effects of intra-operative ischemia on cancer and normal ... · realizing individualized cancer...

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Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut Juhl CEO of Indivumed GmbH and Inostics GmbH Adjunct Professor, Georgetown University and Hamburg University www.indivumed.com [email protected]

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Page 1: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Realizing Individualized Cancer Therapy

Effects of intra-operative ischemia on cancer and n ormal tissueProf. Dr. Hartmut Juhl

CEO of Indivumed GmbH and Inostics GmbHAdjunct Professor, Georgetown University and Hamburg University

[email protected]

Page 2: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Hamburg:

A Federal State of Germany

Germany‘s 2nd largest city

Metropolitan area of ~ 4 Mio people

Highest income/citizen in Europe

„Happiest citizen in Germany“

High density of outstanding cancer clinics

Page 3: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Indivumed GmbHHamburg, Germany

Founded : 2002 Inostics GmbH

B. Vogelstein et al. (JHU)

F.Diehl/H.Juhl

Indivumed Group

IndivuTest GmbH

Employee: 90

Financing: Private

Founded : 2002

Indivumed Inc.Kensington, MD

Founded: 2005

Inostics GmbHHamburg, Germany

Founded: 2009

Inostics Inc.Baltimore, MD

Founded: 2010

IndivuTest GmbHHamburg, Germany

Founded: 2011

Page 4: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Indivumed Vision: Individualizing Cancer Medicine

• Clinical Integration

• Biobanking

Therapy 1 Therapy 2 Therapy 3

•Tissue & Blood Based Research

• Protein Based Dx (Indivumed)

• DNA-Based Dx (Inostics)

• Patient Testing (IndivuTest)

Page 5: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Indivumeds Approach:Integration of Research, Surgery and Patient Care

+ Indivumed Clinical Team

Indivumed Scientific Team++++++

+Biospecimen• Tumor tissue• Normal Tissue• Blood• UrineData• Processing Data• Clinical Data• Follow-up Data

Scientific Team

Tumor-Biobankand

Clinical Data Base

BiospecimenDirect Processing

++++++++++Partner

Hospitals

Page 6: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Indivumed Solution:Integration of Clinical Care, Biobanking and Research

Tumor-Biobankand

Clinical Data Base

Page 7: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Indivumed Standard of Biobanking:

� Exact documented and very short tissue cold ischemi a times of < 12 min (mean 7 min)

� Exact tissue localization and standardized fixation

� Complete biospecimen sets

10 min

Tissue ischemiaand protein

phosphorylation

� Highest tissue quality monitored by visual inspectio n, H&E staining and microscopic assessment

� Native and rapid fluid preparations

� Complete specimen data

� Complete clinical data

� Patients’ confidentiality assured following internat ional standards

10 min

20 min

60 min

Page 8: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Target Validation&

Pathway Analysis

Business Concept

ServiceBusiness

Biomarker(Co-)Development

Understanding Differences between

Patients

Protein-BasedTissue Diagnosis

(Indivumed)

Clinical Integration of

Research

Biobanking

BusinessPatient

Care

DNA-BasedDiagnosis(Inostics)

Page 9: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Understanding the Molecular Basis of Cancer: Status 19 71 (Nixon Declares „War Against Cancer“)

Page 10: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Understanding the Molecular Basis of Cancer: Status 2011

Page 11: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Understanding of Cancer Pathways as Basis for„Targeted Therapeutics“

Page 12: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

„Targeted Therapies“ are the Focus of Current Drug Devel opment

Targeted therapies53%

Vaccines14%

Antihormonaltherapies

1%

Cytotoxics22%

Genetherapies

4%

Immuno-therapies

5%

PDT0.3%

Radio-therapeutics

1%

Cost of 900 Mio $ / new FDA approved compound

90% of compounds fail in clincial phases

Page 13: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Ca. 60 „Targeted Therapies“ are Approved and >800 Compounds are in Clinical Trial

(Status 2012/01)

Page 14: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Analysis of Pathway Activity for Individualizing Cancer T herapy

Targetexpression?

Relevant for cell biology? (aktiv yes/no)

Which Drug?(=Inhibitor)

Page 15: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

DNA mRNA Proteins Protein Modifications(e.g., phosphorylation)

MutationsKRASPIK3CABRAF

Target-ExpressionHER-2EGFRVEGFR

FunctionalitypmTORP-AKTP-MAPK

Predictive Biomarker: High Predictive Value or Robustness?

MutationsOncotypeAmpliChipMammaPrintBRAF

EGFRMany others

VEGFROthersProtein Profiling

P-MAPKMany, many more

Predictive value / Impact of preanalytical factors

MammaPrintOthers

Page 16: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Diagnostic Approaches for Individualizing Cancer Therap y:Tissue and Blood

TumorTissue Blood

• Free Tumor DNA•Tumor cells (CTC)•Proteins•Metabolome•RNA (microRNA)

Frozen orFFPE

Tissue

ViableTissue

Reflects high complexity of tumorDifficult to obtain at time of therapyQuality matters a lot

Only low complexity of tumorStatus at time of therapyAllows monitoring and repeated testing

Page 17: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Specimen is viable and reactive Biomolecules may degrade

Time 0

Cancer Problem #1 for Predictive Biomarker and Drug Dev elopment: :Availability of Tissue that Reflects Reality of Tumor Bio logy

Patient andPresurgical therapy

Medical/SurgicalProcedures

AcquisitionProcessing

StorageTemperatur

Handling, Processingand analytical assays

Expression of Targets (e.g., Pathways) and Biomarker depend on Individual Variables and Tissue Processing

Page 18: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Specimen is viable and reactive Biomolecules may degrade

Time 0

Cancer Problem #1 for Predictive Biomarker and Drug Dev elopment: :Availability of Tissue that Reflects Reality of Tumor Bio logy

Patient andPresurgical therapy

Medical/SurgicalProcedures

AcquisitionProcessing

StorageTemperatur

Handling, Processingand analytical assays

Expression of Targets (e.g., Pathways) and Biomarker depend on Individual Variables and Tissue Processing

Page 19: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Phosphoprotein Expression:pMAPK Immunostaining (Ventana)

10 min

Case BCase A

Change of pMAPK expression

20 min

60 min

Change of pMAPK expressionafter 10-20 min cold ischemia

Without knowledge about tissue processing and rapid tissu e fixation

protein expression data are unreliable and

understanding of pathway activity is impossible

Page 20: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Specimen is viable and reactive Biomolecules may degrade

Time 0

Cancer Problem #1 for Predictive Biomarker and Drug Dev elopment: :Availability of Tissue that Reflects Reality of Tumor Bio logy

Patient andPresurgical therapy

Medical/SurgicalProcedures

AcquisitionProcessing

StorageTemperatur

Handling, Processingand analytical assays

Expression of Targets (e.g., Pathways) and Biomarker depend on Individual Variables and Tissue Processing

Page 21: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Patients receiving left hemicolectomy

Indivumed data base / biobank:

Time (min) between artery ligation

and tumor removal

Retrospective Study:Impact of Time between Ligation of Main Artery and Tumor Resection

on Gene Expression in Colon Cancer(NCI-Indivumed study)

MesentericMesentericartery inferiorartery inferior XX

20 25 30 35 40 45 50

(min)

Time (min) until freezing

10 min

LCM isolation of tumor cells

Gene expression (Affymetrix)

Bioinformatics

Page 22: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Warm ischemia (min)20 25 30 35 40 45 50

PCA mapping: grouping of warm ischemia time points

202530

Impact of Time between Ligation of Main Artery and Tumor Resectionon Gene Expression in Colon Cancer

(NCI-Indivumed study)

35404550

Prospective trial collecting tissue during surgery

Page 23: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Research Studies on the Effect of Intra- and Post-operat ive Ischemia on Gene and Protein Expression Patterns in Liver (Projec t 1) and Colorectal

Tissue (Project 2).An Exploratory Research Study (29XS111)

Partner:

Funded by NCI Contract No. HHSN261200800001E

Partner:

