Identification of a cryptic IRF2BP2-RARA fusion in acute promyelocytic leukemia

Abstract

Acute promyelocytic leukemia (APL) is characterized by fusion of RARA with PML, or rarely other gene partners. We report a patient with APL with a novel fusion between RARA and the interferon regulatory factor 2 binding protein 2 (IRF2BP2) genes. A 19-year-old woman presented with ecchymoses and epistaxis. Bone marrow examination showed morphologic and immunophenotypic features consistent with APL. PML oncogenic domain antibody was positive. Conventional cytogenetics, FISH, RT-PCR and oligonucleotide microarray for PML-RARA and common APL variant translocations were negative. Next generation RNAseq analysis followed by RT-PCR and direct sequencing revealed distinct breakpoints within IRF2BP2 exon 2 and RARA intron 2. The patient received ATRA, arsenic trioxide and gemtuzumab ozogamicin, and achieved complete molecular remission. However, the disease relapsed ten months later, two months after completion of consolidation therapy. This case expands the list of novel RARA partners identified in APL.

Introduction

Acute promyelocytic leukemia (APL) is commonly associated with t(15;17)(q24.1;q21.2) which causes fusion of the retinoic acid receptor alpha (RARA) and promyelocytic leukemia (PML) genes.¹ The fusion accounts for response to all-trans retinoic acid (ATRA) and a favorable prognosis.¹ Rarely, APL cases carry RARA fused with other genes, such as zinc finger and BTB domain containing 16 (ZBTB16, also known as promyelocytic leukemia zinc finger, PLZF),^2,3 nucleophosmin (NPM1),⁴ nuclear mitotic apparatus (NUMA1),⁵ signal transducer and activator of transcription 5b (STAT5B),⁶ cAMP-dependent protein kinase type I alpha regulatory subunit (PRKAR1A),⁷ FIP1-like 1 (FIP1L1),⁸ BCL6 corepressor (BCOR),⁹ and oligonucleotide/oligosaccharide-binding fold containing 2A (OBFC2A).¹⁰ Among these variants, APL associated with PLZF-RARA and STAT5B-RARA are resistant to ATRA.^6,11

We report a patient with APL with a novel RARA fusion partner, interferon regulatory factor 2 (IRF2) binding protein 2 (IRF2BP2) gene. IRF2BP2, located at chromosome 1q42.3, encodes a negative regulator of nuclear factor of activated T-cells (NFAT) transcription factor that regulates genes involved in cell cycle, differentiation, and apoptosis.^12,13 IRF2BP2 is also a muscle-enriched transcription factor required to activate vascular endothelial growth factor A (VEGFA) expression in muscle.^12,13 This is the first report showing involvement of IRF2BP2 in APL.

Methods

Case history

A 19-year-old woman presented with ecchymoses and epistaxis. A complete blood cell count showed white blood cells 4.5K/uL, hemoglobin 9.1g/dL, and platelets 29,000K/uL, with 28% promyelocytes. She had prolonged prothrombin (19.6 seconds) and activated prothrombin (46.3 seconds) times, elevated D-Dimer (>20mcg/mL), and decreased fibrinogen (60mg/dL). Promyelocytes in the peripheral blood and disseminated intravascular coagulopathy (DIC) suggested APL. She was initiated on ATRA therapy. Bone marrow (BM) was hypercellular (100%) with 59% promyelocytes, infrequent Auer rods, strong myeloperoxidase staining, and positive PML oncogenic domain (POD) test.¹⁴ Flow cytometry immunophenotyping showed an aberrant promyelocytic immunophenotype. Cytogenetic analysis showed a diploid karyotype. FISH using a RARA break-apart probe was negative. RT-PCR and oligonucleotide microarray did not detect any translocations commonly seen in APL including t(15;17)(q22;q21)/PMLRARA, t(5;17)(q35;q21)/NPM1-RARA, t(11;17)(q13;q21)/NUMA1-RARA, and t(11;17)(q23;q21)/ZBTB16-RARA.¹⁵ Mutation screen detected a low-level mutation in NRAS (c.35G>A p.Gly12Asp, <5%). Based on the morphologic features and positive POD test, she was diagnosed with APL, and treated with ATRA, arsenic trioxide and gemtuzumab ozogamicin. She responded well, achieved complete molecular remission five weeks later, and received 8 months of consolidation therapy with ATRA and arsenic trioxide.¹⁶ Unfortunately, the disease relapsed two months after completion of consolidation therapy. She is now receiving salvage therapy with ATRA, arsenic trioxide, and idarubicin.

