Utilization of RT-PCR and Optical Genome Mapping in Acute Promyelocytic Leukemia with Cryptic PML::RARA Rearrangement: A Case Discussion and Systemic Literature Review

Giby V George; Murad Elsadawi; Andrew G Evans; Sarmad Ali; Bin Zhang; M Anwar Iqbal

doi:10.20944/preprints202411.0719.v1

Submitted:

09 November 2024

Posted:

11 November 2024

You are already at the latest version

Abstract

Acute promyelocytic leukemia (APL) is characterized by abnormal promyelocytes and t(15;17)(q24;q21) PML::RARA. Rarely, patients may have cryptic or variant rearrangements. All-trans retinoic acid (ATRA)/arsenic trioxide (ATO) is largely curative provided that the diagnosis is established early. We present a case in which the diagnostic work-up suggested a diagnosis of APL; however, FISH, using the PML/RARA dual fusion and RARA breakapart probes, was negative. RT-PCR later revealed a cryptic fusion transcript. Optical genome mapping (OGM) confirmed the nature and orientation of the insertion of RARA into PML. This case underscores the importance of performing alternative testing in FISH-negative cases of APL.

Keywords:

Acute promyelocytic leukemia

;

APL

;

cryptic PML::RARA

;

cryptic APL

Subject:

Medicine and Pharmacology - Hematology

Introduction

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) which accounts for approximately 5-8% of all new AML diagnoses in young adults [1]. Patients present with features of coagulopathy and are at increased risk for disseminated intravascular coagulation (DIC) [1]. Morphologically, APL is characterized by abundant abnormal promyelocytes with a classic immunophenotype demonstrable by flow cytometry [1]. Per the WHO classification, the diagnosis of APL is rendered based on these microscopic findings in conjunction with t(15;17)(q24.1;q21.2). This rearrangement results in the fusion of the 5’ end of the promyelocytic leukemia (PML) gene (exons 1-3) on chromosome 15 with the 3’ end of the retinoic acid receptor alpha (RARA) gene (exons 3-9) on chromosome 17 [1,2,3]. The International Consensus Classification of Myeloid Neoplasms and Acute Leukemias (ICC) requires at least 10% blasts (or blast equivalents, i.e., abnormal promyelocytes) for diagnosis [2]. Morphologic subtypes of APL include the hypergranular (i.e., typical) and hypogranular (i.e., microgranular) forms (with a basophilic variant described in some studies) [2]. Notably, most APL subtypes, including cases with variant RARA translocations, demonstrate similar morphologies [2].

The t(15;17)(q24.1;q21.2) translocation can usually be identified by fluorescence in situ hybridization (FISH) using PML/RARA dual fusion or RARA breakapart probes or chromosomal Giemsa (G)-banding analysis. The reciprocal t(15;17) translocation is present in 90-95% of APL cases, and, depending on the location of the PML breakpoint, three PML::RARA fusion transcript isoforms can be produced: long (bcr1; intron 6), variant (bcr2; exon 6), and short (bcr3; intron 3), with the bcr1 and bcr3 isoforms being the most common [1,4,5]. Rarely, atypical isoforms can be also be detected [1]. The RARA breakpoint consistently occurs within intron 2 [4]. A subset of APL cases (~5-9%) [6] may show cryptic PML::RARA rearrangement with submicroscopic insertion of PML to RARA, or complex rearrangements involving other chromosomes. Other cases of APL (~1-2%) [7] may show variant RARA rearrangements [1,3,8,9,10,11,12,13]. Importantly, cases of APL with cryptic rearrangements may require alternative methods of detection [1].

Herein, we describe a case of cryptically rearranged APL. Fortunately, the patient had been initiated on all-trans retinoic acid (ATRA)/arsenic trioxide (ATO) early based on the morphological/immunophenotypic findings and demonstrated clinical improvement. These findings, in conjunction with those of our systematic literature review, highlight the importance of performing confirmatory testing in FISH-negative cases of suspected APL.

Materials and Methods

Pathological Examination

Standard Wright Giemsa peripheral blood smears were prepared.

Clinical Flow Cytometry Evaluation

Expression of cell surface markers was measured by flow cytometry (Navios, Beckman Coulter) using the ClearLLab 10C 10-color lymphoid and myeloid panels and analyzed using the Kaluza analysis software.