OBBR/NCI

Indivumed GmbH

Department of Surgery, Israelitisches Krankenhaus (D r. Zornig)

Department of Surgery, Diakonieklinikum Alten Eichen (Dr. Dörner)

Department of Hepatobiliary Surgery, University Hospi tal Hamburg (PI: Dr. Nashan)

Page 24: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Cancer -relevant Proteins Gene Expression Profiling

Cancer Normal

Impact of Anesthesia and Surgery on Gene and Protein Exp ression in Colon and Liver Tissue : Study Design

Cancer -relevant Proteins

Protein Quantification Cellular Distribution

Identification of Surgery Dependent Molecules

pMEK1/2

0

1000

2000

3000

4000

5000

MS

D s

igna

l [U

]

Gene Expression Profiling

Specific Genes Comprehensive Analysis

Page 25: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Analysis of Proteins:MSD and Immunohistochemistry

Frozen Tissue:MSD analysis

FFPE TissueIHC (Ventana)

• pEGFR/total EGFR

•pMEK1/2/total MEK1/2 (Ser217/221)

• pEGF-R

• pHER-3•pMEK1/2/total MEK1/2 (Ser217/221)

• pERK1/2/total ERK1/2 (Thr202/Tyr204, Thr185/Tyr187)

• pAkt/total Akt (Ser473)

• pmTOR/total mTOR (Ser 2448)

•pP70S6K/total P70S6K (Thr421/Ser424)

• pGSK-3beta/total GSK-3beta (Ser9)

• control protein x

• Hif-1alpha

• pHER-3

• pMAPK

• pAKT

• pmTOR

Page 26: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Impact of Anesthesia and Surgery on Gene and Protein Exp ression in Colorectal Tissue (Project 2)

Colon Tissue

Normal Cancer

Start of

Blood

Normal

Collected:50 / 40

Surgery

Post-Surgery

10 min

20 min

45 min

BeforeAnesthesia

Before skinincision

Page 27: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

3

HSP27-normal3

HSP27-tumor

Impact of intra- and postsurgical factors:Analysis of Total-control protein X by MSD (Cellular Str ess Marker)

Normal Normal

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

1

2

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

1

2

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 28: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

35

HSP27-normalphosphorylation

30

35

HSP27-tumorphosphorylation

Impact of intra- and postsurgical factors:Analysis of Phospho-control protein X by MSD (Cellular S tress Marker)

Normal Tumor

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

5

10

15

20

25

30

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

5

10

15

20

25

30

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

Page 29: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

P-protein

Analysis of HER-family Pathway in CRC Tissue:Impact of intra- and postsurgical factors

P-protein

P-protein

Page 30: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

4

5

EGFR-normal

4

5

EGFR-tumor

Impact of intra- and postsurgical factors:Analysis of Total-EGFR by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

1

2

3

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

1

2

3

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 31: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

16

18

20

EGFR-normalphosphorylation

16

18

20

EGFR-tumorphosphorylation

Impact of intra- and postsurgical factors:Analysis of Phospho-EGFR by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

2

4

6

8

10

12

14

16

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

2

4

6

8

10

12

14

16

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 32: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

EGFR-%Phospho-normalpe

rcen

tage

0

20

40

60

80

100

EGFR-%Phospho-tumor

perc

enta

ge

0

20

40

60

80

100

Impact of intra- and postsurgical factors:Analysis of Phospho/Total-EGFR by MSD

pre r

esec

tion

10 m

in po

st re

secti

on20

min

post

rese

ction

45 m

in po

st re

secti

on

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

EGFR-%Phospho-normal

0.4648

Gaussian Approximation

ns

No

4

2.559

Difference in rank sum

16.04

6.363

8.900

-9.675

-7.138

2.538

Significant? P < 0.05?

No

No

No

No

No

No

Summary

ns

ns

ns

ns

ns

ns

pre re

secti

on

10 m

in p os

t res

ectio

n

20 m

in p ost

r ese

ction

45 m

in p ost

rese

ction

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

EGFR-%Phospho-tumor

0.4629

Gaussian Approximation

ns

No

4

2.569

Difference in rank sum

-12.15

-14.29

-4.163

-2.138

7.988

10.13

Significant? P < 0.05?

