Celecoxib Pre-treatment in Human Colorectal Adenocarcinoma Patients Is_No Cut Ppt

8/8/2019 Celecoxib Pre-treatment in Human Colorectal Adenocarcinoma Patients Is_No Cut Ppt

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Celecoxib pre-treatment in human

colorectal adenocarcinoma patients is

associated with gene expressionalterations suggestive of diminished

cellular proliferation.Authors:Authors:

James ToddJames Todd Auman Auman; Robert Church;; Robert Church; SooSoo--YounYoun Lee; Mark A Watson; James WLee; Mark A Watson; James W FleshmanFleshman; Howard L; Howard L McleodMcleod

Journal:European journal of cancer (Oxford, England : 1990) Volume: 44

ISO Abbreviation: Eur. J. Cancer Publication Date: 2008 Aug

Presented by :Presented by :Ms. Nipaporn Thipmanee

Code 520531189

Adviser:Adviser:Asst. Prof. Dr.Asst. Prof. Dr. SukonSukon PrasitwattanasarePrasitwattanasare



2

Introduction

Materials and methods

Results

Discussion

Conclusion

ContentsContentsContentsContents

Celecoxib pre-treatment in human colorectal

adenocarcinoma«





Anatomy of colon and rectum

Colorectal cancer

Colorectal cancer involves

Colon

Rectal Anal canal

3rd most common cancers:

men & women

in the United States

4

1

2

Anal canal in rectum



Colorectal cancer [cont.]

Colorectal cancer : high mortality rate of the world

2008 new case In the United States

Colon cancer -> 108,070

Rectal cancer -> 40,740

3rd leading cancer diagnosis

2nd leading cause of cancer death.

Report in Cancer Statistics: Jemal et al; CA Cancer J Clin 2008

5



PolypPolyp (Adenoma)

Definition : Begin tumor but not cancer Polyp begins as a small,

It harmless growth in the wall of the colon

Malignant PolypMalignant Polyp These are cancer !!

Abnormal cells grow

o in the lining of the large intestine(colon) or rectum

Invade surrounding tissue & metastasize to other areas.


Colorectal cancer start

PolypPolyp

MalignantMalignant

PolypPolyp 6

1

2



Hereditary colorectal cancer syndromes

Scientists have discovered several genes contributing

to a susceptibility to two types of colorectal cancer

Hereditary

nonpolyposis

colon cancer) also

called Lynch

Syndrome

Hereditary

nonpolyposis

colon cancer) also

called Lynch

Syndrome

HNPCC

FamilialFamilial

adenomatousadenomatous

polyposispolyposis

FamilialFamilial

adenomatousadenomatous

polyposispolyposis

FAP

FAP susceptibility gene with a specific

chromosomal locus (apc) that is mapped within

5q15-q22{ Chromosome 5 th : ------- At locus query 15-22 } The occurrence within a family of many cases of CRCwithout polyposis,

We are considered HNPCC by the pedigree in apattern consistent with an autosomal-dominant trait

http://www.genome.gov/10000466#al-2



8FAP = familial adenomatous polyposis; GI = gastrointestinal

Source :Vogelstein B, Kinzler KW: Cancer genes and the pathways they control. Nat Med 10 (8): 789-99, 2004.

Hereditary colorectal cancer genes

Table. Show the Hereditary colorectal cancer genes



Pathways Progression of Colorectal cancer

9




Staging for Colorectal cancer

Figure : Staging systems for colorectal cancer

(AJCC, American Joint Committee for Cancer;IUAC,

International Union Against Cancer.)

10

http://www.about.com/

Stage I Cancer has grown through the inner layer of

the colon or rectum

Stage II Cancer has grown through or into the wall of

the colon or rectum: not reached the nearby lymph

nodes yet.

Stage III Cancer has metastasized to nearby organs

and lymph nodes near to the colon or rectum

Stage IV Cancer has metastasized to distant organsspread to other parts of the body such as spread to

liver ,lung, or ovary and brain



11

Nonsteroidal anti-inflammatory drugs

(NSAIDs)

cyclooxygenase-1(Cox-1)

Aspirin

Cyclooxygenase-2(Cox-2)

Celecoxib

Ibuprofen Rofecoxib

Etc. Etc.

InhibitProstaglandin(PG)

Cox-2 (Celecoxib)



12

Selective COX-2 inhibitor

(A) Nonselective inhibitors have access to the binding channels of both isoforms.

(B) The more voluminous residues in COX-1, Ile434, His513, and Ile532, obstruct access of the bulky

side chains of COX-2 inhibitors.