Chromosome G-Banding Karyotype Analysis

Cytogenetic analysis was performed using short-term bone marrow cultures per routine laboratory protocol. For microscopic analysis, metaphase chromosomes were stained using the trypsin-Giemsa technique [14]. For chromosome analysis, 20 cells were analyzed and two to five metaphases were karyotyped. Chromosomal abnormalities were defined according to ISCN (2020) [15].

Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) was performed on 200 interphase nuclei using the AML panel probes (Abbott Molecular/Vysis, Inc.): LSI EGR1 (5q31) SO/D5S23, D5S721 SG, LSI D7S486 (7q31) SO/CEP 7 (D7Z1) SG, LSI RUNX1T1/RUNX1 Dual Color, Dual Fusion Translocation, LSI KMT2A Dual Color, Break Apart Rearrangement, LSI PML/RARA Dual Color, Dual Fusion Translocation, LSI CBFB Dual Color, Break Apart Rearrangement, LSI 13 (13q14) SG/LSI TP53 (17p13.1) SO, and LSI RARA Dual Color, Break Apart Rearrangement probes. Retrospective interphase FISH using the CytoCell (Oxford Gene Technology) PML/RARA dual color dual fusion probe set was performed, followed by confirmatory metaphase FISH using the CytoCell probe.

Mutational Analysis

FLT3 mutational analysis was performed using rapid multiplex PCR. Targeted DNA-based NGS was performed using the 34-gene Illumina TruSight Myeloid Panel.

Optical Genome Mapping

Optical Genome Mapping (OGM) was performed as described previously [16]. Briefly, ultra-high molecular weight DNA was isolated from the patient’s peripheral blood sample using the SP Blood and Cell Culture DNA Isolation Kit. The DLS DNA Labeling Kit was used to fluorescently label long molecules at specific CTTAAG motifs throughout the genome with the enzyme DLE-1 (Bionano Genomics). Labelled DNA was loaded onto a chip and imaged on the Saphyr instrument for the collection of 1500 Gb data with a molecule size of >150 kb. Data analysis was performed using the Bionano Access Software (BAS) Version 1.8.1 and Variant Intelligence Analysis (VIA) version 7.0

Systematic Literature Review

A systematic literature review was conducted through a targeted search (using the following search terms: “APL with cryptic translocation,” “APL with negative FISH,” “APML with negative FISH,” and “APL with RT-PCR”) for case reports and original articles published in English journals from 2013 – 2023 archived in PubMed.

Results

Case Description

A 36-year-old male presented with spontaneous ecchymosis, petechiae, and exertional dyspnea. Laboratory investigations revealed anemia (hemoglobin 11.2 g/dL), thrombocytopenia (platelets 10 THOU/µL), hypofibrinogenemia (70 mg/dL), prolonged prothrombin time (14.7 seconds)/international normalization ratio (INR) (1.3), and elevated D-dimer (26.20 µg/mL), concerning for DIC. Peripheral blood examination demonstrated pancytopenia. Scattered promyelocytes with bilobed nuclei, occasional Auer rods, and variable cytoplasmic granules were noted by microscopy, raising concern for APL (Figure 1A). Consistent with this preliminary diagnosis, flow cytometry evaluation revealed an abnormal cell population within the CD45-dim gate with high side scatter and co-expression of CD13, CD33, CD117, CD38, CD123, CD64, and cytoplasmic myeloperoxidase (MPO), while negative for all other markers tested (Figure 1B).

Stat FISH, however, was negative for the classical t(15;17) rearrangement (Figure 2).

Given the clinical concern for APL, rapid real-time polymerase chain reaction (RT-PCR) was performed, which detected a cryptic PML::RARA gene rearrangement (bcr3 transcript isoform). Despite negative FISH results, the patient had been started on all-trans retinoic acid (ATRA)/arsenic trioxide (ATO) therapy early based on the classical immunomorphology. Targeted DNA-based NGS subsequently revealed an oncogenic mutation in NRAS (c.35G>T, p.G12V) at a variant allele frequency (VAF) of 11%. Retrospective FISH using the CytoCell PML/RARA dual color dual fusion probe set revealed a fusion in 74% of interphase cells, suggestive of an insertion of the RARA gene into the PML gene (Figure 2B). Metaphase FISH also confirmed this aberration (Figure 2C). Further analysis using OGM revealed an insertion in the PML gene at intron 3 (bcr3 transcript) and missing molecules on 17q21.2 from exons 1 – 2 in the RARA gene with the breakpoint in intron 2 (Figure 3). Presently, the patient is in remission and undergoing consolidation therapy.

Literature Review

A total of 71 articles were retrieved, of which 29 articles were selected for further evaluation based on preliminary abstract review. Of these, 17 articles described single case reports of APL with cryptic PML::RARA rearrangements and are summarized in Table 1. The remaining reports are described below.

Discussion

While APL can be suspected based on clinical presentation and pathology evaluation [17], the definitive diagnosis requires the demonstration of the cytogenetic hallmark, t(15;17)(q24;q21) translocation by G-banding or targeted FISH. The chimeric PML::RARA oncoprotein, along with secondarily acquired genetic aberrations (e.g. mutations in FLT3 ITD, WT1, NRAS, or KRAS) [18,19], impair myeloid differentiation and drive leukemogenesis. We describe a case of APL that presented with signs of clinical coagulopathy. Although pathology evaluation in our patient suggested a diagnosis of APL, stat FISH and karyotype analysis were negative for t(15;17)(q24;q21). Concomitant RT-PCR revealed a cryptic PML::RARA gene fusion. Fortunately, the patient had been initiated on ATRA/ATO and demonstrated clinical improvement. Accordingly, we performed a systematic literature review to understand the prevalence, diagnosis, and prognosis of APL with cryptic PML::RARA translocations.

Several individual reports of APL with little-to-no cytogenetic evidence of t(15;17) but with nearly identical clinical presentations and morphologies have been reported in the literature, with RT-PCR later revealing a cryptic PML::RARA fusion (Table 1) [20,21,22]. In an institutional analysis over an 18.5-year timeframe, Gagnon et al found the majority of APL cases (87% [723/831]) to possess balanced PML::RARA translocations by chromosomal banding analysis and PML::RARA dual-color, dual-fusion FISH (Abbott) [6]. Of the remaining 13% of patients, only 0.7% (6/831) were found to harbor a cryptic PML::RARA gene fusion, confirmed by retrospective metaphase FISH in two cases [6]. Goldschmidt et al describe a case of APL with a cryptic PML::RARA gene rearrangement, in which subsequent metaphase FISH revealed an interstitial insertion of RARA into PML [23]. Burns et al present a similar case but with an insertion of PML into RARA.²² Interestingly, in a case of APL with a cryptic PML::RARA translocation, identified by RT-PCR, Koshy et al detected a 49-kilobase duplication of 15q24.1 by SNP microarray, which likely inserted into chromosome 19 or 20; they hypothesize that the RARA insert in this case was too small to detect using FISH or microarray [24]. Other methods of confirmation include sequencing (both Sanger and next-generation).

Likewise, atypical karyotypic findings are present in nearly 52% of cases of cryptically-rearranged APL [25]. Chromosomal aberrations reported in conjunction with cryptic PML::RARA rearrangements include trisomy 8 (most common) [4],⁴ trisomy 9 [23], i(5p) [23], complex karyotypes [26,27], and structural rearrangements involving chromosome 17 [3] (e.g., isochromosome 17q) [27,28,29,30]. Few studies suggest the presence of i(17q) in such cases to confer a worse prognosis, with short complete response durations and high rates of relapse [27]. Similarly, rearrangements involving chromosomes 3, 7, 9, 11, 17, and 22 have also been reported in association with cases of FISH-negative APL [31]. Of note, the presence of structural karyotypes does not seem to affect treatment response to ATRA/ATO [26].

Importantly, all patients with APL resulting from a cryptic t(15;17) achieved remission following treatment with ATRA/ATO [3,20,21]. Greenfield et al compared five patients with cryptically-rearranged APL to eight patients with APL resulting from the reciprocal t(15;17)(q24.1;q21.2) and found no significant difference in disease behavior or prognosis [32]. Furthermore, previous reports observed the bcr3 isoform to be more frequent in cases of cryptic PML::RARA-rearranged APL (61% of cases) [4], as was observed in our case. Although this isoform has been associated with leukocytosis and a poorer prognosis, further studies are needed to understand the association, if any [5,23,25].

