Bone marrow or blood samples were collected from 27 patients at the Uppsala University Hospital, Uppsala, Sweden. All cases were classified according to the 2008 WHO classification  and samples were collected at diagnosis. In addition, a validation cohort consisting of diagnostic bone marrow or blood samples from 12 patients was included. Slides were prepared from the samples for interphase FISH analysis and from cultured cells for karyotyping and metaphase FISH analysis. Total RNA was prepared from all samples at diagnosis. The study was approved by the ethical board at Uppsala University (Dnr: 2013–233).
Karyotyping and fluorescence in situ hybridization (FISH)
Cells were cultured and slides were prepared for G-banding according to standard procedures. When possible, metaphases from two cultures were karyotyped. Interphase FISH analysis was performed for screening of genomic aberrations depending on diagnosis and age at diagnosis using either an AML FISH probe panel (including probes for inv/t(16) Vysis LSI CBFB Break Apart (BA) rearrangement, t(15;17)(q22;q21) Vysis LSI PML/RARA Dual Color, Dual Fusion (DF) Translocation Probe kit, t(8;21)(q21;q22) Vysis LSI AML1/ETO Dual Color, DF Translocation Probe and 11q23-rearrangements Vysis LSI MLL Dual Color, BA Rearrangement probe, Abbott Laboratories, Chicago, Illinois) or the ALL FISH probe panel (including probes for del(9)(p21) Vysis LSI p16/CEP9, t(1;19)(q23;p13) Vysis LSI TCF3/PBX1 Dual Color, DF Translocation Probe, t(12;21)(p13;q22) Vysis LSI ETV6/RUNX1 Dual Color, DF Translocation Probe, t(9;22)(q34;q11) Vysis LSI BCR/ABL Dual Color, DF Translocation Probe, 11q23-rearrangements Vysis LSI MLL Dual Color, BA Rearrangement probe, Abbott Laboratories, Chicago, Illinois). For specific cases, additional probes were used: Poseidon (Kreatech) MLL/MLLT1 t(11;19) Fusion Probe (Leica Biosystems, Wetzlar, Germany), Vysis 4q12 Tri-Color rearrangement Probe, Vysis LSI ETV6 (TEL) Dual Color Probe (Abbott Laboratories,, Chicago, Illinois) and (Kreatech) MLL/MMLT4 t(6;11) DF-probe (Leica Biosystems, Wetzlar, Germany). The analysis was performed using protocols described by the manufacturers. For each sample, at least 200 interphase nuclei were scored for interphase FISH and for metaphase FISH, at least 10 metaphases were analyzed.
RNA was prepared from mononuclear cells using Trizol Reagent Ultra Pure (Invitrogen, ThemoFisher Scientific, Waltham, Massachusetts) according to standard protocols.
Reverse transcriptase (RT)-PCR
RT-PCR was carried out for the fusions outlined in Table 2 and for the TCF3-ZNF384 e10-e3 fusion. cDNA synthesis was performed using 1.5 μg RNA and M-MLV Reverse Transcriptase according to manufacturer’s instructions (Invitrogen, ThemoFisher Scientific, Waltham, Massachusetts). Primer and probe sequences for PCR are given in 5′- > 3′ orientation: PML-RARA e6-e3 (Forward primer (F): TCTTCCTGCCCAACAGCAA, Reverse primer (R): GGCTTGTAGATGCGGGGTAG, Probe (P): TAGTGCCCAGCCCTCC); PML-RARA e3-e3 (F: GACCTCAGCTCTTGCATCACC, R: GGCTTGTAGATGCGGGGTAG, P: TAGTGCCCAGCCCTCC); RBM15-MKL1 e1-e4 (primer-probe mix Hs03024505-ft (Invitrogen, ThemoFisher Scientific, Waltham, Massachusetts)); KMT2A-MLLT4 e8-e2 (F: CCCAAGTATCCCTGTAAAACAAAAA, R: TGCAAAGTTTCCAGCAGCTT); KMT2A-ELL e9-e2 (primer-probe mix Hs03024474-ft (Invitrogen, ThemoFisher Scientific, Waltham, Massachusetts)); KMT2A-AFF1 e8-e4 (F: CCCAAGTATCCCTGTAAAACAAAAA, R: GAAAGGAAACTTGGATGGCTCA, R: CATGGCCGCCTCCTTTGACAG C); KMT2A-MLLT3 e8-e6 (primer-probe mix Hs03296416-ft (Invitrogen, ThemoFisher Scientific, Waltham, Massachusetts)); KMT2A-ARHGEF12 e6-e22 (F:TAAGCCCAAGTTTGGTGGTC, R: GCGCGCCTTCTGTAGTTC); KMT2A-CBL e7-e16 (F: AAAAGCAGCCTCCACCACC, R: AGTTGATTCTCCGCGGGAAT, P: TGAAGGTTCCCAAGTTCCCGAGA); BCR-ABL1 e13-e2 (F: TCCGCTGACCATCAATAAGGA, R: CACTCAGACCCTGAGGCTCAA, P: CCCTTCAGCGGCCAGTAGCATCTGA); ETV6-RUNX1 e5-e3 (F: CTCTGTCTCCCCGCCTGAA, R: CGGCTCGTGCTGGCAT, P: TCCCAATGGGCATGGCGTGC); PBX1-TCF3 e16-e3 (F: CCAGCCTCATGCACAACCA, R: GGGCTCCTCGGATACTCAAAA, P: CCCTCCCTGACCTGTCTCGGCC); and TCF3-ZNF384 e10-e3 (F: CCATCTGCATCCTCCTTCTC, R: GGGGATAGAAGGCCAGAAGT). For breakpoint validation of the KMT2A-ARHGEF12 e6-e22 fusion the following primers were used; F1: TAAGCCCAAGTTTGGTGGTC, F2: GCAGTGCTGCAAGATGAGAA, F3: CCGCCCAAGTATCCCTGTAA, R1: GCGCGCCTTCTGTAGTTC, R2: CCAGCGTCTGTTCCTTCATT, R3: CCCATCTCCCACACATTTTC. For breakpoint validation of the TCF3-ZNF384 e10-e3 fusion the following primers were used; F1: CCATCTGCATCCTCCTTCTC, F2: TACTCCCCGGATCACTCAAG, R1: GGGGATAGAAGGCCAGAAGT, R2: CAGGGACCACCGTGATATTC and R3: CCTCGTCCAGGTGGTCTTC. PCR-protocols are available upon request. The RT-PCR breakpoint validations of the KMT2A-ARHGEF12 and the TCF3-ZNF384 fusions were analyzed using 2200 TapeStation, D1000 ScreenTape and the TapeStation Analysis Software version A.02.01 SR1 (Agilent, Santa Clara, California).
