Childhood cancer survival now exceeds 80%, yet therapy resistance remains a defining clinical challenge, affecting approximately one-third of patients and driving poor long-term outcomes. A central unanswered question is when resistance emerges and whether it can be detected genomically before it becomes clinically manifest. Chemotherapy leaves characteristic mutational footprints in tumor genomes, and because these signatures require cancer cells to survive drug exposure, they represent a direct molecular record of resistance. In this large-scale multi-omics study of hard-to-treat pediatric tumors from three independent precision medicine programs, linked to curated exposure data for 86 therapies, we establish therapy-induced mutational signatures as markers of resistance emergence. Comprehensive signature analysis identified novel signatures exclusive to treated tumors. Platinum drugs were the dominant mutagen, accounting for 10% of all single-base substitutions. Critically, platinum signatures were detectable as early as 91 days after treatment initiation, and their accumulation mirrored resistance kinetics: 35% of platinum-treated patients showed measurable signatures within one year, rising to ~50% by 18 months. Signature-positive tumors exhibited significant overexpression of platinum resistance genes and worse outcomes upon re-exposure, functionally validating signatures as resistance biomarkers. Subclonal analysis further identified hidden platinum signatures in tumors lacking dominant resistance clones, suggesting early detection of emerging resistance before clinical failure. An ensemble machine learning model revealed additional platinum-associated genomic features beyond known signatures. These findings establish a genomic framework for monitoring resistance in real time and informing therapy de-escalation in children with cancer.

Co-authors: Laura Wheaton, Max F. Levine, Katherine E. Miller, Neerav N. Shukla, Michael D. Kinnaman, Dominik Glodzik, Carol Portwine, Sabrina Millson, Alexandra Zorzi, Mariam Mikhail, Conrad Fernandez, Marie Wong-Erasmus, Gunes Gundem, Andrew L. Kung, Uri Tabori, Chelsea Mayoh, Elli Papaemmanuil, Mark J. Cowley, David Malkin, Anita Villani, Ludmil B. Alexandrov, Adam Shlien, Syed Kashif Daud, Eric N. Bergstrom, Mathepan J. Mahendralingam, Nicholas Light, Sasha Blay, Joshua O. Nash, Nathaniel D. Anderson, Jessica N. Au, Scott Davidson, Pedro L. Ballester, Timmy Wen, Marcos Díaz-Gay, Lisa-Monique Edward, S.M. Ashiqul Islam, Azhar Khandekar, Burcak Otlu, Ledia Brunga, Rawan Hammad, Shimaa Nassif, Nirav H. Thacker, Tara Feltham, Noemi A. Fuentes-Bolanos