The present study was approved by the regional ethics committee (Regional Committees for Medical and Health Research Ethics, Oslo, Norway), and written informed consent was obtained from the patient to publish the case details. The ethics committee's approval included a review of the consent procedure. All patient information has been anonymized.

A 39-year-old male was transferred to Oslo University Hospital with a preliminary diagnosis of ALL. He presented with increasing fatigue and a reduced performance status. The clinical examination was unremarkable, except for the presence of an enlarged supraclavicular lymph node on the right side. Blood analysis revealed slight anemia (hemoglobin, 10.5 g/dl), moderate thrombocytopenia (thrombocytes, 55×10⁹/l), moderate leukocytosis (leukocytes, 32.7×10⁹/l) and elevated levels of lactate dehydrogenase, C-reactive protein and creatinine. Examination of a bone marrow aspirate and biopsy showed the complete replacement of normal hematopoiesis by blasts with a high nuclear to cytoplasmic ratio. The percentage of blasts in the blood was 65–75%, as determined by flow cytometric analysis. The immunophenotypic analysis confirmed the B-cell lineage differentiation of the lymphoblasts, which were positive for CD45, the B-cell markers, CD19, CD22 and cytoplasmic CD79a, human leukocyte antigen-antigen D related antigens, CD10 and terminal deoxynucleotidyl transferase. In addition, the B lymphoblasts showed abnormal expression of CD123 and CD58. The molecular genetic analysis was negative for BCR-ABL, TCF3-PBX1 and ETV6-RUNX1, but a clonal immunoglobulin kappa rearrangement was found.

The patient received therapy for ALL based on the Hammersmith 82 regimen (http://oncolex.org/Prosedyrer/TREATMENT/DrugTherapy/Leukemia_ALL?lg=print), which consists of an induction phase of 16 weeks followed by maintenance treatment for 3 years. The patient went into complete morphological remission 6 weeks following the initiation of induction therapy. A reassessment after 20 weeks of therapy confirmed continuous morphological remission and the absence of minimal residual disease (MRD) at a detection level of <0.01%, as assessed by flow cytometry. A G-banding analysis performed at the same time point revealed a normal karyotype. The patient then received a total of 13 maintenance cycles and finished treatment 3 years and 5 months following diagnosis. Bone marrow aspirates taken 24 months following diagnosis and after cessation of therapy revealed complete hematological remission and no MRD.

Bone marrow cells were cytogenetically investigated by standard methods at diagnosis and after 20 weeks of therapy. Chromosome preparations were made from metaphase cells after a 24-h culture. Peripheral blood T lymphocytes stimulated with phytohemagglutinin, M form (PHA-M; Thermo Fisher Scientific, Inc., Waltman, MA, USA) for 72 h were also examined. Chromosome preparations were G-banded using Leishman stain (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and karyotyped according to the International System for Human Cytogenetic Nomenclature 2013 guidelines (13).

Total RNA (3 µg) was extracted from the patient's bone marrow using the miRNeasy Mini kit (Qiagen GmbH, Hilden, Germany) at the time of diagnosis and subjected to high-throughput paired-end RNA-sequencing at the Norwegian Sequencing Centre, Oslo University Hospital. Detailed information about the procedure was described previously (14). The software deFuse (15) was used for detection of fusion transcripts.

For RT-PCR, 1 µg total RNA was reverse transcribed in a 20-µl reaction volume using the iScript™ Advanced cDNA Synthesis kit for RT-qPCR (Bio-Rad Laboratories AB, Oslo, Norway), according to the manufacturer's protocol. The 25-µl PCR volume consisted of 12.5 µl Premix Ex Taq™ DNA Polymerase Hot Start Version (Takara Bio Europe SAS, Saint-Germain-en-Laye, France), 1 µl cDNA and 0.4 µM of each of the forward and reverse primers. The PCR was run on a C-1000 Thermal cycler (Bio-Rad Laboratories AB) with an initial denaturation at 94°C for 30 sec, followed by 35 cycles at 98°C for 7 sec, 64°C for 30 sec and 72°C for 30 sec, and a final extension at 72°C for 5 min. For detection of the FAM53B fusion transcript, the following primer combination was used: Forward FAM53B-1149F1 (5′-ACACCTGGAGCCCTGACCTGCAC-3′) and reverse 19q13-Trans1-R1 (5′-AAGCCTCGCAGCTCTGAAGCCTG-3′). The primers were purchased from Thermo Fisher Scientific, Inc.

The PCR products (3 µl) were stained with GelRed (Biotium, Inc., Hayward, CA, USA), separated by 1.0% agarose gel electrophoresis and photographed. DNA gel electrophoresis was performed using lithium borate buffer (Faster Better Media LLC, Hunt Valley, MD, USA), according to a previous study (16). The remaining PCR products were purified using the Qiagen PCR purification kit (Qiagen GmbH) and directly sequenced using the dideoxy procedure with the ABI Prism BigDye Terminator v1.1 Cycle Sequencing kit (Thermo Fisher Scientific, Inc.) on the Applied Biosystems Model 3500 Genetic Analyzer sequencing system (Thermo Fisher Scientific, Inc.). The basic local alignment search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for computer analysis of sequence data.

At the time of diagnosis, G-banding analysis of the bone marrow cells yielded a karyotype with a single clonal chromosome abnormality: 46,XY,t(10;19)(q26;q13)[15] (Fig. 1A). The G-banding analysis of PHA-M-stimulated peripheral blood mature T lymphocytes yielded a normal 46, XY karyotype. Therefore, the t(10;19)(q26;q13) translocation was found in the leukemic cells only. Notably, cytogenetic examination of the bone marrow cells 6 and 20 weeks following initiation of induction therapy showed a normal 46, XY karyotype (data not shown).

Using deFuse software to analyze the raw sequencing data, a fusion transcript (Fig. 1B) was found in which a coding sequence of the FAM53B gene from band 10q26 (Fig. 1C and D) was fused to a genomic sequence mapping upstream of the SLC7A10 locus (Fig. 1E and F). This corresponded well with the 19q13 breakpoint of the t(10;19)(q26;q13) found in the leukemic cells.

RT-PCR using the FAM53B-1149F1/Trans1-R1 primers amplified a cDNA fragment (data not shown). Direct sequencing of the amplified fragment verified the FAM53B fusion detected with the deFuse program (Fig. 1G).