13

Prostaglandin Biosynthesis

Cox-2 (Celecoxib)



Celecoxib (cox-2 inhibitor)

CelecoxibCelecoxib

14

Coxibs : Pharmacologic Characteristics



Normal colon epitheliumCOX-2 protein staining in colon tumor

15

Cox-2: Overexpressed in colon tissues



Celecoxib (Cyclooxygenase-2)

Immune Function

Carcinogenesis

Prostaglandins (PGE2)

ArachidonicAcid

Free Radical Production

Carcinogen Activation

Proliferation

Angiogenesis

Apoptosis

COX-2

COX

inhibitor

COX

inhibitor XXXX COX-1

Sphingomyelin Ceramide

Non-COX Targets:

qP450s

qPPARF /KoPPARE,K

XX 16



Gene expression profiling

17

GeneGene

ExpressionExpression

ProfilingProfiling

Measurement of the activityMeasurement of the activity (the expression)(the expression)

of thousands of genes at onceof thousands of genes at once

Example for profile :Example for profile : distinguish between cells that are actively dividing

show how the cells react to a particular treatment

A genomic techniqueA genomic technique based on the cell expressed

transcribed into messenger RNA (mRNA).



Microarray

18

What is a microarray?

A tool for analyzing gene expression

Consists small membrane or glass slide

Glass slide contain samples (genes)

22 main types of microarraymain types of microarray

Affymetrix arrays ± single channel

(background-green, foreground-red)

cDNA arrays ± two channel (red, green, yellow)



19

Affymetrix Array

GeneChips

Affymetrix : Prob



20

Collect microarray

data

cDNA microarray method

Affymetrix Array [cont.]

Platform

HG_ U95Av2 platform

HG_U133Av2 platform



21

Materials and methodMaterials and method



Materials and methods

Patients undergoing toWashington University

School of Medicine's

Human colorectal adenocarcinoma patients

22



Materials and methods[cont.]

Eligible Patients

After collect patients

Exclude patients

Take aspirin and NSAIDS

23




EligibleEligible

PatientsPatients((2323 patients)patients)

Random patientsRandom patients

to receiveto receive

celecoxibcelecoxib drugdrug

23 patients

11 patientsReceive celecoxib

drug

12 patientsNot receive

celecoxib drug

21




Review Bords

11patients

12patients

25




Celecoxib drug

26

1st 2nd 7thDay

200200

mgmg

200200

mgmg

400 mg



Identified difference genes

Using GC-RMA algorithm

Gene Expression Omnibus(GEO){GSE11237}

Hierarchical cluster

Volcano plot

Normalization

Microarray raw data

Gene annotation enrichment analysisGene annotation enrichment analysis

Separate groups fromdifference genes

Microarray analysis


27




Microarray raw data

GSEGSE1123711237

DownloadDownloadmicroarray datamicroarray data

Import microarray data intoImport microarray data into GenespringGenespring GXGX77..33

http://www.ncbi.nlm.nih.gov/geo/

http://www.chem.agilent.com/en-US/Products/software/lifesciencesinformatics/genespringgx/Pages/default.aspx 28



Normalization


29

Processing of reduce unwanted variation across chips

May use normalization information from multiple chips

Pre-Normalisation Post-Normalisation

Improve form of RMA(Robust Multiarray Averaging )

GC-RMA use the sequence-specific probe affinities of the GeneChip probes

GeneChip Robust Multiarray Averaging:GC-RMA



30

Improve form of RMA(Robust Multiarray Averaging )

GC-RMA use the sequence-specific probe affinities of the GeneChip probes

GeneChip Robust Multiarray Averaging:GC-RMA


MM data adjusted based on probe affinity, then subtracted from PM.

Does not lose MM data.

Pic MM PM



Volcano Plot


The Scatter plot

showing

differential

genes expression Absolutefold change

(AFC > 1.5) Fisher's exact test(p-value <0.05)

Example : Volcano plot

31

Statistical measure





Used for clustering the different of gene expression data

Hierarchical cluster often good for visualization

32

Outlines

Algorithm for hierarchical cluster

Distance measure



33

Algorithm for hierarchical cluster


Complete Linkage Clustering (or the farthest neighbor method)

Calculating distance between clusters

Calculate distance by linkage function

),(max),( , y xd Y X D Y y X x !

