results grade1 grade2 grade3 (1) can we identify metabolites or metabolic pathways that are...

Resu

lts S co re sca tte rp l o t (t1 vs. t2 )

S ta n d a rd d e via ti o n o f t1 : 4 .9 9 2

S ta n d a rd d e via ti o n o f t2 : 7 .0 8 9

+ /-3 .0 0 0 *S td .De v-1 2 .5 -1 0 .0 -7 .5 -5 .0 -2 .5 0 .0 2 .5 5 .0 7 .5 1 0 .0

t1

-2 0 .0

-1 7 .5

-1 5 .0

-1 2 .5

-1 0 .0

-7 .5

-5 .0

-2 .5

0 .0

2 .5

5 .0

7 .5

1 0 .0

1 2 .5

1 5 .0

t2

grade1grade2

grade3

(1) Can we identify metabolites or metabolic pathways that are associated with breast cancer clinical parameters?

Alex-CIS-GCTOF MS w/ BinBase: 470 detected compounds161 known metabolites, 309 without identified structure.

Partial Least Square (multivariate stats)

grade3

grade2

grade1

breast adipose

0

10

20

30

40

50

60

E+P+H- E+P+H+ E+P-H- E+P-H+ E-P+H- E-P+H+ E-P-H- E-P-H+

grade 1 grade 2 grade 3

Resu

ltsGrade n1 23 tumors show differential up-regulation in Pentose Phosphate Pathway, nucleotides, amino acids, arachidonic acid

(but not free fatty acids, gln)

production of NADPH, ribose units production of DNA, RNA

Resu

ltsGrade n1 23 tumors show differential up-regulation in Pentose Phosphate Pathway, nucleotides, amino acids, arachidonic acid

(but not free fatty acids, gln)

Amino acids

Amino acids

lactate

Ca te g . B o x & Wh iske r P lo t: g l u ta m ic a ci d

n o rm a l 1 2 3

g ra d e

0

1 E 5

2 E 5

3 E 5

4 E 5

5 E 5

6 E 5

glu

tam

ic a

cid

n o rm a l 1 2 3

g ra d e

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

be

ta-a

lan

ine

glu -ala

M e a n M e a n ±S E M e a n ±1 .9 6 *S E

n o rm a l 1 2 3

g ra d e

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

2 0 0 0 0

2 2 0 0 0

2 4 0 0 0

2 6 0 0 0

2 8 0 0 0

3 0 0 0 0

3 2 0 0 0

3 4 0 0 0

ma

late

malate

Ca te g . B o x & Wh iske r P lo t: c i tri c a ci d

n o rm a l 1 2 3

g ra d e

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

3 0 0 0 0

3 5 0 0 0

4 0 0 0 0

4 5 0 0 0

5 0 0 0 0

5 5 0 0 0

citr

ic a

cid

citrate

n 1 2 3 grade n 1 2 3 grade

n 1 2 3 grade n 1 2 3 gradeCa te g . B o x & Wh iske r P lo t: a ra ch id o n i c a ci d

M e a n M e a n ±S E M e a n ±1 .9 6 *S E

n o rm a l 1 2 3

g ra d e

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

arac

hido

nic

acid

C20:4 2HOglutarate

n 1 2 3 graden 1 2 3 gradeIDH1?

Resu

lts

Can we validate biomarkers in a fully independent study?Cohort 2 (113 patients)

Resu

lts

Can we validate lipidomic biomarkers in a fully independent study?

Cohort 2 (113 patients)

Matej Orešič et al. (subm.)

Resu

lts

Can we validate lipidomic biomarkers in a fully independent study? Immunohistochemistry on tissue slides.

Orešič et al. (subm)

Can we get biochemical maps of differential regulation for hormone receptor status?

Met

hods

(1) Up to 20% of the identified metabolites lack enzyme annotations.

(2) Metabolic endpoint metabolites accumulate more than intermediates. ‘Comprehensive maps’ do not work.

Can we get biochemical maps of differential regulation for hormone receptor status?

Met

hods

(1) Up to 20% of the identified metabolites lack enzyme annotations.

(2) Metabolic endpoint metabolites accumulate more than intermediates. ‘Comprehensive maps’ do not work.

Chemical and biochemical mapping of metabolomic resultsM

etho

ds

Resu

ltsGrade 1 tumors vs normal

Red nodes: up-regulated in tumors at p<0.05, size ~ x-foldBlue nodes: down-regulated in tumors at p<0.05, size~x-foldRed edges: enzyme substrate/product in KEGGRpair DBGreen edges: chemical similarity > 0.7

0

10

20

30

40

50

60

E+P+H- E+P+H+ E+P-H- E+P-H+ E-P+H- E-P+H+ E-P-H- E-P-H+

grade 1 grade 2 grade 3

Met

hods

% o

f pati

ents

Estrogen negativeEstrogen positive

triple neg.

ER+ PR+HER2-

ER- PR-HER2-

single neg.

Hormone receptor @ grade 3

Resu

lts

ER- PR- HER- (n=27) Vs ER+PR+ HER- (n=19) P<0.05

Breast Cancer: Grade 3Red : metabolite is enriched in triple negative Blue : metabolite is down regulated in triple negativeNode size= fold change. Largest fold change is 3.

Metabolic phenotype of triple negative tumorsPatients without Estrogen, Herceptin neu2, Progesteron receptor

0

10

20

30

40

50

60

E+P+H- E+P+H+ E+P-H- E+P-H+ E-P+H- E-P+H+ E-P-H- E-P-H+

grade 1 grade 2 grade 3

Met

hods

% o

f pati

ents

Estrogen negativeEstrogen positive

triple neg.

ER- PR-HER2+

ER- PR-HER2- double neg.

Hormone receptor @ grade 3

Resu

ltsEffect of HER2 status on metabolic phenotype

of triple negative tumors

ER- PR- HER- (n=27) Vs ER-PR- HER+ (n=11) P<0.05

Breast Cancer: Grade 3

Red : metabolite is enriched in triple negative Blue : metabolite is down regulated in triple negativeNode size= fold change. Largest fold change is 3.

0

10

20

30

40

50

60

E+P+H- E+P+H+ E+P-H- E+P-H+ E-P+H- E-P+H+ E-P-H- E-P-H+

grade 1 grade 2 grade 3

Met

hods

% o

f pati

ents

Estrogen negativeEstrogen positive

ER+ PR+HER2-

double pos.

ER+ PR+HER2+

triple pos.

Hormone receptor @ grade 2