Digital Multiplex Ligation-Dependent Probe Amplification for Detection of Key Copy Number Alterations in T- and B-Cell Lymphoblastic Leukemia Anne Benard-Slagter,

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Digital Multiplex Ligation-Dependent Probe Amplification for Detection of Key Copy Number Alterations in T- and B-Cell Lymphoblastic Leukemia Anne Benard-Slagter, Ilse Zondervan, Karel de Groot, Farzaneh Ghazavi, Virinder Sarhadi, Pieter Van Vlierberghe, Barbara De Moerloose, Claire Schwab, Kim Vettenranta, Christine J. Harrison, Sakari Knuutila, Jan Schouten, Tim Lammens, Suvi Savola The Journal of Molecular Diagnostics Volume 19, Issue 5, Pages 659-672 (September 2017) DOI: 10.1016/j.jmoldx.2017.05.004 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Target genes included in the D007 ALL digitalMLPA probe mix. Circos plot visualizing all target genes included in the D007-X2-0816 ALL digitalMLPA probe mix. The Circos plot was made using the Progenetix database (arraymap data)8 at http://www.progenetix.org, using subset “Leukemias: immature acute lymphoblastic leukemias,” which at the moment of generation of the figure consisted of 1067 samples from 40 publications. Numbers in parentheses refer to the number of probes included for the respective gene. In blue are the target genes with an expected copy number gain, in red the target genes with an expected copy number loss. Chromosome bands are indicated for each of the genes/regions. In the middle line of the Circos plot, the expected percentages of CNAs in immature ALL, according to the Progenetix database, are indicated (gains in blue and losses in red). The Journal of Molecular Diagnostics 2017 19, 659-672DOI: (10.1016/j.jmoldx.2017.05.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 DigitalMLPA procedure. In digitalMLPA, sequencing is used solely to determine absolute read numbers of the various probe amplicons, not for sequence analysis of the sample DNA. As with conventional MLPA, reference DNA samples, preferably treated and extracted the same way as the DNA samples to be tested, are required to determine the relative copy number of each of the probes. A: After denaturation of the target DNA, the left (LPO) and right probe oligo (RPO) are hybridized to their target sequence overnight (at least 16 hours). B: The next day, the two oligonucleotides are enzymatically ligated and a barcode oligo (BO) is incorporated into the amplicon for sample identification. The Rd1 sequence, as part of the BO, is the Illumina tag required for quantification by Illumina sequencers (Illumina, San Diego, CA). C and D: After PCR (C), amplicons are quantified using Illumina sequencers (D). E: Absolute read counts are compared with reference samples to obtain probe ratios. The Journal of Molecular Diagnostics 2017 19, 659-672DOI: (10.1016/j.jmoldx.2017.05.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Intrachromosomal fusion genes identified in ALL patient samples. DigitalMLPA detects several intrachromosomal fusion genes, including STIL-TAL1 fusion and heterozygous deletion of TAL1 exon 1 and STIL exons 2 to 12 (A), NUP214-ABL1 fusion and gain of one copy of ABL1 exons 3 to 11 and NUP214 exons 2 to 23 (an additional probe for NUP214 exon 29 has been added in D007-X2-0816) (B), and EBF1-PDGRFB fusion and deletion of EBF1 exon 16 and PDGFRB exons 9 to 10 (C). The y axis represents read ratio as compared with the reference samples. The Journal of Molecular Diagnostics 2017 19, 659-672DOI: (10.1016/j.jmoldx.2017.05.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Intragenic and single-exon deletions identified in ALL patient samples. Several intragenic deletions are successfully identified in ALL patient samples, including deletion of ERG exons 5 to 9 (A), deletion of NR3C1 exons 1 and 2 and upstream region on a background of gain of one allele of NR3C1 exons 5 to 8 (B), homozygous deletion of IKZF1 exon 8 (two probes are included for each exon of IKZF1) (C), and homozygous deletion of LEF1 exon 3 (D). All copy number changes were confirmed by other methods. The y axis represents read ratio as compared with the reference samples. The Journal of Molecular Diagnostics 2017 19, 659-672DOI: (10.1016/j.jmoldx.2017.05.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Gain of whole chromosomes or chromosomal regions in ALL patient samples. Gain of whole genes or chromosomal regions as identified by digitalMLPA. A: A case of high hyperdiploidy correctly identified by digitalMLPA showing the characteristic whole chromosome gains. B: Intrachromosomal amplification of chromosome 21 (iAMP21) showing the typical copy number profile along chromosome 21. C: Gain of entire chromosome 21. All copy number changes were confirmed by other methods. The y axis represents read ratio as compared with the reference samples. The Journal of Molecular Diagnostics 2017 19, 659-672DOI: (10.1016/j.jmoldx.2017.05.004) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions