MAGE-TAB Version	1.1						
Investigation Title	Genome-wide DNA Methylation Analysis and Its Contribution to Biological and Clinical Insights in Relapsed Childhood Acute Lymphoblastic Leukemia for the COG ALL TARGET Project						
Experimental Design	disease state design						
Experimental Design Term Source REF	EFO						
Experimental Factor Name							
Experimental Factor Type							
Experimental Factor Term Source REF							
Person Last Name	NCI Office of Cancer Genomics (OCG)	NCI Center for Biomedical Informatics and Information Technology (CBIIT)	Geng	Melnick	Li	Hunger	Loh
Person First Name			Huimin	Ari	Yushan	Stephen	Mignon
Person Mid Initials				M		P	
Person Email	ocg@mail.nih.gov	ncicbiit@mail.nih.gov	huimin.geng@ucsf.edu	amm2014@med.cornell.edu	yul2008@med.cornell.edu	hungers@chop.edu	lohm@peds.ucsf.edu
Person Phone	+1 301 451 8027	+1 888 478 4423	415 502 2137	212-746-7643	212-746-2517		415-476-3831
Person Fax	+1 301 480 4368		212-746-8866	212-746-8866	212-746-8866		
Person Address	31 Center Dr, Rm 10A07, Bethesda, MD 20892	9609 Medical Center Dr, Rockville, MD 20850	513 Parnassus Ave, MSB Rm. S-1457A, San Francisco, CA 94143 	1300 York Ave, Rm C-640,New York, NY 10021	515 E 71st Street, S-222, New York, NY 10021	3401 Civic Center Blvd Philadelphia, PA 19104	Box 0106, UCSF
Person Affiliation	National Cancer Institute	National Cancer Institute	Department of Laboratory Medicine, University of California, San Francisco	Department of Medicine, Weill Cornell Medical College	Epegenomic Core Facility, Weill Cornell Medical College	Children's Hospital of Philadelphia	UCSF Benioff Children's Hospital
Person Roles	funder	data coder;curator	investigator;data analyst;submitter	investigator	investigator	investigator	investigator
Person Roles Term Source REF	EFO	EFO;EFO	EFO	EFO	EFO	EFO	EFO
Quality Control Type							
Quality Control Term Source REF							
Replicate Type							
Replicate Term Source REF							
Normalization Type	median normalization log2 ratio of HpaII/MspI						
Normalization Term Source REF							
Date of Experiment	2012-02-01						
Public Release Date							
PubMed ID							
Publication DOI							
Publication Author List	Huimin Geng, Mignon L. Loh, Richard Harvey, I-Ming Chen, Meenakshi Devidas, Tanja Davidsen, Jaime M. Guidry Auvil, Leandro C. Hermida, Daniela S. Gerhard, Malcolm A. Smith, Andrew J. Carroll, Nyla A. Heerema, Julie M. Gastier-Foster, Cheryl L.Willman, Charles G. Mullighan, Stephen P. Hunger and Ari Melnick						
Publication Title	Genome-wide DNA Methylation Analysis Reveals Biological and Clinical Insights in Relapsed Childhood Acute Lymphoblastic Leukemia: A Report from the COG ALL TARGET Project						
Publication Status							
Publication Status Term Source REF							
Experiment Description	Although survival of children with B-cell acute lymphoblastic leukemia (B-ALL) has improved substantially over time, 15% to 20% of patients will relapse, and most of those who experience a bone marrow relapse will die. A better understanding of genetic and epigenetic aberrations in relapsed ALL will facilitate new strategies for risk stratification and targeted therapy. In this collaborative study with the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) project, we performed high resolution genome-wide DNA methylation profiling using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) array on a total of 178 (110 diagnosis, 68 relapse) leukemia samples from 111 patients with childhood B-ALL enrolled on the Children’s Oncology Group (COG) clinical trials who experienced relapsed, and 12 normal preB samples isolated from the bone marrows of 12 healthy individuals. The HELP array covers 117,521 CpG sites, annotated to ~22,000 gene promoters. For eight diagnosis/relapse pairs, base-pair resolution DNA methylation using the eRRBS (enhanced Reduced Representation Bisulfite Sequencing) method was also performed on Illumina HiSeq2000. 

The median relapse time for the 111 patients was 21.8 months (range 2.1 to 56.2). Unsupervised clustering analysis using the HELP data revealed seven clusters: one cluster contained only the 12 normal preB samples; four clusters were enriched with MLLr, ETV6/RUNX1, Trisomy 4+10, and TCF3/PBX1 samples, respectively. The sixth cluster was not enriched for specific cytogenetic cases, but interestingly, all cases in this cluster were NCI High Risk (age&gt;10 years or WBC&gt;=50,000; p&lt;0.0001, Fisher’s Exact test) while the seventh cluster has a mixture of other cases. 

Supervised analysis of HELP profiles between paired relapse/diagnosis samples (n=67) revealed a markedly aberrant DNA methylation signature (1011 probesets, 888 genes, FDR&lt;0.01 and methylation difference dx &gt;25%, paired t-test), with 70% of the genes hyper- and 30% hypo-methylated in relapse samples. Using a Bayesian predictor and leave-one-out cross validation, this methylation signature could predict a sample as diagnosis or relapse with 95.3% accuracy. When comparing early (&lt;36 months; n=50) versus late relapses (&gt;=36 months; n=18), we detected a profound hypermethylation signature in early relapse (96.6% of the 610 probesets, 544 genes, FDR&lt;0.01, dx &gt;25%). 