RNAseq analysis

Library construction was performed using 700ng total RNA and TruSeq RNA sample prep kit v2 (Illumina). The library was selectively enriched by 12 cycles of PCR followed by size selection per manufacturer’s protocol. The resulting size-fractionated library was sequenced using a 75bp paired-end reads protocol on an Illumina HiSeq2000 instrument. The resultant BCL files were converted to FASTQ files using Illumina offline basecalling software VAVASA (http://support.illumina.com/sequencing/sequencing_software/casava.ilmn). After standard QC using RNA-SeQC (http://www.broadinstitute.org/cancer/cga/rna-seqc) FASTQ files were processed using TopHat_TopHat-Fusion-Post (http://tophat.cbcb.umd.edu) to detect potential fusions. Supporting evidence (number of split reads, mate pairs, mate pairs with split ends) was manually examined to remove false positive calls.

RT-PCR and DNA sequencing

Total RNA was reverse transcribed using random hexamers. PCR was performed using primers designed to amplify IRF2BP2-RARA fusion transcripts from cDNA and genomic DNA (Figure 1A–B). The PCR product was sequenced and analyzed using the EMBLEBI Multiple sequence alignment tool CLUSTAL OMEGA.

Figure 1. Molecular characterization of IRF2BP2 and RARA fusion.

(A). Primer sequences for the detection of IRF2BP2-RARA fusion. Primers were designed in exonic and intronic regions of the IRF2BP2 and RARA genes to cover putative fusion sequences from cDNA and genomic DNA. To facilitate sequencing of PCR amplicons, primers were tagged with M13 universal sequences. (B). Primer design covering IRF2BP2 exon 2 and RARA exon 3 or intron 2 for detection of IRF2BP2-RARA fusion in cDNA and genomic DNA, respectively. FP, forward primer; RP: reverse primer. (C). Results from RT-PCR and genomic PCR for detection of IRF2BP2-RARA fusion. RT-PCR using forward primer from IRF2BP2 exon 2 and reverse primer from RARA exon 3 gives rise to an amplicon of 310 bp. Genomic PCR using forward primer from IRF2BP2 exon 2 and reverse primer from RARA intron 2 gives rise to an amplicon of 323 bp. (D). Direct sequencing of genomic PCR products reveals a IRF2BP2-RARA fusion with a distinct breakpoint and part of sequences from IRF2BP2 exon 2 and RARA intron 2. (E). Alignments of genomic sequences of IRF2BP2 exon 2, IRF2BP2-RARA fusion and RARA intron 2 using EMBL-EBI Multiple sequence alignment tool CLUSTAL OMEGA demonstrate breakpoints in exon 2 of IRF2BP2 gene and intron 2 of RARA gene.

Results and Discussion

We report a case of APL with a novel IRF2BP2-RARA fusion. The clinical course was similar to that of typical APL; however, the clinical onset was insidious with the patient having a two-month history of bleeding disorder before DIC. Morphologically, the promyelocytes showed distinct cytoplasmic granules, strong myeloperoxidase positivity and rare Auer rods (Figure 2). POD stain was only weakly positive in a subset of cells. Although this case fits APL clinically, morphologically and immunophenotypically, testing for t(15;17)(q24.1;q21.2)/PML-RARA and common variant translocations was negative.

Figure 2. Morphologic findings.

(A). Bone marrow biopsy specimen showing sheets of leukemic cells with irregular nuclei and abundant eosinophilic cytoplasm (H&E, 200x). (B). Bone marrow aspirate smear showing promyelocytes with distinct cytoplasmic granules. Auer rods, however, are infrequent (Wright-Giemsa, 1000x). (C). The promyelocytes are strongly positive for myeloperoxidase (1000x). (D). Promyelocytic oncogenic domain (POD) antibody shows that the promyelocytes are positive (1000x).

RNAseq data from BM aspirate were analyzed for fusion transcripts involving RARA. We identified 87 sequence-unique mate-pair reads that defined a translocation joining intron 1 of IRF2BP2 gene on chromosome 1 to intron 2 of RARA gene on chromosome 17. Sixteen sequence unique split-reads (spanning putative breakpoints) were identified that further confirmed and refined the IRF2BP2-RARA fusion. The data suggest that multiple transcripts are likely driven off the fusion. Given the strength of the RNAseq data and involvement of RARA, a novel IRF2BP2-RARA fusion is likely involved in the pathogenesis of this APL case (Supplement Data).

We performed RT-PCR and genomic DNA PCR to detect the IRF2BP2-RARA fusion, and obtained amplicons of 310bp and 323bp, respectively (Figure 1C). Sanger sequencing of genomic DNA PCR products revealed that the IRF2BP2-RARA fusion involved exon 2 of IRF2BP2 gene and intron 2 of RARA gene with breakpoints at position 1687bp in IRF2BP2 and 41620bp in RARA (Figures 1D–E). These findings confirm the fusion partners and breakpoints detected by RNA sequencing.

IRF2BP2 encodes a nuclear protein that contains an N-terminal zinc finger and a Cterminal RING finger domain that interacts specifically with the C-terminal transcriptional repression domain of IRF2.¹⁷ IRF2BP2 acts as a transcriptional corepressor and represses transactivation of NFAT-responsive promoters.¹² IRF2BP2 is also an ischemia-induced coactivator of VEGFA expression that may contribute to revascularization of ischemic muscle.¹³ IRF2BP2 is a direct target gene of TP53 and its overexpression inhibits apoptosis by impeding TP53-mediated transactivation of the TP21 and BAX genes.¹⁸ IRF2BP2 has been identified as a antiapoptotic factor in breast cancer cell lines¹⁹ and a tumor-associated antigen in monoclonal gammopathy of undetermined significance.²⁰ Whole transcriptome sequencing identified a novel IRF2BP2-CDX1 fusion as a result of t(1;5)(q42;q32) in mesenchymal chondrosarcoma.²¹

RARA functions by binding to retinoic acid response elements (RAREs) as a heterodimer with retinoids X receptor (RXR).²² This RARA-RXR complex is required for promyelocyte differentiation. Various X-RARA fusion proteins could have a dominant negative effect on wild-type RARA and X proteins, or form heterodimers sequestering RXR and recruiting corepressors and histone deacetylase complex to repress genes implicated in myeloid differentiation.²³ Similarly, IRF2BP2-RARA may promote leukemogenesis by serving as a dominant negative regulator of RARA and IRF2BP2. The nature of the RARA fusion partner correlates with response to ATRA. In our case, the patient initially responded well to ATRA and arsenic trioxide, and therefore APL with IRF2BP2-RARA appears to be sensitive to ATRA. However, early relapse suggests that APL with IRF2BP2-RARA may have a more aggressive clinical course and require more intensive therapy.

In summary, we report a case of APL with a novel IRF2BP2-RARA fusion. The patient initially responded to ATRA and arsenic trioxide, however, the disease relapsed shortly after completion of consolidation therapy.

Key Points.

• A novel fusion gene IRF2BP2/RARA in variant APL with in vivo sensitivity to all-trans retinoic acid and arsenic trioxide

• A clinical course with complete molecular remission followed by early relapse 2 months after completion of consolidation therapy