No

No

No

No

No

No

Summary

ns

ns

ns

ns

ns

ns

Page 33: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

pEGFR normalH

-sco

re

pre re

secti

on

10 min

post

rese

ction

20 min

pos t r

ese cti

on

45 min

post

rese

ction

0

50

100

150

pEGFR tumor

H-s

core

pre r

esec

tion

10 m

in po

s t res

ection

20 m

in po

st re

secti

on

45 m

in po

s t res

ection

0

50

100

150

200

Analysis of Phospho-EGFR by IHC

pre re

secti

on

10 min

post

rese

ction

20 min

pos t r

ese cti

on

45 min

post

rese

ction

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

pEGFR normal

0.0268

Gaussian Approximation

*

Yes

4

9.194

Difference in rank sum

16.45

16.45

20.400.0

3.950

3.950

Significant? P < 0.05?

No

No

YesNo

No

No

Summary

ns

ns

*ns

ns

ns

pre r

esec

tion

10 m

in po

s t res

ection

20 m

in po

st re

secti

on

45 m

in po

s t res

ection

Table Analyzed

Kruskal-Wallis test

P valueExact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection20 min post resection vs 45 min post resection

pEGFR tumor

0.0020Gaussian Approximation

**

Yes

4

14.80

Difference in rank sum

22.53

20.58

25.10

-1.950

2.5754.525

Significant? P < 0.05?

Yes

Yes

Yes

No

NoNo

Summary

*

*

**

ns

nsns

Page 34: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

pHer3 normal

H-s

core

pre re

secti

o n

10 m

in pos t r

esecti

on

20 m

in po

st res

ectio

n

45 m

in pos

t rese

ction

0

50

100

150

pHer3 tumor

H-s

core

pre

resec

tion

10 m

in po

st re

sect

ion

20 m

in pos

t res

ectio

n

45 m

in p

ost r

esec

tion

0

50

100

150

Analysis of Phospho-HER3 by IHC

Table Analyzed

One-way analysis of variance

P value

P value summary

Are means signif. different? (P < 0.05)

Number of groups

F

R square

Bartlett's test for equal variances

Bartlett's statistic (corrected)

P value

P value summary

Do the variances differ signif. (P < 0.05)

ANOVA Table

Treatment (between columns)

Residual (within columns)

Total

Bonferroni's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

pHer3 normal

0.1131

ns

No

4

2.056

0.07508

2.045

0.5631

ns

No

SS

2550

31415

33965

Mean Diff.

6.550

10.10

15.55

3.550

9.000

5.450

df

3

76

79

t

1.019

1.571

2.419

0.5522

1.400

0.8477

MS

850.0

413.4

Significant? P < 0.05?

No

No

No

No

No

No

Summary

ns

ns

ns

ns

ns

ns

95% CI of diff

-10.87 to 23.97

-7.317 to 27.52

-1.867 to 32.97

-13.87 to 20.97

-8.417 to 26.42

-11.97 to 22.87

10 m

in po

st re

sect

ion

20 m

in pos

t res

ectio

n

45 m

in p

ost r

esec

tion

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

pHer3 tumor

0.0187

Gaussian Approximation

*

Yes

4

9.990

Difference in rank sum

17.75

13.73

21.03

-4.025

3.275

7.300

Significant? P < 0.05?

No

No

Yes

No

No

No

Summary

ns

ns

*

ns

ns

ns

Page 35: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

P-protein

Analysis of HER-family Pathway in CRC Tissue:Impact of intra- and postsurgical factors

P-protein

Page 36: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

120

140

160

Akt-normal

120

140

160

Akt-tumor

Impact of intra- and postsurgical factors:Analysis of Total-AKT by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

20

40

60

80

100

120

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

20

40

60

80

100

120

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 37: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

4

Akt-normalphosphorylation

4

Akt-tumorphosphorylation

Impact of intra- and postsurgical factors:Analysis of Phospho-AKT by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

1

2

3

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

1

2

3

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 38: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Akt-%Phospho-normal

perc

enta

ge

0

20

40

60

80

100

Akt-%Phospho-tumor

perc

enta

ge

0

20

40

60

80

100

Impact of intra- and postsurgical factors:Analysis of Phospho/Total-AKT by MSD

pre re

secti

on

10 m

in p os

t res

ectio

n

20 m

in p ost

rese

ction

45 m

in po

st re

secti

on

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

Akt-%Phospho-normal

< 0.0001

Gaussian Approximation

****

Yes

4

54.76

Difference in rank sum

53.74

61.75

68.26

8.013

14.53

6.513

Significant? P < 0.05?

Yes

Yes

Yes

No

No

No

Summary

***

***

***

ns

ns

ns

pre re

secti

on

10 m

in p os

t res

ectio

n

20 m

in p ost

r ese

ction

45 m

in p ost

rese

ction

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

Akt-%Phospho-tumor

< 0.0001

Gaussian Approximation

****

Yes

4

80.39

Difference in rank sum

82.64

71.80

67.11

-10.84

-15.53

-4.688

Significant? P < 0.05?

Yes

Yes

Yes

No

No

No

Summary

***

***

***

ns

ns

ns

Page 39: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

pAkt normal

H-s

core

pre re

secti

on

10 m

in p

ost re

secti

on

20 m

in p ost

rese

ction

45 min

post

r esec

tion

0

50

100

150

pAkt tumor

H-s

core

pre

res ec

tion

10 m

in po

st re

secti

on

20 m

in po

st re

secti

on45

min

pos

t res

ectio

n

0

50

100

150

200

250

Analysis of Phospho-AKT by IHC

10 m

in p

ost re

secti

on

20 m

in p ost

rese

ction

45 min

post

r esec

tion

Table Analyzed

One-way analysis of variance

P value

P value summary

Are means signif. different? (P < 0.05)

Number of groups

F

R square

Bartlett's test for equal variances

Bartlett's statistic (corrected)

P value

P value summary

Do the variances differ signif. (P < 0.05)

ANOVA Table

Treatment (between columns)

Residual (within columns)

Total

Bonferroni's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

pAkt normal

0.6336

ns

No

4

0.5745

0.02217

4.075

0.2534

ns

No

SS

641.2

28279

28920

Mean Diff.

5.750

4.600

7.700

-1.150

1.950

3.100

df

3

76

79

t

0.9426

0.7541

1.262

0.1885

0.3197

0.5082

MS

213.7

372.1

Significant? P < 0.05?

No

No

No

No

No

No

Summary

ns

ns

ns

ns

ns

ns

95% CI of diff

-10.78 to 22.28

-11.93 to 21.13

-8.825 to 24.23

-17.68 to 15.38

-14.58 to 18.48

-13.43 to 19.63

10 m

in po

st re

secti

on

20 m

in po

st re

secti

on45

min

pos

t res

ectio

n

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

pAkt tumor

0.0010

Gaussian Approximation

**

Yes

4

16.21

Difference in rank sum

16.63

17.98

29.30

1.350

12.68

11.33

Significant? P < 0.05?

No

No

Yes

No

No

No

Summary

ns

ns

***

ns

ns

ns

Page 40: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

18

20

mTOR-normal

18

20

mTOR-tumor

Impact of intra- and postsurgical factors:Analysis of Total-mTOR by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

2

4

6

8

10

12

14

16

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

2

4

6

8

10

12

14

16

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

Page 41: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

2

mTOR-normalphosphorylation

2

mTOR-tumorphosphorylation

Impact of intra- and postsurgical factors:Analysis of Phospho-mTOR by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

1

1

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

1

1

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

Page 42: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

mTOR-%Phospho-normalpe

rcen

tage

50

60

70

80

90

100

mTOR-%Phospho-tumor

perc

enta

ge

50

60

70

80

90

100

Impact of intra- and postsurgical factors:Analysis of Phospho/Total-mTOR by MSD

pre r

esec

tion

10 m

in po

st re

secti

o n

20 m

in po

st re

secti

on

45 m

in po

st re

secti

on

50

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

mTOR-%Phospho-normal

0.5191

Gaussian Approximation

ns

No

4

2.266

Difference in rank sum

11.78

7.588

-1.663

-4.188

-13.44

-9.250

Significant? P < 0.05?

