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Diagnostic primary leukemia samples from 162 pediatric T-ALL patients were used for this study ( Fig 1 ; S1 Table ). In the discovery phase, we used DNA isolated from matched pre-treatment (diagnostic) and post-treatment (remission) sample pairs from 13 pediatric T-ALL patients who enrolled in the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol between 2004 and 2012. In the expansion phase of this study, we used DNA from diagnostic, pre-treatment patient material from 69 patients who enrolled in the German Co-operative Study Group for Childhood Acute Lymphoblastic Leukemia (COALL) protocol between 1997 and 2003 (COALL-97). Initial findings were then confirmed in a larger cohort of diagnostic, pre-treatment T-ALL patient materials including DNA from the previously mentioned 69 COALL patients plus that of five additional patients who also enrolled in the COALL-97 protocol and 72 additional T-ALL patients who enrolled in the DCOG protocols ALL-7/8 ( n = 30) or ALL-9 ( n = 42). Functional validation was done on viably frozen pre-treatment leukemia cells from peripheral blood or bone marrow from eight of the 74 patients who enrolled in the COALL-97 protocol (as mentioned above) plus two additional patients who enrolled in the COALL-03 study and one patient who enrolled in the DCOG ALL-10 study. Median follow-up for patients who enrolled in the DCOG ALL-7/8/9 (1988–2004) and COALL-97 protocols was 67 and 52 mo, respectively. The patients’ parents or legal guardians provided informed consent to use leftover diagnostic material for research with approval from the institutional review board of the Erasmus MC Rotterdam and in accordance with the Declaration of Helsinki. Leukemia cells were harvested from blood or bone marrow samples and enriched to a purity of at least 90% as described previously [ 20 ].

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