PAN3–PSMA2 fusion resulting from a novel t(7;13)(p14;q12) chromosome translocation in a myelodysplastic syndrome that evolved into acute myeloid leukemia

Background Acquired primary chromosomal changes in cancer are sometimes found as sole karyotypic abnormalities. They are specifically associated with particular types of neoplasia, essential in establishing the neoplasm, and they often lead to the generation of chimeric genes of pathogenetic, diagnostic, and prognostic importance. Thus, the report of new primary cancer-specific chromosomal aberrations is not only of scientific but also potentially of clinical interest, as is the detection of their gene-level consequences. Case presentation RNA-sequencing was performed on a bone marrow sample from a patient with myelodysplastic syndrome (MDS). The karyotype was 46,XX,t(7;13)(p14;q12)[2]/46,XX[23]. The MDS later evolved into acute myeloid leukemia (AML) at which point the bone marrow cells also contained additional, secondary aberrations. The 7;13-translocation resulted in fusion of the gene PAN3 from 13q12 with PSMA2 from 7p14 to generate an out-of-frame PAN3–PSMA2 fusion transcript whose presence was verified by RT-PCR together with Sanger sequencing. Interphase fluorescence in situ hybridization analysis confirmed the existence of the chimeric gene. Conclusions The novel t(7;13)(p14;q12)/PAN3–PSMA2 in the neoplastic bone marrow cells could affect two key protein complex: (a) the PAN2/PAN3 complex (PAN3 rearrangement) which is responsible for deadenylation, the process of removing the poly(A) tail from RNA, and (b) the proteasome (PSMA2 rearrangement) which is responsible for degradation of intracellular proteins. The patient showed a favorable response to decitabine after treatment with 5-azacitidine and conventional intensive chemotherapy had failed. Whether this might represent a consistent feature of MDS/AML with this particular gene fusion, remains unknown.


Background
Hematologic malignancies, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), often carry visible acquired chromosomal aberrations which may be either primary or secondary in leukemogenesis [1]. According to Heim and Mitelman [1] "Primary aberrations are frequently found as the sole karyotypic abnormalities in cancer and are often specifically associated with particular tumor types. The term primary not only refers to the fact that these are the first changes we see in neoplastic cells, but also reflects their causal role in tumorigenesis; they are essential in establishing the neoplasm". In the same reference secondary aberrations are described as "rarely or never found alone; as the name implies, they develop in cells already carrying Full list of author information is available at the end of the article a primary abnormality. In later disease stages, however, they may be so numerous as to completely dominate the karyotypic picture. Although less specific than primary changes, secondary aberrations nevertheless demonstrate nonrandom features with distribution patterns that appear to be dependent both on which primary abnormality is present and on the type of neoplasm". The report of a new primary chromosomal aberration in a given cancer is of scientific as well as clinical interest. The aberration may give rise to a novel fusion protein, alternatively abrogation of an otherwise normal gene product, and thus define a new genetic subgroup in such malignancies [2]. An example is the cryptic t(7;21) (p22;q22)/RUNX1-USP42 chromosomal translocation which was first described in an AML patient and currently is considered a rare but nonrandom feature of myeloid malignancies where it is frequently found together with del(5q) [3][4][5][6][7].
We here present the molecular genetic and clinical features of a case of myelodysplastic syndrome with a novel primary t(7;13)(p14;q12) chromosome translocation that recombined the proteasome subunit alpha 2 (PSMA2) gene on 7p14 and the PAN3 poly(A) specific ribonuclease subunit (PAN3) gene on 13q12 generating a PAN3-PSMA2 fusion gene. The disease later evolved into AML at which point also secondary chromosome abnormalities could be seen.

Case presentation
A 74-year-old female patient was in April 2016 referred to our hospital because of thrombocytopenia. Blood analysis showed hemoglobin 10.8 g/dL, thrombocytes 31 × 10 9 /L, and white blood cells 3.8 × 10 9 /L with a normal differential count. Examination of a bone marrow biopsy showed 16% CD34-positive cells and dysplasia affecting mainly the megakaryocytic lineage, and the patient was diagnosed with myelodysplastic syndrome with excess of blasts 2 (MDS-EB 2). Cytogenetic analysis at this time showed the karyotype 46,XX,t(7;13)(p14;q12) (see below). No treatment was given.
One month later, examination of a new bone marrow aspirate showed 55% blasts and she was diagnosed with AML. Treatment with 5-azacytidine 5 days every 4th week was begun. However, a bone marrow aspirate after 6 monthly courses of 5-azacytidine showed 70% blasts, and because of disease progression she was now changed to intensive chemotherapy with age adjusted "3+7" (daunorubicin 50 mg/m 2 day 1-3 and cytarabine 200 mg/m 2 day 1-7), followed by one course of consolidation chemotherapy with mitoxantrone 10 mg/m 2 day 1-5 and etoposide 100 mg/m 2 day 1-5. In April 2017, 2 months after consolidation therapy, a new bone marrow aspirate again showed more than 50% blasts indicating AML recurrence. She was therefore started on decitabine 20 mg/m 2 for 5 days every 4th week and after four courses, a new bone marrow biopsy showed 3-5% CD34+ cells. After another 2 months, a bone marrow biopsy still showed 5% CD34+ cells. Decitabine is continued and the patient is at the time of writing enjoying an active life in remission from her leukemia.
Bone marrow cells were cytogenetically investigated by standard methods [8,9] and karyotyped according to the International System for Human Cytogenomic Nomenclature guidelines [10].
Total RNA was extracted from the patientʼs bone marrow at the time when MSD was diagnosed using miRNeasy Mini Kit (Qiagen Nordic, Oslo, Norway). The RNA quality was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and 1 µg of total RNA was sent to the Genomics Core Facility at the Norwegian Radium Hospital, Oslo University Hospital (http://genomics.no/oslo/) for high-throughput pairedend RNA-sequencing. The RNA sequencing data were analyzed using the FusionCatcher software in order to discover fusion transcripts [11,12] (https://github.com/ ndaniel/fusioncatcher).
The primers used for PCR amplification and sequencing are listed in Table 1. The procedures of reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing of the PCR products were previously described [13]. For amplification of the PAN3-PSMA2 fusion transcript, the primer sets PAN3-957F1/PSMA2-206R1 and PAN3-1005F1/PSMA2-168R1 were used. For amplification of the putative reciprocal PSMA2-PAN3 fusion transcript, the primer set PSMA2-21F1/PAN3-1241R1 was used. For amplification of the PAN3 and PSMA2 transcripts, the primer sets PAN3-957F1/PAN3-1241R1 and PSMA2-21F1/PSMA2-206R1 were used, respectively. The PCR cycling involved initial denaturation at 94 °C for 30 s, followed by 35 cycles of 7 s at 98 °C, 30 s at 55 °C, 30 s at 72 °C, and a final extension for 5 min at 72 °C.
Using the FusionCatcher software on the fastq files of RNA sequencing data, 24 fusion genes were found (data not shown), among them fusion of PAN3 from chromosome band 13q12 with the PSMA2 gene from 7p14.
A green (PAN3 probe), a red (PSMA2 probe), and two yellow fusion signals were seen in 48 out of 68 examined nuclei (Fig. 1i).
PSMA2 is ubiquitously expressed and encodes a peptidase which is a component of the alpha subunit of the 20S core proteasome complex [23]. The proteasome is a multi-catalytic proteinase complex. It is distributed throughout eukaryotic cells at a high concentration and cleaves peptides in an ATP/ubiquitin-dependent process as part of a non-lysosomal pathway [24,25]. It plays a central role in the control of numerous cellular activities including regulation of the cell cycle [24,25]. Inhibition of proteasome was found to be an effective therapeutic strategy in many hematologic malignancies [26][27][28][29].
The PAN3-PSMA2 fusion transcript codes for a putative PAN3 truncated protein which contains amino acid residues 1-333 from PAN3 protein (accession number NP_787050 version 6) corresponding to exons 1-3 of the gene, and 15 novel amino acid residues (ARLVNLSRL-NMLWLL) stemming from the out-of-frame fusion of PSMA2. This putative protein would therefore contain the zinc finger and a PABP interacting motif 2 (PAM2) but would lack the normal C-terminal part which contains the pseudokinase, the coiled coil, and the C-terminal knob domain of PAN3. The precise role of the truncated PAN3 protein in the development of myelodysplasia/leukemia cannot be predicted without functional studies. However, an anomaly in deadenylation, which is fundamental to the regulation of gene expression, can be assumed. Alternatively, loss of a functional PAN3 and/ or PSMA2 allele might be the important factor in pathogenesis. Whether any functional similarity exists between the present translocation and abrogation case and ALLs with deletion of an entire PAN3 allele [22], is a moot point.
Chromosomal rearrangements resulting in gene truncation have been described repeatedly for the RUNX1 and ETV6 genes [30,31]. The aberrations generate a premature stop codon in the open reading frames leading to expression of C-terminal truncated forms of the RUNX1 or ETV6 proteins [30,32]. Truncated RUNX1 proteins were shown to interfere with normal RUNX1 [33][34][35]. Truncated forms of ETV6 were found to have a dominant-negative effect on normal ETV6 function and disrupt both primitive and definitive hematopoiesis in the zebrafish model [36]. Chromosome translocations resulting in gene truncation have also been reported for other genes. For example, a t(3;21)(q22;q22) leading to truncation of RYK was seen in atypical chronic myeloid leukemia [37]. In a case of AML transformed from myelodysplastic syndrome, a t(2;7)(p24.3;p14.2) generated an out-of-frame NBAS-ELMO1 fusion transcript coding for a truncated NBAS protein [38]. Recently, in a case of AML, a t(3;5)(p24;q14) translocation was found to result in fusion of SATB1 with an expression sequence tag. The SATB1-fusion transcript would code for a SATB1 protein lacking the C-terminal DNA-binding homeodomain [39].
The patient in this report did not respond to treatment with 5-azacitidine, daunorubicin plus cytarabine, or mitoxantrone plus etoposide. She did, however, have a very favorable response to decitabine. It is possible that the 7;13-translocation is causatively involved in this difference, but in the absence of other cases with the same genetic change, one cannot tell. The case anyway illustrates that some patients with MDS or AML who do not respond favorably to standard treatment, may benefit from a change to decitabine [40,41]. It may be particularly noteworthy from a clinical point of view that some patients who do not respond to 5-azacitidine, may do so to decitabine in spite of the fact that both drugs are hypomethylating agents.