No

No

No

No

No

No

Summary

ns

ns

ns

ns

ns

ns

pre r

ese ct

ion

10 m

in po

st re

sec

tion

20 m

in po

st re

sectio

n

45 m

in po

st re

sec tio

n

50

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

mTOR-%Phospho-tumor

< 0.0001

Gaussian Approximation

****

Yes

4

31.84

Difference in rank sum

43.81

54.80

38.54

10.99

-5.275

-16.26

Significant? P < 0.05?

Yes

Yes

Yes

No

No

No

Summary

***

***

**

ns

ns

ns

Page 43: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

pmTOR tumor

H-s

core

0

100

200

300

pmTOR normal

H-s

core

0

50

100

150

200

Analysis of Phospho-mTOR by IHC

pre re

secti

on

10 m

in po

st re

secti

on

20 m

in po

st re

secti

on

45 m

in po

st re

secti

on

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resectionpre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

pmTOR tumor

0.5953

Gaussian Approximationns

No

4

1.891

Difference in rank sum

-7.250

-4.975-9.675

2.275

-2.425

-4.700

Significant? P < 0.05?

No

NoNo

No

No

No

Summary

ns

nsns

ns

ns

ns

pre re

sec tio

n

10 m

in po

st re

secti

on20

min

post re

secti

on

45 m

in po

s t res

ectio

n

0

Table Analyzed

Kruskal-Wallis testP value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groupsKruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resectionpre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection20 min post resection vs 45 min post resection

pmTOR normal

0.0026

Gaussian Approximation

**

Yes

414.21

Difference in rank sum

-6.100-10.80

-26.40

-4.700

-20.30-15.60

Significant? P < 0.05?

NoNo

Yes

No

YesNo

Summary

nsns

**

ns

*ns

Page 44: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

100

120

p70s6k-normal

100

120

p70s6k-tumor

Impact of intra- and postsurgical factors:Analysis of p70s6k by MSD

0

20

40

60

80

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

20

40

60

80

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

Page 45: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

3

4

p70s6k-normalphosphorylation

3

4

p70s6k-tumorphosphorylation

Impact of intra- and postsurgical factors:Analysis of Phospho-p70s6k by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

1

2

3

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

1

2

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

getime

Page 46: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

p70s6K-%Phospho-normal

perc

enta

ge

0

20

40

60

80

100

p70s6K-%Phospho-tumor

perc

enta

ge

0

20

40

60

80

100

Impact of intra- and postsurgical factors:Analysis of Phospho/Total-p70s6k by MSD

pre re

secti

on

10 m

in po

st re

secti

on

20 m

in po

st r es

ectio

n

45 m

in po

st r es

ectio

n

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

p70s6K-%Phospho-normal

0.0188

Gaussian Approximation

*

Yes

4

9.969

Difference in rank sum

24.31

5.488

-6.750

-18.83

-31.06

-12.24

Significant? P < 0.05?

No

No

No

No

Yes

No

Summary

ns

ns

ns

ns

*

ns

pre r

ese cti

on

10 m

in po

st re

sectio

n

20 m

in po

st re

sectio

n

45 m

in po

st r e

secti

on

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

p70s6K-%Phospho-tumor

< 0.0001

Gaussian Approximation

****

Yes

4

50.80

Difference in rank sum

69.29

40.74

16.88

-28.55

-52.41

-23.86

Significant? P < 0.05?

