Download MLL: ALL

MLL translocations specify a
distinct gene expression
profile that distinguishes a
unique leukemia
Armstrong et al, Nature Genetics
30, 41-47 (2002)
E. Canaani: A.L.L. LEUKEMIA
27 PATIENTS, 14 with MLL translocation, (“MLL”)
13 without (ALL)
log2 E, center, normalize
expression matrix E
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-4
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3250 genes passed filter
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E. Canaani: A.L.L. LEUKEMIA
27 PATIENTS, 14 with MLL translocation, (“MLL”)
13 without (ALL)
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MLL
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ALL
25
AIM: FIND THE GENES THAT ARE EITHER
ACTIVATED OR SUPPRESSED BY THE
t(4,11) CHIMERIC MLL PROTEIN
THESE GENES MAY BE THE CAUSE OF
CANCER AND TARGETS FOR THERAPY
Rozovskaia, Ravid, Getz, ....., Canaani:
PNAS (2003)
SUPERVISED ANALYSIS: HYPOTHESIS TESTING
USING CLINICAL INFORMATION (MLL/ALL=NO TRANS.)
IDENTIFY DIFFERENTIATING GENES
HYPOTHESIS: THE EXPRESSION LEVELS OF GENE g
IN SAMPLES WITH THE MLL TRANSLOCATION AND
WITHOUT – ARE DRAWN FROM THE SAME DISTRIBUTION
t(4:11) MLL
ALL without t(4:11)
USE STANDARD STATISTICAL TESTS,
FOR ONE GENE AT A TIME TO CALCULATE
Pg = probability that the 27 expression
levels of gene g (14 from MLL, 13 ALL)
are taken from the same distribution
Cluster Incl. U70321:Human herpes virus entry mediator mRNA
MLL: -1.0522 -0.5557 -1.0522 -0.6297 -1.0522 -0.7643 -0.4200
-0.7931 -1.0522 -0.7548 -1.0522 -0.5799 0.0225 -0.8326
mean = -0.76
std = 0.3
ALL: 2.1513 0.5143 0.0709 0.0421 0.9885 1.1993 2.0765
-0.0126 1.2574 0.2553 0.5241 -0.2726 1.7740
mean = 0.81
std = 0.82
histograms
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4.5
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0.5
0
-2.5
-2
-1.5
-1
-0.5
0
mean = -0.76
0.5
1
1.5
2
std = 0.3
0
2.5 -2.5
-2
-1.5
-1
-0.5
0
mean = 0.81
0.5
1
1.5
2
std = 0.82
2.5
T = -6.6
P = 6e-7
Cluster Incl. AB005298:Homo sapiens BAI 2 mRNA
MLL: 0.3449 1.1137 0.2628 -0.5576 0.3116 1.4970
0.0126 -1.2401 -1.2401 -1.2401 -0.6049 -1.2401
0.0520 -1.2401
mean = -0.27
ALL:
std = 0.92
1.5279 2.0361 -0.2097 0.4142 -1.2401 0.5464
0.8569 -0.9042 0.8856 0.5590 -0.4396 -1.2401
0.9760
mean = 0.29
std = 1.00
histograms
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NORMALIZED (FREQUENCIES)
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0.25
0.35
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mean = -0.27 std = 0.92
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mean = 0.29 std = 1.00
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85%
T = -1.48
P = 0.15
Sorted P-values
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0.9
0.9
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0.7
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0.6
P-value
P-value
Sorted P-values
1
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0.4
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0.3
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0.2
0.1
0
0.5
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0
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929 genes with p < 0.05
ordered by difference of
means (ALL –MLL)
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300
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400
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RANDOM DATA
ALL
MLL
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40
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80
-1
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-2
120
-3
200
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300
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140
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500
143 genes with p < 0.05
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929 genes with p < 0.05
(OUT OF 3250 GENES TESTED)
(OUT OF 3250 GENES TESTED)
RANDOM DATA
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143 genes with p < 0.05
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0
(OUT OF 3250 GENES TESTED)
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BEWARE OF MULTIPLE
COMPARISONS!!!
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Bonferroni: reject H0 only
for genes with
p < 0.05 / N
p < 0.05/3250 = 1.5 10-5
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929 genes with p < 0.05
Bonferroni – 30 pass at 0.05
This list of 30 genes is
“error free” with prob. 0.95
Sorted P-values
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0.9
0.9
0.8
0.8
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0.7
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0.6
P-value
P-value
Sorted P-values
1
0.5
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0
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0
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929 genes with p < 0.05
ordered by difference of
means (ALL –MLL)
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4
200
3
300
2
1
400
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500
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600
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143 WITH P-value<0.05
RANDOM DATA
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929 genes with p < 0.05
how many genes at FDR=0.05?
