially lethal complication, traditionally treated with dexamethasone. We conducted a three-institution retrospective study, aiming to evaluate the role of short-term adjuvant chemotherapy in managing moderate DS for patients with low- or intermediate-risk APL initially treated with ATRA/ATO only protocols. We evaluated the difference in incidence and duration of moderate DS in APL patients who were treated with ATRA/ATO with or without adjuvant chemotherapy. 57 low- or intermediate-risk APL patients were retrospectively identified and included for this study; 36 patients received ATRA/ATO only induction treatment, and 21 patients received ATRA/ATO/adjuvant chemotherapy combination induction therapy. Similar proportions of patients experienced DS in both groups (66.7% vs. 81.0%, P = 0.246). The median duration of DS resolution in patients receiving ATRA/ATO only was 17 days (n = 23), and in patients receiving combination therapy was 8 days (n = 16) (P = 0.0001). The lengths of hospital stay in patients receiving ATRO/ATO only was 38 days (n = 7), and in patients receiving combination therapy was 14 days (n = 17) (P = 0.0007). In conclusion, adding adjuvant chemotherapy to ATRA/ATO only protocol may reduce the duration of DS and the length of hospital stay during APL induction treatment.

Vitamin D Derivatives in Acute Myeloid Leukemia: The Matter of Selecting the Right Targets.

Acute myeloid leukemia (AML) is an aggressive and often fatal hematopoietic malignancy. A very attractive way to treat myeloid leukemia, called “differentiation therapy”, was proposed when in vitro studies showed that some compounds are capable of inducing differentiation of AML cell lines. One of the differentiation-inducing agents, all-trans-retinoic acid (ATRA), which can induce granulocytic differentiation in AML cell lines, has been introduced into clinics to treat patients with acute promyelocytic leukemia (APL) in which a PML-RARA fusion protein is generated by a chromosomal translocation. ATRA has greatly improved the treatment of APL. Since 1,25-dihydroxyvitamin D (1,25D) is capable of inducing monocytic differentiation of leukemic cells, the idea of treating other AMLs with vitamin D analogs was widely accepted. However, early clinical trials in which cancer patients were treated either with 1,25D or with analogs did not lead to conclusive results. Recent results have shown that AML types with certain mutations, such as isocitrate dehydrogenase (IDH) mutations, may be the right targets for differentiation therapy using 1,25D, due to upregulation of vitamin D receptor (VDR) pathway.

MicroRNA-125b Accelerates and Promotes PML-RARa-driven Murine Acute Promyelocytic Leukemia

ObjectiveMost acute promyelocytic leukemia cases are characterized by the PML-RARa fusion oncogene and low white cell counts in peripheral blood. MethodsBased on the frequent overexpression of miR-125-family miRNAs in acute promyelocytic leukemia, we examined the consequence of this phenomenon by using an inducible mouse model overexpressing human miR-125b. ResultsMiR-125b expression significantly accelerates PML-RARa-induced leukemogenesis, with the resultant induced leukemia being partially dependent on continued miR-125b overexpression. Interestingly, miR-125b expression led to low peripheral white cell counts to bone marrow blast percentage ratio, confirming the clinical observation in acute promyelocytic leukemia patients. ConclusionThis study suggests that dysregulated miR-125b expression is actively involved in disease progression and pathophysiology of acute promyelocytic leukemia, indicating that targeting miR-125b may represent a new therapeutic option for acute promyelocytic leukemia.