Yes

Yes

No

Yes

Yes

No

Summary

***

***

ns

*

***

ns

Page 47: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

20

25

GSK3beta-normal

20

25

GSK3beta-tumor

Impact of intra- and postsurgical factors:Analysis of Total-GSK3beta by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

5

10

15

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

5

10

15

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 48: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

3

GSK3beta-normalphosphorylation

3

GSK3beta-tumorphosphorylation

Impact of intra- and postsurgical factors:Analysis of Phospho-GSK3beta by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

1

2

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

1

2

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 49: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

GSK3beta-%Phospho-normalpe

rcen

tage

0

20

40

60

80

100

GSK3beta-%Phospho-tumor

perc

enta

ge

0

20

40

60

80

100

Impact of intra- and postsurgical factors:Analysis of Phospho/Total-GSK3beta by MSD

pre r

e sect

ion

10 m

in pos

t res

ectio

n

20 m

in po

st re

secti

on45

min

pos

t rese

ction

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

GSK3beta-%Phospho-normal

0.0123

Gaussian Approximation

*

Yes

4

10.89

Difference in rank sum

14.39

18.84

33.88

4.450

19.49

15.04

Significant? P < 0.05?

No

No

Yes

No

No

No

Summary

ns

ns

**

ns

ns

ns

pre re

secti

on

10 m

in pos

t re se

ctio

n

20 m

in po

st re

sect

ion

45 m

in po

st re

sec

tion

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

GSK3beta-%Phospho-tumor

< 0.0001

Gaussian Approximation

****

Yes

4

44.38

Difference in rank sum

64.91

51.25

45.79

-13.66

-19.13

-5.463

Significant? P < 0.05?

Yes

Yes

Yes

No

No

No

Summary

***

***

***

ns

ns

ns

Page 50: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

P-protein

Analysis of HER-family Pathway in CRC Tissue:Impact of intra- and postsurgical factors

P-protein

P-protein

Page 51: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

10

12

MEK-normal

10

12

MEK-tumor

Impact of intra- and postsurgical factors:Analysis of Total-MEK by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

2

4

6

8

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

2

4

6

8

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 52: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

7

MEK-normalphosphorylation

6

7

MEK-tumorphosphorylation

Impact of intra- and postsurgical factors:Analysis of Phospho-MEK by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

1

2

3

4

5

6

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

1

2

3

4

5

6

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 53: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

MEK-%Phospho-normalpe

rcen

tage

0

20

40

60

80

100

MEK-%Phospho-tumor

perc

enta

ge

0

20

40

60

80

100

Impact of intra- and postsurgical factors:Analysis of Phospho/Total-MEK by MSD

pre re

secti

on

10 m

in p os

t res

ectio

n

20 m

in p ost

r ese

ction

45 m

in p ost

rese

ction

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

MEK-%Phospho-normal

< 0.0001

Gaussian Approximation

****

Yes

4

27.13

Difference in rank sum

52.61

17.30

19.04

-35.31

-33.58

1.738

Significant? P < 0.05?

Yes

No

No

Yes

Yes

No

Summary

***

ns

ns

**

**

ns

pre re

secti

on

10 m

in p os

t res

ectio

n

20 m

in p ost

r ese

ction

45 m

in p ost

rese

ction

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

MEK-%Phospho-tumor

< 0.0001

Gaussian Approximation

****

Yes

4

27.93

Difference in rank sum

52.68

38.94

32.39

-13.74

-20.29

-6.550

Significant? P < 0.05?

Yes

Yes

Yes

No

No

No

Summary

***

**

*

ns

ns

ns

Page 54: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

7

8

ERK-normal

7

8

ERK-tumor

Impact of intra- and postsurgical factors:Analysis of Total-ERK (MAPK) by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

1

2

3

4

5

6

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

1

2

3

4

5

6

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

Page 55: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

6

7

ERK-normalphosphorylation

6

7

ERK-tumorphosphorylation

Impact of intra- and postsurgical factors:Analysis of Phospho-ERK (pMAPK) by MSD

Case NC7 Case NC13 Case NC17 Case NC27 Case NC35 Case NC39 Case NC40 Case NC41 Case NC42 Case NC47

Case NC9 Case NC11 Case NC12 Case NC18 Case NC20 Case NC21 Case NC28 Case NC30 Case NC33 Case NC36

