metavectumRecklinghausen

Modern Moleculare Diagnosticin Oncology

Aims of Molecular Oncology

Early Diagnosis of Tumors

Detection of disseminated Tumor-Cells and Micrometastasis

Deciding of therapeutic Aims und Therapy Resistance

Monitoring of Therapy

Individualisised Therapy

Tumorprogression by genetic Alterations

Dissemination/Metastasis

Cancer is caused by genetic Alteration

cancer cells obtain unnormal features

Loss of Apoptosis

Unkontrolled Growth Signal

Unresponsive against growth inhibitors

Metastasis creation

Unlimited Replication

Angiogenesis

Genes for Growth Factors

Genetic Changes in Tumor-Cells

Erb-b2, EGFR, c-Kit, PDGFR, K-ras

Replications facilitating Genes

Telomerase, c-myc

Angiogenesis facilitating Genes

VEGF

Apoptosis facilitating Genes

Bcl-2, p53

Circulating Tumor-cells (CTC)

Risk-Factor for recurrance and metastasis.

indicate risk for micro-metastasis.

Easier indentification of patients profiting from adjuvant therapy.

Suggested further reading:

1. Cristofanilli M. et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004 Aug 19;351(8):781-91.

2. Allard WJ et al.: Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases Clin Cancer Res. 2004 Oct 15;10(20):6897-904.

Molekular Charakterisation of CTCs

Isolation of mRNA from CTCs

Measuring different Gene-Expression

Quantitative real-time PCR of molekular Tumor-Marker (e.g.):

• Cytokeratine

• PSA (Prostata), G250 (kidney), AFP (liver)

• Growth / Cell-cycle-Regulating Gene (c-myc, erbb2,

Telomerase)

Effect of Soy on Gene-Expressiondisseminated Tumor-Cells

Five Patients per group suffering from metastastastic Prostate-, Ovary- and Breast -Ca received Chemo + fermented soy

Clinical response in all patients

Question: Can molecular factors predict clinical outcome of Ca-Therapy by fermented Soy ?

Clincial : Bestimmung der modulierende Wirkung von Soja auf die Gen-Expression vor und nach 100 Tagen Einnahme des Soja-Extraktes

ER-beta

ER-alpha

DNA-Doppelstrang

DNA-Doppelstrang

Funktion der Östrogenrezeptoren(ER) in der Zelle

Zellteilungsaktivität wird gesteigert (Wucherung)

Zellteilungsaktivität wird verringert

a

ß

ERE

ERE

a

ß

Normale Zellen

Tumorzellen

Tumorzellen mit Soja-Extrakt

ER-beta ER-alpha ER-beta ER-beta ER-alphaER-alpha

Expression der Östrogenrezeptoren(ER) alpha und beta

ß

a ß ßa

a

konstantes Verhältnis

Absinken

Anstieg Absinken

Anstieg

unbehandeltBrust / Ovar Prostata

Estrogen-receptor beta is inducedby Haelan 951

Haelan increases ER2 expression in circulating tumor cells of P-CA patients (n=5)

0

100

200

300

400

500

600vor Hnach H

cases

rela

tive

gene

ex

pres

sion

Modulation of estrogen-receptor expression by Haelan 951

Circulating Tumor Cells (CTCs) of prostate cancer patients were analysed for expression of ER-alpha and ER-beta

vor H nach H vor H nach H

0

100

200

300

400

500

600

ER 1 ER 2

rela

tive

gene

ex

pres

sion

-> Expression of ER-beta was significantly induced by Haelan 951

relative Genexpression

Mamma-Ca. patients n= 5

all shown

breast cancer patients

are

ER-beta positive +

EstrogenreceptorER-beta

Modulation of estrogen-receptor expression by Haelan 951

Estrogenreceptor-beta (ER-beta)Expression

Relative G

enexpression

Mamma-Ca.Patients

with Haelan951

= No Haelan951 = Haelan951 application

Healthy persones n=7 Mamma-Ca. patients n=5

33,9

6,2

Healthy persones

without Haelan951

Estrogen-receptor beta is inducedby Haelan 951

Haelan increases ER2 expression in circulating tumor cells of ovarian cancer patients (n=5)

0

50

100vor Hnach H

cases

rela

tive

gene

ex

pres

sion

Modulation of estrogen-receptor expression by Haelan 951

Circulating Tumor Cells (CTCs) of ovarian cancer patients were analysed for expression of ER-alpha and ER-beta