Given the aggressive coagulopathy associated with APL (85% of cases) [4], it is imperative that cryptic PML::RARA translocations are identified early to initiate ATRA/ATO given the resistance of APL to standard induction chemotherapy regimens. FISH is an invaluable tool to detect the classical t(15;17) translocation with a resolution of nearly 200 kb; however, cryptic cases may be missed due to submicroscopic insertions unable to be visualized by fluorescent microscopy [33]. Although smaller custom FISH probes can be applied [33], alternative testing modalities, such as PCR, OGM, or sequencing, may be required to establish a diagnosis of APL [5,20,21,24,34,35]. Seldom, RT-PCR may also be negative in the case of an atypical PML::RARA rearrangement with a new breakpoint in PML that is not readily identifiable using routine primers [23]. Finally, both conventional cytogenetics and RT-PCR may fail to yield a positive result, such as cases with a submicroscopic deletion of 3’ RARA or cases with no RARA rearrangement [23,36,37].

Regardless, a high degree of clinical suspicion and prompt recognition are required in suspected cases of APL for immediate initiation of ATRA/ATO [4,38]. Although rare, cryptically-rearranged APL poses a diagnostic challenge and may require alternative, parallel testing modalities to establish the diagnosis [33,38].

Author Contributions

GVG wrote the manuscript with input from MUE. AGE, SA, BZ, and MAI provided edits. All authors reviewed and approved the final version of the manuscript.

Funding

This research received no funding.

Data Availability

No new data were created or analyzed in this study.

Conflicts of Interest

The authors declare no potential conflicts of interest.

Patient Consent

Informed consent was not required for this study as no patient identifying information has been included.

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Figure 1. Peripheral Smear Findings. A. Morphologic examination revealed scattered abnormal promyelocytes with variable cytoplasmic granules and occasional Auer rods. B. An abnormal cell population (gray) with high SSC comprising 80% of total events is observed, co-expressing CD13, CD33, CD117 (not shown), CD38, CD123, CD64 and cytoplasmic-MPO, while negative for CD34, HLA-DR, and all other markers tested.

Figure 2. A. Interphase FISH performed using the Vysis dual color dual fusion t(15;17) probe, showing normal signals for both PML (SpectrumOrange) and RARA (SpectrumGreen). B. Retrospective FISH using the CytoCell PML/RARA dual color dual fusion probe set, which targets smaller regions, revealed a fusion in 74% of interphase cells, suggestive of an insertion of the RARA gene into the PML gene. C. Metaphase FISH also confirmed this cryptic rearrangement.

Figure 3. Optical Genome Mapping Results. A. OGM analysis using the BAS software showed an insertion in the PML gene at breakpoints 73995446 and 74023755 marked in red. B. Further analysis using the VIA software confirmed the insertion in PML at intron 3 (bcr3 region). C. OGM analysis using the BAS software showed the missing alignment of molecules on the RARA gene highlighted using red. D. The low coverage of OGM molecules from exon 1 and 2 and the breakpoint in intron 2 of the RARA gene is highlighted in red, confirm the missing alignment of molecules. E. The possible S-isoform with type A translocation/fusion was constructed based on the available breakpoints and intron 3 involvement on the PML gene.

Table 1. Previously reported single cases of APL with cryptic t(15;17).

