Complex rearrangement in acute myeloid leukemia M2 with RUNX1/RUNX1T1 fusion involving chromosomes 8, 17 and 21

The translocation t(8;21)(q22;q22) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML) sub type M2. About 3–5 % of cases with additional chromosomal abnormalities, including structural and numerical ones, are reported to include a complex translocation t(8;21;N). Here we report a chromosome rearrangement observed in a 19 years-old female diagnosed with AML-M2. When subjected to (molecular) cytogenetic analyses a complex three-way translocation involving chromosomes 8, 17 and 21 was detected, forming not a t(8;21;17) as one would expect. Real time-polymerase chain reaction analysis using 6 AML specific markers showed the presence of RUNX1/RUNX1T1 fusion gene transcripts identical to those found in classical translocation t(8;21) coupled with presence of FLT3-ITD mutation identified by fragment analysis. The present case highlights importance of complex rearrangements rarely encountered in AML, suggesting that all involved regions harbor critical candidate genes regulating the pathogenesis of AML, leading to novel as well as well-known leukemia associated chromosomal aberrations.


Background
It is well known that karyotyping and molecular cytogenetics play very important roles in establishing diagnosis and prognosis in acute myeloid leukemia (AML). Even though banding cytogenetics is limited to detect chromosomal rearrangements at a low resolution, it remains the gold standard for basic genetic analyses in AML. Among cytogenetic rearrangements predictive of most favorable outcomes in AML is the translocation t(8;21)(q22;q22), being detectable in approximately 7-8 % of all AML cases. This translocation results in a fusion gene located on derivative chromosome 8, composed of RUNX1T1 (also known as ETO) on 8q22 and RUNX1 gene (also known as AML1) on 21q22 [1]. RUNX1 gene has also been reported to fuse with different other genes in both de novo and therapy related acute leukemia [2]. The 2016 revision of World Health Organization (WHO) classification for hematolymphoid neoplasms underscores that AML associated with translocation t(8;21) constitutes a distinct AML subtype and is associated with favorable prognosis [3]. However, approximately 3-5 % of AML cases with translocation t(8;21) have variant chromosome partners involved [1]. The clinicopathological features of variant t(8;21) is contradictory discussed in literature and some studies have even reported a favorable outcome for patients with variant translocation t(8;21) [4,5].

Case presentation
In the year 2018, a 19-years-old female of Asian ethnicity belonging to Jammu and Kashmir, India was presented with bleeding gums for the past 8 months and with high fever for 15 days. On physical examination, spleen was palpable with lymphadenopathy in the cervical and inguinal regions. At admission, blood analyses revealed white blood cell (WBC) count of 64.30 × 10 9 /l with 48 % blasts, and platelet count of 95 × 10 9 /l. A bone marrow (BM) examination characterized hypercellular marrow with 65 % leukemic blasts being positive for CD3, CD 15, CD19, CD34, CD-45 and HLA-DR, a clinical statues being compatible diagnosis of AML (Fig. 1a). The distinct morphologic features of marrow blasts, suggested that the patient has CBF-AML. The patient was initially treated with standard induction chemotherapy, including 7 days of infusion with cytarabine (100 mg/m 2 ) and 3 days of daunorubicin (60 mg/m 2 ). However, complete remission was not achieved after two cycles of chemotherapy. Afterwards she received 12 cycles of low-dose cytarabine and blood transfusions. Following bone marrow biopsy revealed a normal karyotype with ongoing morphologic remission. The patient then received another cycle of cytarabine followed by 2 cycles of 5-azacitidine (75 mg/ m 2 for 7 doses) before discontinuing any further therapy. As the situation stands with over 2 years of diagnosis, the patient remains in complete remission with no evidence of cytogenetic anomalies.

Banding cytogenetics and fluorescencein situhybridization (FISH)
A BM aspirate collected during initial diagnosis was studied by GTG (Giemsa using trypsin) banding and  [6]. Fluorescence in situ hybridization (FISH) using the RUNX1/RUNX1T1 probe (dual-color dual-fusion translocation probe) and subtelomeric probes (ST) 17pter, 17qter and 21qter (Vysis/ Abbott Molecular, Des Plaines, IL, USA) were also conducted. In-house whole chromosome painting probes (WCP) for chromosomes 8, 17 and 21 and a multicolor banding probe set for chromosome 8 were used to characterize the nature of the complex rearrangement [7].

DNA extraction
Genomic DNA was extracted from bone marrow samples using QIAsymphony DNA mini kit, Qiagen according to manufacturer's instruction.

RNA preparation and cDNA synthesis
Total RNA from the leukemic cells was isolated using QIAamp RNA blood Mini Kit (Qiagen). Total RNA (1-5 µg) was converted to cDNA using High Capacity cDNA reverse transcription kit using random primers as per the established protocols [8].

Real time detection of AML ETO fusion product
AML-ETO (or RUNX1-RUNX1T1) was detected by Real Time PCR method. The negative and positive controls were analysed in duplicate using ABI 7500 (Applied Biosystems, USA). AML-ETO transcripts were amplified in 20 µl reaction containing 300 nM of each primer, 100 nM of probe and 5 µl of cDNA. PCR master mix was prepared with Premix Ex TaqTM (Probe qPCR) and custom reaction mix containing 0.5 µl of ROX (ROX Reference Dye II; Takara). The PCR program consisted of an initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min and extension. Similarly, endogenous control corresponding to ABL transcripts were amplified in order to compensate for differences in RNA integrity and cDNA synthesis efficiency. The AML-ETO transcripts were amplified using previously published primer probe sequences according to EAC protocol [8]. Standard TaqManTM PCR parameters (ABI 7500) were applied to all AML-ETO and ABL amplifications.

Fragment analysis
FLT3 gene exon 11 was amplified using the Veriti ™ 96 well thermal cycler (Applied Biosystems ™ ) in a 20 µl PCR mixture containing 50 ng of extracted DNA, 0.5 µmol/l each of forward primer with FAM (5′ end-labeled with carboxyfluorescein) labelled and unlabeled reverse primer [8]. The PCR program consisted of an initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 1 min and extension at 72 °C for 10 min. Afterwards, 2 µl of PCR products was mixed with 9 µl HIDI formamide and 0.25 µl ROX500 internal size standard. The mixtures were denatured at 94 °C for 5 min, chilled at 4 °C and then subjected to ABI PRISM 3500Dx Genetic Analyzer. The results were analyzed using the Gene Mapper software (Applied Biosystems).
Further, molecular studies by Real Time PCR confirmed fusion of RUNX1/RUNX1T1 and fragment analysis characterized a FLT3-ITD mutation in 13q12.2 (Fig. 2a, b).
In addition, Real Time-PCR analysis revealed the presence of RUNX1/RUNX1T1 fusion transcripts coupled with presence of FLT3-ITD mutation. The FLT3 gene mutations are the most frequent genetic abnormality associated with AML. The rapid identification of these mutations is therefore significant to determine the prognostic impact against the overall genetic background of the leukemic cells. The insertion of tandem duplication into exon 11 and exon 12 in the wild-type FLT-3 produces internal tandem duplication (ITD), which results in constitutive activation of the negative regulation of the juxtamembrane domain [12]. Activating the mutation of D835 within the activation loop of FLT-3 in human hematologic malignancies leads to persistent activation of the FLT-3 receptor. In pediatric patients, FLT3 mutations have been associated with poor prognosis [13]. It has been reported that in adults with CBF (core binding factor) AML the substantial prognostic markers for the outcome are minimal residual disease levels, rather than the FLT3-ITD mutations [14].

Conclusions
The translocation t(8;21)(q22;q22) is a frequent chromosome abnormality seen in AML-M2 which is easily identified by standard chromosome analysis. The present study illustrates a unique complex translocation t(8;21;17), which reinforces that the region on chromosome 17 may harbor a critical candidate gene that could