multi-dimensional genomic profiling of acute leukemias characterized by mll gene rearrangements...
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Multi-dimensional Genomic Profiling of Acute Leukemias Characterized by
MLL gene rearrangements
Eunice S. Wang MD (Medicine) and
Norma J. Nowak PhD (Cancer Genetics)
Roswell Park Cancer Institute
2008 Estimated US Cancer Cases*
*Excludes basal and squamous cell skin cancers and in situ carcinomas except urinary bladder.Source: American Cancer Society, 2008.
Men745,180
Women692,000
26% Breast
14% Lung & bronchus
10% Colon & rectum
6% Uterine corpus
4% Non-Hodgkin lymphoma
4% Thyroid
4% Melanoma of skin
3% Ovary
3% Kidney & renal pelvis
3% Leukemia
23% All Other Sites
Prostate 25%
Lung & bronchus 15%
Colon & rectum 10%
Urinary bladder 7%
Non-Hodgkin5% lymphoma
Melanoma of skin 5%
Kidney & renal pelvis 4%
Oral cavity 3%
Leukemia 3%
Pancreas 3%
All Other Sites 20%
Jordan C et al. N Engl J Med 2006;355:1253-1261
Blood cancers are normal blood cells gone “bad”
Acute Lymphocytic Leukemia (ALL)
Clonal expansion of immature lymphoblasts
Acute Lymphocytic Leukemia (ALL)
• Blood cancer characterized by uncontrolled growth of immature abnormal white blood cells.
• The most common pediatric cancer with 75% of cases occurring in children < 6 yrs old
• Comprise up to 20% of acute leukemia in adults
• Cure rates following standard chemotherapy and bone marrow transplantation depend on the particular genetics of the ALL cells.
Diagnosis of blood cancers
Cancer cell
1. Appearance (morphology)
Prote
in
2. Abnormal protein expression (flow cytometry) (histochemistry)
Pro
tein
3.Chromosome abnormalities (cytogenetics)
XX
4. Specific gene mutations
Chromosomes, genes, and DNA
22 chromosomes,X and Y
Mixed Lineage Leukemia (MLL) Gene Rearrangement
4 and 11
11 and 19
Transfer of genes fromchromosome 11 to another one
Acute leukemias associated with MLL gene rearrangements do poorly with therapy
No MLL
MLL positive
MLL fusion proteins function to modify DNA transcription and induce leukemia
Need for new therapies for ALL
Multi-dimensional genomic profiling of acute leukemia characterized by MLL
gene rearrangements
The goal of this project is to identify and characterize the key genes and pathways underlying MLL-associated leukemia for
future translation into novel clinical therapies.
Multi-dimensional genomic profiling
Using cutting-edge multi-dimensional genomic profiling, we will perform in depth genetic analyses of MLL leukemia patient cells from individuals treated at RPCI:
- Change in expression of all known human genes
- Submicroscopic DNA gains/losses (aCGH)
- Alterations in non-coding RNAs regulating genes (microRNA or miRs)
- Single nucleotide DNA sequence variations (SNPs)
- Attachment of DNA methyl groups (methylation)
Studies of Copy Number Variation (CNV)
Differences in the number of copies of large segments of DNA have been found between individuals at many sites of the human genome.
Studies of CNV have revealed that:
– CNV can drive genetic disease;
– parts of the genome are polymorphic for copy number.
Cataloging of these variants in disease and in reference populations is underway, using both sequencing and microarray hybridization.
Traditional CGH Array CGH
Control Test Control Test
Picture courtesy of Brynn Levy, Ph.D.
Metaphase CGH to aCGH
Oligonucleotide arrays (25 – 85mer)
BAC arrays (175Kb)
G-Banding
Diagnostics
Discovery
Resolution Coverage
>10Mb 100%
>175Kb 96%
>40Kb <1%
Single Nucleotide Polymorphisms
Illumina HumanCytoSNP-12 BeadChip
All Markers 299,671
Markers within 10kb of a Gene 148,666
nsSNPs 3,480
MHC / ADME / Indel SNPs 761 / 2,382 / 0
Spacing kb (Mean/Median) 9.6 / 6.2
SNPs in Mitochondrial Genome 0
X-chr / Y-chr / PAR Loci 15,056 / 2,679 / 10
DNA Input Requirement 200 ng
Number of samples per BeadChip 12
Content Number
A Wide Spectrum of Aberrations Can Be Detected
A
C
B
D
MicroRNAs (Mirs)
Single-strandedRNAs capable of
regulating gene expression in cells
Illumina MicroRNA Profiling
Human– 1146 human mature miRNA
sequences are targeted
Content– Sanger miRBase v12
(September 2008 Release) and recent literature
– Targeted
Sample– 200ng total RNA input– R2 >0.99 even for FFPE
samples
24 MLL Leukemia Patient Samples
Frozen Samples
RNA & DNA extraction
-aCGH
-microRNA
-SNPs
-methylation
Pts
24 MLL Patient CNV/SNP Data
24 MLL Patient MicroArray Data
Statistical Analysis
Results of these arrays will be- Compared with normal cells (when
available) from the same patients- Subjected to statistical analysis using
specialized software in order to determine which corresponding target genes and pathways represent potential therapeutic targets.
Jacquie Hirsch for ALL Foundation Research