No.	Authors/ PMID	Age/Sex	Clinical Presentation	Microscopic Findings	Flow Cytometry Expression	Karyotype Findings	Interphase FISH	Mutational Analysis	RT-PCR Results	Confirmatory Testing
1	Avgerinou et al/ 32909480	12/F	Multiple ecchymoses	Leukocytosis Abnormal promyelocytes with bilobed nuclei and cytoplasmic granules Anemia thrombocytopenia	MPO, CD34 CD123, CD64, CD33, CD117, CD9, HLA-DR and CD2, while negative for other markers	46, XX	Abbott Molecular LSI PML/RARA dual-color dual-fusion translocation probe: Negative Cytocell (Cambridge, UK) positive for PML::RARA fusion	FLT3-ITD	bcr3-PML/RARA transcript	Sanger sequencing: in-frame fusion of PML exon 3 and RARA exon 3
2	Blanco et al/ 24561214	17/M	Gum bleeding, multiple ecchymoses, abdominal pain, and fever	Leukocytosis Abnormal promyelocytes with bilobed nuclei and cytoplasmic granules	CD117, CD45 (dim), CD13, CD33, CD15 (weak), and CD64 while negative for HLA-DR, CD34, and other markers	46, XY	Negative	FLT3-ITD	bcr1-PML/RARA transcript	Sequencing: in-frame fusion of PML exon 6 and RARA exon 3
3	Burns et al/ 30030569	23/F	Epistaxis and easy bruising	Leukocytosis Abnormal myeloblasts/promyelocytes with ovoid nuclei and cytoplasmic granules (rare Auer rods) Anemia thrombocytopenia	CD13, CD33 (partial), CD56 (partial, dim), CD64, and MPO, while negative for HLA-DR and CD34	46, XX,+8	Abbott Molecular LSI PML/RARA dual-color dual-fusion translocation probe: Negative	Not mentioned	Cryptic PML::RARA fusion	Metaphase FISH: interstitial insertion of PML into the RARA gene
4	El-Hajj Ghaoui et al/ 29550828	8/M	Bruising and bleeding gums	Leukocytosis Blasts and abnormal promyelocytes with large irregularly folded or bi-lobed nuclei and abnormal granulation (rare Auer rods) Anemia thrombocytopenia	CD13, CD33, CD117, CD123, and CD45, while negative for other markers	46,XY,der(17)ins(17; 15) (q21;q24q24)?del(17)(p11.2)add(17)(q21)	MetaSystems, Germany dual colour dual fusion PML-RARA probe: single fusion signal and 2 copies of PML and RARA; second expected reciprocal fusion signal not present, and one each of the PML and RARA signals was of diminished intensity	FLT3-ITD	bcr3-PML/RARA transcript and a faint ∼ 350-bp product of unknown origin	Metaphase FISH (using RARA break-apart probe [Abbott Molecular, USA], 15q11.2 control locus [RP11-160D9 from Australasian Genome Research Facility, Melbourne, Australia], subtelomere clones for chromosome 15q [GS-154P1], 17p [cosmid 2111b1], and 17q [PAC GS-362K4], and NF1 within chromosome band 17q11.2): single fusion with diminished RARA signals on the derivative chromosome 17; i.e., der(17) 850K SNP chromosome microarray: no clinically relevant chromosome copy number abnormality across the tumor genome
5	Fan et al/ 24673420	61/F	Fatigue and easy bruising	Leukocytosis Blasts/promyelocytes 40% Anemia thrombocytopenia	Dim CD45, CD13, CD33,CD117, variable CD34, and lacking HLA-DR	46, XX, +8 (17/20 cells)	Peripheral blood: variant abnormal signal pattern with 1fusion (1F1O2G) in 52.5% of the nuclei Bone marrow: variant abnormal signal pattern with 1fusion (1F1O2G) in 42% of the nuclei	Not mentioned	Cryptic PML::RARA fusion without reciprocal RARA-PML fusion transcripts in either the diagnostic or follow-up samples	Metaphase FISH: Non-reciprocaltranslocation with the fusion signal on chromosome 15 and absence of the fusion signal on chromosome 17 Metaphase FISH using whole chromosome paint: RARA(green) signal on chromosome15, without the corresponding PML (orange) signal on chromosome 17, demonstrating an insertion
6	Mai et al/ 32366568	17/M	Seizure (with recent history of nausea, blood-tinged vomiting, lethargy, and right-sided weakness)	Leukocytosis Blasts/promyelocytes 83% Anemia thrombocytopenia	CD2 (partial), CD4 (partial), CD13, CD33, CD38, CD45, CD64, CD117 (partial), HLA-DR (small subset), and MPO (bright), while negative for other markers	46, XY	Negative	Not mentioned	Cryptic PML::RARA fusion	Not performed
