DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia.
BACKGROUND
We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA methylation as a complement to current diagnostic methods. A secondary aim was to gain insight into the functional role of DNA methylation in ALL.


RESULTS
We used the methylation status of ~450,000 CpG sites in 546 well-characterized patients with T-ALL or seven recurrent B-cell precursor ALL subtypes to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived that consisted of only 246 CpG sites. The mean sensitivity and specificity of the classifier across the known subtypes was 0.90 and 0.99, respectively. We then used DNA methylation classification to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations ('other' subtype). Nearly half (n = 106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative polymerase chain reaction (PCR) and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5.


CONCLUSIONS
Our findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant biological characteristics in otherwise unclassified ALL patients.
Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Box 1432, BMC, SE-751 44 Uppsala, Sweden.
Jessica
J
Nordlund
Department of Medical Sciences, Cancer Pharmacology and Computational Medicine, Uppsala University, Uppsala University Hospital, Entrance 40, SE-751 85 Uppsala, Sweden.
Christofer L
CL
Bäcklin
Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
Vasilios
V
Zachariadis
Department of Immunology, Genetics and Pathology, Uppsala University, Rudbecklaboratoriet, SE-751 85 Uppsala, Sweden.
Lucia
L
Cavelier
Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Box 1432, BMC, SE-751 44 Uppsala, Sweden.
Johan
J
Dahlberg
Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
Ingegerd
I
Öfverholm
Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
Gisela
G
Barbany
Department of Molecular Medicine and Surgery, Karolinska Institutet, SE-171 76 Stockholm, Sweden.
Ann
A
Nordgren
Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Box 1432, BMC, SE-751 44 Uppsala, Sweden.
Elin
E
Övernäs
Department of Pediatrics, Queen Silvia Children's Hospital, Rondvägen 10, SE-416 85 Gothenburg, Sweden.
Jonas
J
Abrahamsson
Department of Pediatrics, Tromsø University and University Hospital, Sykehusveien 38, N-9038 Tromsø, Norway.
Trond
T
Flaegstad
Childhood Cancer Research Unit, Karolinska Institutet, Astrid Lindgren Children's Hospital, Karolinska University Hospital, Q6:05, SE-171 76, Stockholm, Sweden.
Mats M
MM
Heyman
Pediatric Hematology-Oncology, Children's Hospital, Barnaspitali Hringsins, Landspitali University Hospital, Norðurmýri, 101, Reykjavik, Iceland.
Ólafur G
ÓG
Jónsson
Division of Hematology-Oncology and Stem Cell Transplantation, Children's Hospital, Helsinki University Central Hospital and University of Helsinki, Box 281, FIN-00029 Helsinki, Finland.
Jukka
J
Kanerva
Department of Medical Sciences, Cancer Pharmacology and Computational Medicine, Uppsala University, Uppsala University Hospital, Entrance 40, SE-751 85 Uppsala, Sweden.
Rolf
R
Larsson
Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Box 1432, BMC, SE-751 44 Uppsala, Sweden.|Department of Women's and Children's Health, Pediatric Oncology, Uppsala University, Uppsala University Hospital, Entrance 95, SE-751 85 Uppsala, Sweden.
Josefine
J
Palle
Pediatrics and Adolescent Medicine, Rigshospitalet, and the Medical Faculty, Institute of Clinical Medicine, University of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.
Kjeld
K
Schmiegelow
Department of Medical Sciences, Cancer Pharmacology and Computational Medicine, Uppsala University, Uppsala University Hospital, Entrance 40, SE-751 85 Uppsala, Sweden.
Mats G
MG
Gustafsson
Department of Women's and Children's Health, Pediatric Oncology, Uppsala University, Uppsala University Hospital, Entrance 95, SE-751 85 Uppsala, Sweden.
Gudmar
G
Lönnerholm
Department of Medical Biosciences, University of Umeå, SE-901 85 Umeå, Sweden.
Erik
E
Forestier
Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Box 1432, BMC, SE-751 44 Uppsala, Sweden.
Ann-Christine
AC
Syvänen
450 k array; CpG site; Cytogenetics; DNA methylation; Pediatric acute lymphoblastic leukemia; RNA-seq; Subtyping; epigenetics