Sorted P-values
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Sorted P-values
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P-value
P-value
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297*0.05=15 false separating genes
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15 out of 297 - false
Sorted P-values
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P-value
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15 - false
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MLL translocations specify a
distinct gene expression
profile that distinguishes a
unique leukemia
Armstrong et al, Nature Genetics
30, 41-47 (2002)
PROBLEM:
HIDDEN, CONFOUNDING
VARIABLES( (FACTORS)
HEMATOPOIESIS: Differentiation from STEM CELLS to mature cells
LEUKEMIA: MALIGNANCY, INDUCED BY MUTATIONS, TRANSLOCATION,..
differentiation
STEM CELLS: immortal,
(unlimited number of divisions)
multipotent (differentiation
into many targets)
Mature cells: finite lifetime, finite number of divisions
fixed class (B stays B etc)
B
T
NK
ACUTE LYMPHOID LEUKEMIA
(ALL)
ACUTE MYELOID LEUKEMIA
(AML)
Red
Hematopoiesis: HSCs can be categorized into long-term self-renewing HSCs, short-term self-renewing HSCs and multipotent progenitors (red arrows indicate self renewal). HSC give rise to common
lymphoid progenitors (CLPs) and to common myeloid progenitors (CMPs). CMPs mature into red blood cells, megakaryocyte (cells producing platelets), granulocytes, dendritic cells, and
macrophages. The CLP differentiate into B and T cell lymphocytes, natural killer cells and dendritic cells. (adapted from Reya et al., 2001)
LEUKEMIA: MALIGNANCY INDUCED BY MUTATION OR TRANSLOCATION
normal differentiation
•UNCONTROLED PROLIFERATION
•OVERCROWDING & DEATH OF
NORMAL CELLS
•ARREST OF DIFFERENTIATION
PRO B-cell ALL
PRE B-cell ALL
PRE T-cell ALL
EARLY
LATE
LATE
TRANSLOCATION: DURING DNA REPLICATION TWO STRANDS, FROM
TWO DIFFERENT CHROMOSOMES, CROSS
MLL – GENE ON BAND 23
OF CHROMOSOME 11
AF4 – GENE ON BAND 21
OF CHROMOSOME 4
AF4
t(4,11)
MLL TRANSLOCATIONS
ARE IMPLICATED IN 10%
OF ALL AND IN UP TO
80% OF INFANT LEUKEMIA
partner gene
MLL fusion protein
AIM: FIND THE GENES THAT ARE EITHER
ACTIVATED OR SUPPRESSED BY THE
t(4,11) CHIMERIC MLL PROTEIN
THESE GENES MAY BE THE CAUSE OF
CANCER AND TARGETS FOR THERAPY
Rozovskaia, Ravid, Getz, ....., Canaani:
PNAS (2003)
EXPRESSION DATA: 27 ALL samples, 14 with MLL TRANS. t(4,11)
13 without TRANS.
t(4:11) MLL
ALL without t(4:11)
3060 GENES PASSED FILTER
There is another factor (differentiation)
that separates these two groups of
samples! Which of the 230 genes
responds to the MLL translocation?
t(4:11) MLL
ALL without t(4:11)
??
USE STANDARD STATISTICAL
TEST TO LOOK FOR GENES THAT
SEPARATE t(4,11) MLL FROM ALL:
PROBLEM OF MULTIPLE COMPARISONS
SOLVED BY CONTROLLING THE
FALSE DISCOVERY RATE (FDR)
230 genes differentially expressed between ALL with t )4:11(and ALL without,
at FDR = 0.05.
Translocation
CD10-
Late
Differentiation
MLL
Yes
No
No
CD10-
No
Yes
No
ALL
No
No
Yes
Attribute
Group
MLL vs ALL
FDR = 5%
448 genes
WHERE ARE THE MLLs
THAT LOOK LIKE ALL?
MLL
CD10-
M LL
CD10-
T, Pre-B ALL
T, Pre-B ALL
3
20
Differentiation
2
Sensitive to Trasloc.
and/or Differentiation
40
1
60
0
MLL vs CD10Sensitive to
FDR = 12%
Translocation
144 genes
and/or CD10-
80
Translocation
46
Differentiation
sensitive
Sensitive to
Differentiation ALL vs CD10FDR = 12%
and/or CD10167 genes
CD10-
Translocation
sensitive
80
-1
100
120
-2
140
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-3
AIM: FIND THE GENES THAT ARE EITHER
ACTIVATED OR SUPPRESSED BY THE
t(4,11) CHIMERIC MLL PROTEIN
FINDING: WE IDENTIFIED 46 GENES THAT
ARE ACTIVATED OR SUPPRESSED BY
THE MLL ONCOGENE. TARGETS OF
NEXT STAGE EXPERIMENTS ON MICE
SPINOFF: SOME MLLS ARE LATE DIFFERENTIATORS
Rozovskaia et al. PNAS 2003
E2
MLL
 = E1- 2E2 < 0
E1-2E2= 0
E1
 = E1- 2E2 > 0
ALL
E2
MLL
w
E1
ALL
+/- PROJECTIONS ON w – DO SEPARATE ALL FROM MLL
E2
MLL
w’
E1
ALL
+/- PROJECTIONS ON w’ – DO NOT SEPARATE ALL FROM MLL
E2
E1
WELL SEPARATED CENTERS OF MASS NO SEPARATION OF THE TWO CLOUDS
WEAK SEPARATION OF
CENTERS OF MASS –
GOOD SEPARATION OF
THE TWO CLOUDS
E2
E1
E2
MLL
PERCEPTRON
FISHER
E1
ALL
OPTIMAL LINE
TO PROJECT ON