Cancer Care Cancer Care Engineering Colorectal Engineering Colorectal CancerCancer

Gabriela Chiorean, Gabriela Chiorean, M.D.M.D.

June 26, 2009June 26, 2009

Rationale in colorectal Rationale in colorectal cancercancer PerformPerform OMICs of healthy, polyps, cancer OMICs of healthy, polyps, cancer

Compare OMICs between cancer, polyps and Compare OMICs between cancer, polyps and healthy: develop new healthy: develop new screeningscreening and and riskrisk assessment toolsassessment tools

Analyse changes in OMICs with treatment and Analyse changes in OMICs with treatment and correlate with response/toxicity: correlate with response/toxicity: predictive predictive markersmarkers

Mathematical modeling and Mathematical modeling and bio-mappingbio-mapping

Cancer Cancer care deliverycare delivery

Rationale: CCE nowRationale: CCE now

METABOLOMICS

GLYCO-PROTEOMICS LIPIDOMICS

BIOMAP

CRC

GENOMICS

Mathematicalmodeling

Schema IUCRO-0221Schema IUCRO-0221CCE in CRC CCE in CRC active April 2009active April 2009

SAMPLES

Blood (Serum)7 mL red topMetab, vit D

Blood (Plasma)21 mL purple top

Genomics, lipidomics, glycoproteomics

N=270

Stratification:

-Healthy (n=90)

-Polyps (n=90)

-Cancer (n=90)

stg 1/2

stg 3

stg 4 metastatic

Tissue10 mg polyp or50 mg cancer /

50 mg normal tissue

SHIP

DRY

ICE

8-hr fasting

Samples CollectionSamples Collection Healthy ControlsHealthy Controls

Screening Colonoscopy – GI Clinic

Sign ICS (RN)

Label specimensHealthy

if no polyps/tumor

Collect by RN/processing CRS Blood 1x 7 mL glass red top 3 x 7 mL plastic lavender

Questionnaires diet/environmental exposures

N= 5

Samples CollectionSamples Collection Adenomatous PolypsAdenomatous Polyps

Screening Colonoscopy – GI Clinic

Sign ICS (RN)

Label specimensPolyp

Polyps identified

Tissue procurement/Research specialist -Polyp cut in ½

-Place in tube with no preservative-Freeze at -70oC

Collect by RN/processing CRS Blood 1x 7 mL glass red top 3 x 7 mL plastic lavender

Questionnaires diet/environmental exposures

N= 3

Samples CollectionSamples Collection CancerCancer

Surgery

Sign ICS (RN)

Call tissue procurement -Tumor tissue ~ 50 mg

-Normal mucosa ~ 50 mg-Place in tube with no preservative

-Freeze at -70oC

Collect by RN/processing tissue procurementBlood: 1 x 7 mL red top glass tube 3 x 7 mL lavender plastic tubes

Questionnaires: diet/environmental exposures

Chemotherapy Follow-up

Every 3 months Up to 24 months

N= 8

CCE Blood Acquisition Protocols

Glass Red Top Tube (1)Volume = 7mL

Glass Purple Top Tubes (EDTA) (3)Volume = 7mL /tube

0.2 mL (2) Whole Blood into freezing tubes containingcomet assay solution, mix, place on dry ice, FREEZE (-80oC)

Centrifuge: 1750g, RT, 15 min

Plasma (~ 6mL), place on wet ice

SILICONIZED EPPENDORF TUBES0.2 mL (2) FREEZE (-80 oC) Lipidomics

1.5 mL (1) LONG TERM STORAGE (LIQUID N2); Regular Eppendorf Tubes0.2 mL (12) LONG TERM STORAGE (LIQUID N2); Siliconized Eppendorf Tubes

Centrifuge: 1500g, RT, 15 min

Serum ( ~ 3mL), place on wet ice

REGULAR EPPENDORF TUBES0.3 mL (2) FREEZE (-80 oC) Metabolomics NMR0.2 mL (2) FREEZE (-80 oC) Metabolomics MS0.5 mL (2) FREEZE (-80 oC) Vitamin D Analysis

0.5 mL (2) LONG TERM STORAGE (LIQUID N2)

Page: Amber Allen (page #) for transport to laboratory (RT) and processing

Maximum time at RT from draw to centrifugation: 45-60 min.

Maximum time at RT from draw to centrifuge: 30 min.

Remaining whole blood

Following blood draw, patients and care givers administered diet and life style questionnaire

Maximum time at RT from draw to Whole Blood Removal: 20 min.

Pellets (2); resuspend (1), combine with second pellet, re-centrifuge 1750g RT 5 min, decant, place

on dry ice: FREEZE (-80 oC) SNP

REGULAR EPPENDORF TUBES

1.5 mL (1) FREEZE (-80 oC)Glycoproteomics0.2 mL (1) FREEZE (-80 oC) Proteomics

MetabolomicsMetabolomics

Typical 2D GCxGC/MS data from a colon cancer patient serum sample. After derivitization, approximately 800 metabolites are observed (many of the lower intensity peaks are not evident in this figure). Dan Raftery-Purdue

MetabolomicsMetabolomics

Combination of the GC PCA data with NMR PCA data improves the classification to 95%. In the figure, 2 PCs from the GCxGC/TOF dataset are combined with 1 PC from the NMR data. Oblong shapes are used to indicate 95% confidence limits.

Schema IUCRO-0198Schema IUCRO-0198Metabolomics in CRCMetabolomics in CRC

SAMPLES

Blood (Serum)7 mL red top

Urine10 mL

N=150

Stratification:

-Healthy (n=30)

-Polyps (n=30)

-Cancer (n=90)

stg 1/2

stg 3

stg 4 metastatic

Tissue10 mg polyp or50 mg cancer /

50 mg normal tissue

SHIP

DRY

ICE

8-hr fasting

Principle Component Analysis of Metabolites in serum in IUCRO-0198

Dan Raftery, Lingyan Liu - Purdue

Investigators:Investigators:

Indiana UniversityIndiana UniversityGabriela Chiorean - Gabriela Chiorean -

OncologyOncologyPat Loehrer – OncologyPat Loehrer – OncologyStephen Williams - Stephen Williams -

OncologyOncologyYan Xu - LipidomicsYan Xu - LipidomicsJim Klaunig - GenomicsJim Klaunig - GenomicsBruce Robb - SurgeryBruce Robb - SurgeryEric Wiebke - SurgeryEric Wiebke - SurgeryDoug Rex - GIDoug Rex - GIMike Chiorean - GIMike Chiorean - GICharles Kahi - GICharles Kahi - GIPeter Johnstone – Rad OncPeter Johnstone – Rad OncOscar Cummings - Oscar Cummings -

PathologyPathology

Purdue UniversityPurdue UniversityMarietta Harrison - ChemistryMarietta Harrison - ChemistryDaniel Raftery – MetabolomicsDaniel Raftery – MetabolomicsFred Regnier – ProteomicsFred Regnier – Proteomics - Glycoproteomics- GlycoproteomicsDorothy Teegarden – Vitamin Dorothy Teegarden – Vitamin

DDMin Zhang – Statistical Min Zhang – Statistical

ModelingModelingJake Chen – Biological Jake Chen – Biological

Modeling Modeling