Duplex Sequencing Uncovers Recurrent Low-frequency Cancer-associated Mutations in Infant and Childhood KMT2A-rearranged Acute Leukemia
Mattias Pilheden,
Louise Ahlgren,
Axel Hyrenius-Wittsten,
Veronica Gonzalez-Pena,
Helena Sturesson,
Hanne Vibeke Hansen Marquart,
Birgitte Lausen,
Anders Castor,
Cornelis Jan Pronk,
Gisela Barbany,
Katja Pokrovskaja Tamm,
Linda Fogelstrand,
Olli Lohi,
Ulrika Norén-Nyström,
Johanna Asklin,
Yilun Chen,
Guangchun Song,
Michael Walsh,
Jing Ma,
Jinghui Zhang,
Lao H. Saal,
Charles Gawad,
Anna K. Hagström-Andersson
Affiliations
Mattias Pilheden
1 Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden
Louise Ahlgren
1 Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden
Axel Hyrenius-Wittsten
1 Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden
Veronica Gonzalez-Pena
2 Division of Pediatric Hematology/Oncology, Stanford University, School of Medicine, Stanford, CA, USA
Helena Sturesson
1 Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden
Hanne Vibeke Hansen Marquart
3 Department of Clinical Immunology, National University Hospital, Rigshospitalet, Copenhagen, Denmark
Birgitte Lausen
4 Department of Paediatrics and Adolescent Medicine, Rigshospitalet, University of Copenhagen, Denmark
Anders Castor
5 Childhood Cancer Center, Skane University Hospital, Lund, Sweden
Cornelis Jan Pronk
5 Childhood Cancer Center, Skane University Hospital, Lund, Sweden
Gisela Barbany
6 Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden
Katja Pokrovskaja Tamm
7 Department of Oncology and Pathology, Karolinska Institutet, Stockholm, Sweden
Linda Fogelstrand
8 Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden
Olli Lohi
10 Tampere Center for Child, Adolescent and Maternal Health Research and Tays Cancer Center, Tampere University and Tampere University Hospital, Tampere, Finland
Ulrika Norén-Nyström
11 Department of Clinical Sciences, Pediatrics, Umeå University, Umeå, Sweden
Johanna Asklin
12 SAGA Diagnostics, Lund, Sweden
Yilun Chen
12 SAGA Diagnostics, Lund, Sweden
Guangchun Song
13 Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Michael Walsh
13 Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Jing Ma
13 Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Jinghui Zhang
14 Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Lao H. Saal
12 SAGA Diagnostics, Lund, Sweden
Charles Gawad
2 Division of Pediatric Hematology/Oncology, Stanford University, School of Medicine, Stanford, CA, USA
Anna K. Hagström-Andersson
1 Division of Clinical Genetics, Department of Laboratory Medicine, Lund University, Lund, Sweden
Infant acute lymphoblastic leukemia (ALL) with KMT2A-gene rearrangements (KMT2A-r) have few mutations and a poor prognosis. To uncover mutations that are below the detection of standard next-generation sequencing (NGS), a combination of targeted duplex sequencing and NGS was applied on 20 infants and 7 children with KMT2A-r ALL, 5 longitudinal and 6 paired relapse samples. Of identified nonsynonymous mutations, 87 had been previously implicated in cancer and targeted genes recurrently altered in KMT2A-r leukemia and included mutations in KRAS, NRAS, FLT3, TP53, PIK3CA, PAX5, PIK3R1, and PTPN11, with infants having fewer such mutations. Of identified cancer-associated mutations, 62% were below the resolution of standard NGS. Only 33 of 87 mutations exceeded 2% of cellular prevalence and most-targeted PI3K/RAS genes (31/33) and typically KRAS/NRAS. Five patients only had low-frequency PI3K/RAS mutations without a higher-frequency signaling mutation. Further, drug-resistant clones with FLT3D835H or NRASG13D/G12S mutations that comprised only 0.06% to 0.34% of diagnostic cells, expanded at relapse. Finally, in longitudinal samples, the relapse clone persisted as a minor subclone from diagnosis and through treatment before expanding during the last month of disease. Together, we demonstrate that infant and childhood KMT2A-r ALL harbor low-frequency cancer-associated mutations, implying a vast subclonal genetic landscape.