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Genomic landscape of B-other ALL
Figure 2. Frequency of acute lym- phoblastic leukemia (ALL) subtypes among 410 B-cell precursor (BCP) ALL patients consecutively diagnosed and uniformly treated in the Czech Republic from December 2010 to December 2017. In addition to the five established B-other ALL subtypes, frequencies of specific PAX5 aberrations which were mutually exclusive with these subtypes are shown. Aberrations which are not mutually exclusive with the established subtypes [e.g. CRLF2r, dic(9;20)] are not shown. Infants and BCR-ABL1-posi- tive patients were treated according to different protocols and are not included. Chr: chromosomes. PAX5 fusions do not include ZCCHC7-PAX5.
cific (absent in healthy mononuclear blood cells) are reported.
To analyze the presence of IGH-DUX4 and IGH-CRLF2 fusion transcripts that may not be revealed by TopHat/deFuse, reads
mapped to fusion partner genes were analyzed again manually.
Analysis of genomic variants
Read pairs were aligned to hg19 using BWA35 (WES) and STAR36 (RNA-sequencing) and further processed by Picard tools (http://broadinstitute.github.io/picard/). Variant calling was performed using VarScan37 and Samtools (http://samtools.sourceforge.net/). To distinguish somatic and germline variants, WES results from ALL diagnosis were compared to the remission sample.
Gene expression profiling, hierarchical clustering analysis, DUX4 gene expression analysis
Genome-wide gene expression analysis was performed using RNA-sequencing data of the 110 B-other study patients plus two BCR-ABL1-positive and nine ETV6-RUNX1-positive patients. Alignment and counting were performed as described previous- ly.38 Data normalization and hierarchical clustering analysis (HCA) based on the expression of the most variably expressed genes or of genes belonging to particular gene sets were performed using R package Deseq239 (vst normalization, ward.D method and Euclidean distance linkage for HCA). The list of gene sets and included genes used for HCA are shown in Online Supplementary Tables S4 and S5.
To assess DUX4 gene expression, reads mapped to the DUX4 reference were counted as described previously.3 To correct for uneven sequencing depth, counts were normalized using size fac- tors computed by Deseq2.
Flow cytometry
Routine flow cytometry was used for the diagnosis of BCP-ALL and mixed-phenotype acute leukemia (MPAL) using criteria of the
World Health Organization and/or of the European Group for Immunophenotyping of Leukemia. CRLF2 expression was assessed using PE anti-human TSLPR Antibody (clone 1D3; BioLegend, USA). In general, a 50% (10%) cut-off was used to assign strong (weak) positivity. A small CRLF2-positive subclone (<10%), distinct from the major negative population and clearly distinguishable from the technical background, was reported in onepatient.
For more details of the methods used see the Online Supplementary Methods.
Results
Frequency of the B-other-derived acute lymphoblastic leukemia subtypes and associations with clinical parameters
To classify B-other patients into iAMP21, ZNF384r, MEF2Dr, DUX4r, ETV6-RUNX1-like and BCR-ABL1-like subtypes, we used data from SNPa and RNA-sequencing. Our cohort included six cases with ZNF384 fusions (EP300-ZNF384, n=4; TCF3-ZNF384, n=1; TAF15- ZNF384, n=1), four cases with iAMP21, and two cases with MEF2Dr (MEF2D-BCL9, n=2). The BCR-ABL1-like, ETV6-RUNX1-like and DUX4r ALLs were classified using HCA based on the expression of gene sets representative of subtype-specific gene expression signatures (subtype- specific gene sets) from published studies4,10,11 (Figure 1A- C). Using the DUX4r gene set, 30 cases were classified as DUX4r ALL. In accordance with published studies, DUX4r patients had significantly higher DUX4 expression com- pared to remaining ALLs (Online Supplementary Figure S1) and fusion transcripts supporting the presence of DUX4r were found in 29 out of 30 patients. To verify co-clustering
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