Library preparation was performed with the Archer™ FusionPlex™ Heme Panel v1 with Archer™ Universal RNA Fusion Detection v1 for the Illumina Platform according to the protocols described by the manufacturer (ArcherDX, Boulder, Colorado) (Fig. 1). 200 ng RNA was used as input material. Libraries were purified using Agencourt AMPure Beads on a Life Technologies™ DynaMag™ and quantified with the KAPA Biosystem Library Quantification Kit (Illumina, San Diego, California). Libraries were sequenced by combining four samples, at a concentration of 18pM, using the sequencing kit version 2 and the MiSeq instrument (Illumina, San Diego, California). 10% PhiX was used. Given the size of our clinical laboratory, simultaneous runs of four samples would meet the need to routinely perform the analysis once a week. For the validation cohort, Archer™ FusionPlex™ Heme Panel v2 (ArcherDX, Boulder, Colorado) was used and samples were sequenced in batches of six, using the sequencing kit version 3 and the MiSeq instrument (Illumina, San Diego, California). The Heme Panel v2 was used due to the fact that the v1 panel was no longer commercially available, however, the targets examined are included in both versions.
Data analysis of sequencing results
Sequencing data were analyzed in the Archer™ Analysis 3.1.1 Software (ArcherDX, Boulder, Colorado). For the validation cohort Archer™ Analysis 126.96.36.199 Software (ArcherDX, Boulder, Colorado) was used as the 3.1.1 Software was not compatible with the Heme Panel version 2. The fusion detection algorithm of strong candidate fusions included mapping of reads to a control region followed by mapping to target regions, the remaining reads were mapped to the human genome (hg19 (GRCh37)). Reads spanning two separate genes were considered fusion candidates if at least 23 bp were mapped on either side of the breakpoint. Each fusion candidate read that spanned the same breakpoint between two reads were binned and a final consensus sequence was compared to the human genome to annotate fusion partners. The following criteria were used in order to qualify a candidate fusion as a strong evidence fusion: i) candidate had a minimum coverage of 5 unique reads; ii) candidate was present in Quiver (if found in Quiver this overrode all subsequent criteria and was reported as a strong evidence fusion); iii) percent of breakpoint-spanning reads of gene-specific primer 2 (GSP2, used in gene-specific PCR 2, see Fig. 1) that supported the candidate relative to the total number of RNA reads spanning the breakpoint was at least 10%; and iv) candidate had at least 3 unique start sites (unique start sites refer to a subset of the unique reads and represent the total number of unique fragment lengths extracted from the sample). The candidate was not considered as a strong evidence fusion if it fulfilled any of the following conditions: i) if it was an exon-intron fusion; ii) if there was evidence of mispriming; iii) if the candidate aligned to known paralogs; iV) if the alignment to the human genome was poor; or v) if cross-contamination to a fusion in the same analysis was present. For a more thorough description of the fusion filters we refer to the Archer Analysis user manual. The QC settings used were: minimum unique reads for valid fusion = 5, minimum average unique RNA start sites per GSP2 controls = 10 (GSP2 control refers to gene-specific primers that target genes that are reliably expressed in any tissue type), minimum unique start sites for valid fusions = 3, fusion percent of GSP2 reads = 10, minimum average unique RNA reads per GSP2 = 0. All filters and cutoffs used were standard settings in the Archer Analysis software.