-> Expression of ER-beta was significantly induced by Haelan 951

Induction of ER1 and ER2 by Haelan

vor H nach H vor H nach H

0

100

200

ER 1 ER 2

rela

tive

gene

ex

pres

sion

Stoffwechselweg der Apoptose

c-mycmitogens

proliferation

apoptosis

Bcl2

(survival signals)

blocking

NF - kB Bax

Duales Signal von c-myc:• Der apoptotische Stoffwechselweg wird blockiert durch bcl2 / bax und c-myc fördert Proliferation

• Nach Verlust des bcl2 Überlebenssignals, fördert c-myc die Apoptose

Apoptotic conditioning inducedby Healan 951

Healan induces pro-apoptotic condition in circulating tumor cells of P-CA patients

0

5

10

50

60

70

nach Hvor H

cases

bax

/bcl

2 ra

tio

CTCs of prostate cancer patients showed an increased gene expression ratio of apoptosis-triggering genes bax/bcl2 after Haelan951-therapy

without Haelan951 Haelan951 application 100 days

relative Genexpression

Mamma-Ca. patients n= 5

BAX/BCL2no significantchange

1,2 1,1

Apoptotic conditioning inducedby Healan 951

Ovarian-Ca. patients n=6

Apoptotic conditioning inducedby Healan 951

vor H nach H

0

5

10

Bax/Bcl2 Bax/Bcl2

rela

tive

gene

ex

pres

sion

Apoptotic conditioning inducedby Healan 951

Haelan increases BAX expression in circulatingtumor cells of ovarian cancer patients (n=5)

0

10

20

30vor Hnach H

cases

rela

tive

gene

ex

pres

sion

The p53 Signaling Pathway

Bax

Bcl-2

p21

MDM2p53

Caspases

Apoptosis

Cell CycleProgression

Cyclin D1

CDK4

Cyclin ECDK2

block

block

Cell cycle inhibitor

block

(Ovarian Ca.)

M

G1

p21 Involvement in Cell Cycle

p53

p21CIP1

Cyclin A/B CDK1CDK2

M-phaseentry

block

Cell cycle inhibitorMitosis

Prolife

ratio

n

DNA-Synthesis

S

Chromosomes Duplication

G2

37,8

Cellcycle Inhibitor P21 is inducedby Haelan951

without Haelan951 Haelan951 (100) Days)

1,9 0,5 0,9 1,0 0,6

relative Gen

expressio

n

Prostata-Ca. patients n=5

cases

56,3

7,112,4

0,9

14,3

Cellcycle Inhibitor P21 is inducedby Haelan951

without Haelan951 Haelan951 (100 days)

4,5

0,11 1,11

332,5

2,28

relative Genexpression

Mamma-Ca. patients n=5

cases

scale unit progressive

15,815,0

7,36

211,9

without Haelan951 Haelan951 application 100 days

relative Genexpression

Mamma-Ca. patients n= 5

p21 CIPincreased

2,0 13,1

Cellcycle Inhibitor P21 is inducedby Haelan951

Cellcycle Inhibitor P21 is inducedby Haelan951

Haelan increases P21expression in circulating tumor cellsof ovarian cancer patients (n=5)

0

50

100

150vor Hnach H

cases

rela

tive

gene

ex

pres

sion

Schlussfolgerungen

• Alle Patienten zeigten auf die Gabe fermentierten Soja-Extraktes klinischen Response

• Fermentiertes Soja-Extrakt fördert den pro-apoptotischen Zustand der Prostata-Karzinomzellen durch Herunterregulation von Bcl2. Dies dürfte die Zellen sensitiver machen für eine Chemotherapie und somit für eine Kombinationstherapie mit konventionellen Chemotherapeutika sprechen.

• Zugabe von fermentiertem Soja induziert die Expression des antiproliferativen ER-beta bei Prostata- und Ovar-Ca Patienten.

• Fermentiertes Soja-Extrakt induziert die Transkription des p21, welches zu einem Stillstand des Zellzyklus führt und damit das Krebswachstum unterdrückt, sowohl bei Prostata-, Ovar- als auch bei Mamma-Ca Patienten.

The classical view of biological organization is to consider the flow of information from the genome to the transcriptome, to the proteome and then the metabolome. The metabolome can be used to monitor changes in the genome or to measure the effects of downregulation or upregulation of a specific gene transcript.

metavectum

Interaction

Invluence of Haelan951 toVEGF Expression

without Haelan951 Haelan951 (100 Days)

relative Genexpression

Prostata-Ca. patients n=5 Mamma-Ca. patients n=5

Vascular EndothelialGrowth Factor

No alteration

0,97 0,971,46

37,5