Case NC2 Case NC3 Case NC4 Case NC5 Case NC8 Case NC10 Case NC15 Case NC16 Case NC22 Case NC23

Case NC25 Case NC26 Case NC29 Case NC32 Case NC34 Case NC37 Case NC38 Case NC43 Case NC46 Case NC48

0

1

2

3

4

5

6

pre resection 10 min post resection 20 min post resection 45 min post resection

rela

tive

chan

ge

time

0

1

2

3

4

5

6

pre resection 10 min post resection 20 min post resection 45 min post resectionre

lativ

e ch

ange

time

Page 56: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

ERK-%Phospho-normal

perc

enta

ge

0

20

40

60

80

100

ERK-%Phospho-tumor

perc

enta

ge

0

20

40

60

80

100

Impact of intra- and postsurgical factors:Analysis of Phospho/Total-ERK (pMAPK) by MSD

pre re

secti

on

10 m

in p os

t res

ectio

n

20 m

in p ost

r ese

ction

45 m

in p ost

rese

ction

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

ERK-%Phospho-normal

0.0002

Gaussian Approximation

***

Yes

4

19.30

Difference in rank sum

44.76

17.23

16.41

-27.54

-28.35

-0.8125

Significant? P < 0.05?

Yes

No

No

Yes

Yes

No

Summary

***

ns

ns

*

*

ns

pre re

secti

on

10 m

in p os

t res

ectio

n

20 m

in p ost

r ese

ction

45 m

in p ost

rese

ction

0

Table Analyzed

Kruskal-Wallis test

P value

Exact or approximate P value?

P value summary

Do the medians vary signif. (P < 0.05)

Number of groups

Kruskal-Wallis statistic

Dunn's Multiple Comparison Test

pre resection vs 10 min post resection

pre resection vs 20 min post resection

pre resection vs 45 min post resection

10 min post resection vs 20 min post resection

10 min post resection vs 45 min post resection

20 min post resection vs 45 min post resection

ERK-%Phospho-tumor

< 0.0001

Gaussian Approximation

****

Yes

4

42.23

Difference in rank sum

63.91

49.81

37.58

-14.10

-26.34

-12.24

Significant? P < 0.05?

Yes

Yes

Yes

No

No

No

Summary

***

***

**

ns

ns

ns

Page 57: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Liver

Normal Met‘s

ArteryClamping

Impact of Anesthesia and Surgery on Gene and Protein Exp ression in Liver Tissue (Project 1)

Blood

Normal

Collected:42 / 40 (still continued)

Clamping

0min

10 min

Post-Surgery

10 min

20 min

45 min

BeforeAnesthesia

Before skinincision

Page 58: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Cancer-relevant Proteins Gene Expression Profiling

Cancer Normal

Impact of Anesthesia and Surgery on Gene and Protein Exp ression in Colon and Liver Tissue : Next Steps

Protein Quantification Cellular Distribution

pMEK1/2

0

1000

2000

3000

4000

5000

MS

D s

igna

l [U

]

Specific Genes Comprehensive Analysis

Colon:Completed

Liver:First set of 20 completedFinal set of 20 shortly

Colon:Completed by end of March

Liver:Completed shortly

Page 59: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Summary

• Positive control markers demonstrate that cold ischemia a s well as surgery can

have a significant impact on expression and activity of fu nctional proteins

• Cancer and corresponding normal tissue react different ( normal<cancer)

• High variability of functional protein expression and eff ects of intra- and

postsurgery between patients

• Overall, MSD data and IHC analyses are constistent altho ugh in some markers

(e.g., p-mTOR) differences can be seen

• A comprehensive data analysis – including gene expression data – will follow when

results of the liver study are completed. This will includ e correlative analysis of

clinical and anesthesiological data

Page 60: Effects of intra-operative ischemia on cancer and normal ... · Realizing Individualized Cancer Therapy Effects of intra-operative ischemia on cancer and normal tissue Prof. Dr. Hartmut

Thank you very much for your attention!

Greetings from Hamburg