Finally, we identified 1800 probesets (1658 genes) as differentially methylated within all cytogenetic subtypes described above compared to the normal preB samples (Dunnett’s test with normal preB as reference, FDR&lt;0.01, dx&gt;25%). Again the majority (70%) of those genes were hypermethylated in relapse as compared to diagnostic and normal preB.

The base-pair resolution and more comprehensive eRRBS methylation analysis for the eight pairs of samples identified 39,679 CpG sites as differentially methylated (dx &gt;25%, FDR&lt;0.01), with 78.3% CpG sites hyper- and 21.7% hypo-methylated in relapse samples. Remarkably, the hypermethylated CpGs are primarily in promoter regions (50%, defined as +/-1kb to TSS), followed by intergenic (26%), then intragenic (14%), and exonic (10%) regions. In contrast, the hypomethylated CpGs are mainly in intragenic (48%), followed by intergenic (31%), exonic (14%) and promoter (7%) regions. The hypermethylated CpGs were mainly in CpG islands (86%) or CpG shores (10%), while hypomethylated CpGs were not (CpG islands: 8%, CpG shores: 27%). We further identified 3040 differentially methylated regions (DMRs) with a median size 426 bp. 78.4% of those DMRs were hyper- (1362 gene promoters) and 21.6% hypo-methylated (98 promoters) in relapse compared to diagnostic samples. Gene set enrichment and Ingenuity pathway analysis showed epigenetically disrupted pathways that are highly involved in cell signaling, and embryonic and organismal development. 

Taken together, our genome-wide high resolution DNA methylation analysis on a large cohort of relapsed childhood B-ALL from the COG trial identified unique methylation signatures that correlated with relapse and with specific genetic subsets. Those methylation signatures featured prevailing promoter hypermethylation and to a lesser extent, intrageneic hypomethylation. Epigenetically dysregulated gene networks in those relapse samples involved cell signaling, and embryonic and organismal development. 						
Protocol Name	ucsf.edu:Protocol:DNA-Extraction-Nimblegen:01	ucsf.edu:Protocol:MethylationArray-Labeling-Nimblegen:01	ucsf.edu:Protocol:MethylationArray-Hybridization-Nimblegen:01	ucsf.edu:Protocol:MethylationArray-Scanning-Nimblegen:01	ucsf.edu:Protocol:MethylationArray-DataNormalization-Nimblegen:01	nci.nih.gov:CBIIT.OCGDCC.Protocol:MethylationArray-GeneAnnotation:01	
Protocol Type	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol 	array scanning protocol	normalization data transformation protocol	data transformation protocol	
Protocol Term Source REF	EFO	EFO	EFO	EFO	EFO	EFO	
Protocol Description	"As described in NimbleGen Manufacturer protocol, details in "NG_DNA_Methylation_Guide_v7p1.pdf""	"As described in NimbleGen Manufacturer protocol, details in "NG_DNA_Methylation_Guide_v7p1.pdf""	"As described in NimbleGen Manufacturer protocol, details in "NG_DNA_Methylation_Guide_v7p1.pdf""	"As described in NimbleGen Manufacturer protocol, details in "NG_DNA_Methylation_Guide_v7p1.pdf""	"Median normalized log2 ratio of signal intensity of HpaII and MspI, as detailed in Thompson et al, Bioinformatics 2008;24:1161-7. Software: NimbleScan version 2.5.26, Value: median normalized log2-transformed HpaII/MspI ratios, "NA": if MspI signal intensity &lt; 1 mean absolute deviatin (MAD) above the median of random probe signals"	"Transformation of L2 data matrix into per sample L3 files with probe set gene annotations added"
Protocol Parameters					cutoff_value_NA_1_MAD		
Protocol Hardware	Qiagen Puregene Core Kit A	NimbleGen Dual-color DNA Labeling Kit	NimbleGen Hybridization System 4	NimbleGen MS 200 Microarray Scanner			
Protocol Software				NimbleScan version 2.5.26	NimbleScan version 2.5.26, and R software as described in Thompson et al, Bioinformatics 2008;24:1161-7.		
Protocol Contact	Yushan Li	Yushan Li	Yushan Li	Yushan Li	Huimin Geng	Leandro C. Hermida	
SDRF File	TARGET_ALL_MethylationArray_Phase2_20160812.sdrf.txt						
Term Source Name	NCBITaxon	NCIt	MO	EFO	OBI		
Term Source File	http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html	http://ncit.nci.nih.gov/	http://mged.sourceforge.net/ontologies/MGEDontology.php	http://www.ebi.ac.uk/efo	http://purl.obolibrary.org/obo/obi		
Term Source Version							
Comment[Quality Control Info]	1, array image: no severe regional artifacts affecting more than 1/6 of the area; 2, no severe intensity bias (Loess curve displaced beyond +0.5 to -0.5); 3, "NA" values if the MspI signal intensity &lt; 1 mean absolute deviatin (MAD) above the median of random probe signals						