7	Zhang et al/ 31959056	66/M	Petechiae and bruises	Leukopenia Blasts/promyelocytes 68% (irregular nuclear shapes, misty nucleoli) Anemia thrombocytopenia	CD34, CD7, CD13, CD33, CD117, CD38, HLA-DR and MPO, while negative for other markers	46, XY	Negative	Biallelic CEBPA mutation	bcr2-PML/RARA transcript (nested RT-PCR)	Not performed
8	Gu et al/ 33052080	62/M	Pharyngalgia, fatigue, and gum bleeding	Anemia Leukopenia Hypercellular bone marrow with abnormal promyelocytes	CD117, CD33, myeloperoxidase (MPO), CD13, CD58, CD38, and CD81	46,XY, add(11) (p15), and ?t(13;20)(q12;q11.2)	Atypical PML::RARA fusion signal in 91% of nuclei	FLT3, WT1, and KRAS mutations	Major PML/RARα transcript harbored the three type breakpoints	Not performed
9	Kim et al/ 21156244	18/M	Hematuria and hematochezia	Leukocytosis Blasts/promyelocytes 84% Anemia thrombocytopenia	CD13, CD33, CD45, and CD117 and negative for HLA-DR and CD34	46,XY	Negative	Not mentioned	Three PML::RARA fusion transcripts: bcr2, bcr1, and novel transcript (exon 4 of PML and exon 3 of RARA)	Long-distance DNA-PCR: rearrangement between PML (intron 6) and RARA (intron 2)
10	Goldschmidt et al/ 20863428	52/F	Bleeding tendency	Anemia Leukopenia Thrombocytopenia	Strongly positive for CD33 and CD13 while negative for HLA-DR	47,XX,zi(5)(p10)[20]/48,idem,z9[2]/46,XX[6]	Negative	Not mentioned	Bcr1-PML/RARa transcript	Metaphase FISH: interstitial insertion of RARa gene into PML gene (low signal retrospectively identified on interphase FISH)
11	Koshy et al/ 22982005	29/M	Progressive fatigue Bruises	Pancytopenia 26% abnormal promyelocytes	Positive for CD117, CD33, and CD13 but negative for HLA-DR and CD34	46,XY	Negative with a small PML signal present on another chromosome (20% of cells)	Not mentioned	Positive for PML::RARA transcript	Sanger sequencing across the PML-RARA breakpoint demonstrated a BCR1-type fusion. Whole genome SNP microarray: intragenic duplication of PML on chromosome 15q24.1 (30% of cells).
12	Karlin et al/ 35572917	54/M	DVT	Pancytopenia 77% abnormal promyelocytes in the BM	Positive for MPO and CD117 while negative for CD34 and HLA-DR	46,XY	Negative	FLT3 p.D835Y variant	Bcr1-PML/RARA transcript	Not performed
13	Mahmud et al/ 32924730	68/F	Dizziness Fatigue Acute on chronic PE	Pancytopenia 70% abnormal promyelocytes/blasts	Not mentioned	46, XX	Two copies of chromosome 15, but absence of the reciprocal translocation on the two copies of chromosome 17	Not mentioned	Positive for bcr3-PML::RARA transcript	Metaphase FISH: insertion of a RARA segment into chromosome 15 at the location of PML. Whole-genome sequencing: complex t(15;17) with a possible intrachromosomal rearrangement of chromosome 15.
14	Schultz et al/ 31809670	57/F	Bruising Gingival bleeding	Anemia Neutropenia Thrombocytopenia Abnormal promyelocytes/blasts	Positive for CD13, CD33, CD34 (partial), CD117, MPO, and aberrant partial CD2 expression	46, XX	Negative	Not mentioned	PML-RARA fusion in 53% of cells	Mate-pair sequencing: cryptic insertional translocation resulting in PML-RARA fusion with breakpoints located within intron 6 of PML and intron 2 of RARA.
15	Shepshelovich et al/ 26471811	53/F	Not mentioned	Not mentioned	Not mentioned	46XX, iso(17)(q11)	Using the Cytocell probe: negative	Not mentioned	Major PML−RARA transcript harbored the BCR-1 breakpoint. BCR-2 and BCR-3 were demonstrated as spliced variants.	FISH using the Vysis probe: several clones detected
16	Tang et al/ 26823883	21/M	Melena Bleeding tendency	Not mentioned	Not mentioned	Complex karyotype with isochromosome 17q	Two PML/RARA fusion signals	Not mentioned	Not mentioned	Not performed
17	Venci et al/ 28599418	73/F	Diagnosed incidentally (pre-operatively)	Anemia Thrombocytopenia Abnormal promyelocytes/blasts	Positive for CD13, CD33, MPO, CD2 and CD9, while negative for other markers	46,XX	Two fusion signals on the two copies of chromosome 15, but absence of the reciprocal on the two copies of chromosome 17	Not mentioned	Bcr3/short form PML-RARA fusion transcript	Metaphase FISH: two PML/RARA fusion signals (one on each copy of chromosome 15, and two normal RARA signals on the two copies of chromosome 17), raising the possibility of uniparental isodisomy.

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