Design and Feasibility of a Novel, Rapid, and Simple Fluorescence 26-Plex RT-PCR Assay for Simultaneous Detection of 24 Fusion Transcripts in Adult Acute Myeloid Leukemia  Marie-Pierre Laforêt, Pascal Turlure, Eric Lippert, Pascale Cornillet-Lefebvre, Arnaud Pigneux, Rachel Pradeau, Jean Feuillard, Nathalie Gachard  The Journal of Molecular Diagnostics  Volume 15, Issue 2, Pages 186-195 (March 2013) DOI: 10.1016/j.jmoldx.2012.11.004 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Effect of spacers on size of PCR products from BCR/ABL1 e13a2 and CBFB/MYH11 type K and I fusion transcripts. A: Capillary electrophoresis profile of the multiplex positive control (24 chimeric RNAs) focused on peaks corresponding to the BCR/ABL1 e13a2 fusion transcript of t(9;22)(q34;q11) and the CBFB/MYH11 type K and I fusion transcripts of inv(16)(p13.1q22) [t(16;16)(p13.1;q22)] after RT-PCR with primers without spacers (Table 1). An observed 2-bp difference with some overlapping between peaks was found for BCR/ABL1 e13a2 and CBFB/MYH11 type K fusion transcripts, whereas the theoretical size difference between these peaks was expected at 11 bp. B: Capillary electrophoresis profile of multiplex positive control focused on the same area after PCR with primers that included a 3-bp sequence spacer in forward primer for all CBFB/MYH11 transcripts. No overlapping between peaks for BCR/ABL1 e13a2 and CBFB/MYH11 type K fusion transcript was found. Arrows point from each peak to the corresponding size and fusion transcript. The Journal of Molecular Diagnostics 2013 15, 186-195DOI: (10.1016/j.jmoldx.2012.11.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Capillary electrophoresis profile after 26-plex RT-PCR of the multiplex positive control. Each peak corresponds to a specific transcript at a specific size, separated from other peaks by at least 5 bp (observed). Corresponding transcripts and translocations are indicated at the top of each peak. Internal controls are highlighted in bold type. This control was run for each series of patients. Data shown are representative of >10 independent experiments. The Journal of Molecular Diagnostics 2013 15, 186-195DOI: (10.1016/j.jmoldx.2012.11.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Blast cell dilution experiments. A: Kasumi-1 and K562 cells, containing t(8;21)(q22;q22) (RUNX1/RUNX1T1 fusion transcript, with a 335-bp amplification product) and t(9;22)(q34;q11) (BCR/ABL1 fusion transcript, with a 530-bp amplification product) respectively, were diluted in the L-363 human leukemia cell line from 100% to 0.15% blast cells, with a final amount of 100 ng total RNA for each dilution. For each dilution, background was calculated as the mean height of the five highest nonspecific peaks for each translocation tested (10 peaks altogether). B: Representative fluorescence traces for three consecutive dilutions of Kasumi-1 cells diluted in the L-363 cell line. Percentages of Kasumi-1 cells are indicated in the upper left corner of each capillary electrophoresis profile. The lowest positive dilution (1.5%) is underlined. The RUNX1/RUNX1T1 fusion transcript peak is indicated by an arrow. The detection threshold, defined as background level + 2 SD, is indicated by a dashed line. Data in A are expressed as means ± SD of three independent experiments. The Journal of Molecular Diagnostics 2013 15, 186-195DOI: (10.1016/j.jmoldx.2012.11.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Representative electropherograms after 26-plex RT-PCR of RNA from AML adult patients. A: DEK/NUP214 t(6;9)(p23;q34) fusion transcript. B: PICALM1944/MLLT10.241 t(10;11)(p13;q14) fusion transcript. C: RUNX1/RUNX1T1 t(8;21)(q22;q22) fusion transcript. D: CBFB/MYH11 type A fusion transcript of inv(16)(p13.1q22) [t(16;16)(p13.1;q22)]. E: A patient specimen without any of the 24 fusion transcripts. Peaks for the Kanr, ABL1, and B2M internal controls are connected by dotted lines across fluorescence traces in all five electropherograms, demonstrating the reproducibility of electrophoresis. The Journal of Molecular Diagnostics 2013 15, 186-195DOI: (10.1016/j.jmoldx.2012.11.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions