Validation of DNA methylation biomarkers for diagnosis of acute lymphoblastic leukemia.
BACKGROUND
DNA methylation biomarkers capable of diagnosis and subtyping have been found for many cancers. Fifteen such markers have previously been identified for pediatric acute lymphoblastic leukemia (ALL). Validation of these markers is necessary to assess their clinical utility for molecular diagnostics. Substantial efficiencies could be achieved with these DNA methylation markers for disease tracking with potential to replace patient-specific genetic testing.


METHODS
We evaluated DNA methylation of promoter regions of TLX3 (T-cell leukemia homeobox) and FOXE3 (forkhead box E3) in bone marrow biopsies from 197 patients classified as leukemic (n = 95) or clear of the disease (n = 102) by MALDI-TOF. Using a single nucleotide extension assay (methylSABER), we tested 10 bone marrow biopsies collected throughout the course of patient chemotherapy. Using reference materials, diagnostic thresholds and limits of detection were characterized for both methods.


RESULTS
Reliable detection of DNA methylation of TLX3 and FOXE3 segregated ALL from those clear of disease with minimal false-negative and false-positive results. The limit of detection with MALDI-TOF was 1000-5000 copies of methylated allele. For methylSABER, the limit of detection was 10 copies of methylated TLX3, which enabled monitoring of minimal residual disease in ALL patients.


CONCLUSIONS
Mass spectrometry procedures can be used to regionally multiplex and detect rare DNA methylation events, establish DNA methylation loci as clinically applicable biomarkers for disease diagnosis, and track pediatric ALL.
Cancer and Disease Epigenetics and Department of Paediatrics, University of Melbourne, Melbourne, Australia; current address: Fishberg Department of Neuroscience and Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY; zac.chatterton@mssm.edu.
Zac
Z
Chatterton
National Measurement Institute, Sydney, Australia;
Daniel
D
Burke
National Measurement Institute, Sydney, Australia;
Kerry R
KR
Emslie
Developmental Epigenetics, Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Australia;
Jeffery M
JM
Craig
Cancer and Disease Epigenetics and Department of Paediatrics, University of Melbourne, Melbourne, Australia;
Jane
J
Ng
Andrew Love Cancer Centre, Deakin University, Victoria, Australia;
David M
DM
Ashley
Children's Cancer Centre, Royal Children's Hospital, Victoria, Australia;
Francoise
F
Mechinaud
Cancer and Disease Epigenetics and Department of Paediatrics, University of Melbourne, Melbourne, Australia;
Richard
R
Saffery
Cancer and Disease Epigenetics and Department of Paediatrics, University of Melbourne, Melbourne, Australia; current address: Ludwig Institute of Cancer Research, Olivia Newton-John Cancer and Wellness Centre, Austin Hospital, Heidelberg, Victoria, Australia.
Nicholas C
NC
Wong
D000293:Adolescent; D016022:Case-Control Studies; D002648:Child; D002675:Child, Preschool; D019175:DNA Methylation; D005188:False Negative Reactions; D005189:False Positive Reactions; D005260:Female; D051858:Forkhead Transcription Factors / Q000235:genetics; D018628:Gene Dosage; D005819:Genetic Markers; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D007223:Infant; D057230:Limit of Detection; D008297:Male; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis* / Q000188:drug therapy / Q000235:genetics; D011401:Promoter Regions, Genetic; D012015:Reference Standards; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C404285:FOXE3 protein, human; D051858:Forkhead Transcription Factors; D005819:Genetic Markers; D018398:Homeodomain Proteins; C489494:TLX3 protein, human