(1) Can we identify metabolites or metabolic pathways
that are associated with breast cancer clinical parameters?
Partial Least Square
(multivariate stats)
Score scatterplot (t1 vs. t2)
Standard deviation of t1: 4.992
Standard deviation of t2: 7.089
15.0
grade2
12.5
grade1
grade1
10.0
grade3
7.5
5.0
2.5
0.0
t2
Results
Alex-CIS-GCTOF MS w/ BinBase: 470 detected compounds
161 known metabolites, 309 without identified structure.
breast
adipose
grade2
-2.5
-5.0
-7.5
-10.0
-12.5
grade3
-15.0
-17.5
-20.0
-12.5 -10.0 -7.5
-5.0
-2.5
t1
0.0
2.5
5.0
7.5
10.0
60
Grade
n1  23 tumors show differential up-regulation in
Pentose Phosphate Pathway, nucleotides, amino acids, arachidonic acid
(but not free fatty acids, gln)
50
40
grade 1
Results
30
grade 2
grade 3
20
10
0
E+P+H-
E+P+H+
E+P-H-
E+P-H+
production of NADPH, ribose units
E-P+H-
E-P+H+
E-P-H-
E-P-H+
production of DNA, RNA
Grade n1  23 tumors show differential up-regulation in
Pentose Phosphate Pathway, nucleotides, amino acids, arachidonic acid
(but not free fatty acids, gln)
Categ. Box & Whisker Plot: beta-alanine
Categ. Box & Whisker Plot: glutamic acid
14000
6E5
12000
5E5
glu
10000
b-ala
beta-alanine
glutamic acid
4E5
3E5
2E5
2000
n 1
normal
2 3 grade
1
2
Categ. Box & Whisker Plot: malate
34000
3
grade
n 1
2 3 grade
0 Mean
Mean±SE normal
1
2
3
Mean±1.96*SE
Categ. Box & Whisker
Plot: citric acid
grade
Mean
Mean±SE
Mean±1.96*SE
55000
32000
30000
28000
26000
malate
50000
45000
citrate
24000
22000
lactate
40000
citric acid
20000
18000
malate
16000
14000
35000
30000
12000
10000
25000
8000
6000
4000
2000
20000
n 1
2 3 grade
15000
1 Plot: arachidonic
2
3
Categ. normal
Box & Whisker
acid
18000
16000
Amino
acids
n 1
Mean
Mean±SE
normal
Mean±1.96*SE
2 3 grade
1
2
3
grade
grade
C20:4
2HOglutarate
14000
arachidonic acid
Results
6000
4000
1E5
0
8000
Amino
acids
12000
10000
8000
6000
4000
2000
n 1
normal
1
2 3 grade
2
3
Mean
Mean±SE
Mean±1.96*SE
n 1
IDH1?
2 3 grade
Mean
Mean±SE
Mean±1.96*SE
Results
Can we validate biomarkers in a fully independent study?
Cohort 2 (113 patients)
Matej Orešič et al. (subm.)
Results
Can we validate lipidomic biomarkers in a fully
independent study?
Cohort 2 (113 patients)
Orešič et al. (subm)
Results
Can we validate lipidomic biomarkers in a fully independent
study? Immunohistochemistry on tissue slides.
Methods
Can we get biochemical maps of differential regulation
for hormone receptor status?
(1) Up to 20% of the identified metabolites lack enzyme annotations.
(2) Metabolic endpoint metabolites accumulate more than intermediates.
‘Comprehensive maps’ do not work.
Methods
Can we get biochemical maps of differential regulation
for hormone receptor status?
(1) Up to 20% of the identified metabolites lack enzyme annotations.
(2) Metabolic endpoint metabolites accumulate more than intermediates.
‘Comprehensive maps’ do not work.
Methods
Chemical and biochemical mapping of metabolomic results
Results
Grade 1 tumors vs normal
Red nodes: up-regulated in tumors at p<0.05, size ~ x-fold
Blue nodes: down-regulated in tumors at p<0.05, size~x-fold
Red edges: enzyme substrate/product in KEGGRpair DB
Green edges: chemical similarity > 0.7
Hormone receptor @ grade 3
60
% of patients
Methods
50
single neg.
ER+ PR+
HER2-
40
grade 1
30
grade 2
grade 3
20
triple neg.
ER- PRHER2-
10
0
E+P+H-
E+P+H+
E+P-H-
Estrogen positive
E+P-H+
E-P+H-
E-P+H+
E-P-H-
Estrogen negative
E-P-H+
Metabolic phenotype of triple negative tumors
Results
Patients without Estrogen, Herceptin neu2, Progesteron receptor
Breast Cancer: Grade 3
ER- PR- HER- (n=27) Vs ER+PR+ HER- (n=19)
Red : metabolite is enriched in triple negative
Blue : metabolite is down regulated in triple negative
Hormone receptor @ grade 3
60
% of patients
Methods
50
40
grade 1
30
grade 2
grade 3
20
triple neg.
ER- PRHER2-
10
double neg.
ER- PRHER2+
0
E+P+H-
E+P+H+
E+P-H-
Estrogen positive
E+P-H+
E-P+H-
E-P+H+
E-P-H-
Estrogen negative
E-P-H+
Results
Effect of HER2 status on metabolic phenotype
of triple negative tumors
Breast Cancer: Grade 3
Red : metabolite is enriched in triple negative
Hormone receptor @ grade 2
60
% of patients
Methods
50
40
30
double pos.
ER+ PR+
HER2-
grade 1
grade 2
grade 3
20
10
triple pos.
ER+ PR+
HER2+
0
E+P+H-
E+P+H+
E+P-H-
Estrogen positive
E+P-H+
E-P+H-
E-P+H+
E-P-H-
Estrogen negative
E-P-H+