),( y xd X Y

§

!

n

i y x

ii y y x x

n y xd

1

))((

1

1),(

W W

Pearson

Correlation



34

Calculate all pair-wise distances

Join the closest pair

Agglomerative method

Calculate pair¶s distance to all others

Repeat from 2 until all joined

1

2

3

4







35







36





37





38





39





40



Gene annotation enrichment analysis

41http://david.abcc.ncifcrf.gov/content.jsp?file=functional_annotation.html

The tool suite introduced in theThe tool suite introduced in the

first version of DAVIDfirst version of DAVID

Highlight genes most relevantHighlight genes most relevant

GO termGO term

Analysis gene have enrichmentAnalysis gene have enrichment

in batch annotation and genein batch annotation and gene--GOGO

Annotation coverage

Analyze genes

In different biological

process

Aspects their genes in

a single space

(chart report or pathway)



42

Gene annotation enrichment analysis [cont.]

FunctionalFunctional

Anannotation Anannotation

Analysis AnalysisConclude:Using fisher µs exact test for adopt to measure the

gene-enrichment in annotation terms

Using Fisher¶s exact test

Determine the proportion of those genes

Proportion value fall into each category

differs by group



43

Fisher¶s exact test

The score for a GO term is the degree of independence between the two

properties:

A = {gene is in the list of significant genes}

B = {gene is found in the GO term}.

Testing the independence of two groups in the above contingency table corresponds

to Fisher¶s exact test [Khatri and Draghici, 2005]

Gene annotation enrichment analysis [cont.]



44

Fisher¶s exact test (cont.)


Define variables

N = genes are onmicroarray

Bio is a GO term

M genes in Bio

N íM genes notin Bio

K be the number of significant genes

probability of getting exactly x by chance

(not what we want)

Text in hereCompute the significance of a gene set :Compute the significance of a gene set : HypergeometricHypergeometric testtest



45

ResultsResults



3

Hierarchical

clustering

of 190 genes

4

Gene annotation

enrichment

analysisVolcano plot

N=190 geneso 35 higher expression

o 155 lower expression

21

Unsupervised

hierarchical clustering

N=10,083 genes

46

Results



Results[cont.]

Volcano plot

Fig 2 A.

Volcano plot of gene expression changes between colorectal adenocarcinomas from patients pre-treated

and not pre-treated with celecoxib.

Increase expression

Decrease expression

No difference

Excel File

47



Results[cont.]

Fig 2 B. Clustering using the 190 differentially expressed genes.

In sub-cluster A

8/ 9 tumour samples** receiving no celecoxib pre-treatment

6/7 tumour sample

** receiving celecoxib pre-treatment

4 patients receiving celecoxib 3 patients receiving no drug.

In sub-cluster B

In sub-cluster C

Results[cont.]

Hierarchical clustering

Black

to the

Results

48



Results[cont.]


Genes alter for Genes alter for celecoxibcelecoxib

prepre--treatment for treatment for 77 daysdays

23 genes,

involve in cellular

lipid metabolism

4 genes involve in

glutathionemetabolism

19 genes involve in

cell adhesnion

Black

to the

Results

49



50

Discussion and ConclusionDiscussion and Conclusion



No obviousAgreement with

previous studyLow stringency cut-off

Usefulness Celecoxib & Time

for treat

Conclusion

Gender

Tumour site

Clinical stage

Celecoxib

pre-treatment status

Celecoxib

decrease expression of

glutathione metabolism

Achieve the goal of this

pilot study

Identify gene expression

alterations associate with

celecoxib

7 days vs 12 months

190 and over 1,000 genes

difference expression for time

respectively

No genes alteration involve

in transforming growth

factor- signaling

Pharmacology develop adjuvant

chemotherapy by using celecoxib

drug

But previous research reported

celecoxib increase risk for

cardiovascular side effect

Recent phase II study : combine

celecoxib and 5-fiuorourcil

chemotherapy

mediate cytotoxicity

Discussion

ene alteration (in ol e)

Li i + l t t i

t li

ll i

51



Conclusion

Results suggest

Gene expression alterations celecoxib drug

Research team suggests

Celecoxib reduces the proliferative capacity of colorectal adenocarcinomasin short-term study

Indicative decreased cellular lipid and glutathione metabolism

Should provide the impetus for larger studies in the future

52

Either using celecoxib treatment alone or in combinationwith other chemotherapeutic regimens.



53



54

Adjusts for background intensities

Reduces noise

More accurate gene expression

values.

Exclude genes expression standard

deviation < 0.04 across all sample

Advantages of GC-RMA method


Documents

Celecoxib Pre-treatment in Human Colorectal Adenocarcinoma Patients Is_No Cut Ppt