Presenter:Presenter: Venkat Rao Katkoori., Ph.D.Venkat Rao Katkoori., Ph.D.

AuthorsAuthors:: Venkat R. Katkoori1, Xu Jia1, Tom Callens2, Ludwine Messiaen2, Wen Wan3, Sreelatha Meleth3, Harvey Bumpers4 and Upender Manne1.

Departments of Pathology1 and Genetics2 and Medicine3, University of Alabamaat Birmingham;

Department of Surgery 4, Morehouse School of Medicine, Atlanta, GA.

Prognostic Significance of p53 Codon 72Polymorphism Differs with Race in Colorectal

Adenocarcinoma

Normal

Epithelium

Abnormal

Crypts Foci

Early

Adenoma

Late

Adenoma

In situ

Carcinoma

Invasive

Carcinoma

Metastatic

Carcinoma

APC,

5q Loss

DCC/Smad4

18q Loss

p53, BAT-26

17p Loss

Many genes

8p Loss

Many genesCell

Adhesion

Many decades 2 - 5 years

2 - 5 years

A:A: Chromosomal Instability (CIN) Pathway (Gene mutations, Chromosomal deletions)

K-Ras, β-CateninMMR (hMSH2)

B: Microsatellite Instability (MSI) Pathway(Mismatch Repair Inactivation)

Multi-Step Molecular Pathways of Colorectal Multi-Step Molecular Pathways of Colorectal CarcinogenesisCarcinogenesis

Prognostic Molecular Markers of SporadicPrognostic Molecular Markers of SporadicColorectal AdenocarcinomaColorectal Adenocarcinoma

Promising Prognostic MarkersPromising Prognostic Markers: : Microsatellite Instability &Microsatellite Instability &18q deletions18q deletions

Candidate Prognostic Markers:p53, p27kip-1, Bcl-2, Bax, MUC1 & RPH3AL (Rabphillin-3A-like),

Potential Prognostic Makers: MUC4, MUC17, VEGF,EGFR, c-erb-2, TGF-α, COX-2,TP, TS, DPD, 17p deletions17p deletions

STUDY RATIONALESTUDY RATIONALE

PREMISEPREMISE: Several studies from ourlaboratory as well as others have demonstratedthat prognostic value of molecular makers differbased on tumor location, tumor stage and/orpatient race/ethnicity in colorectal cancer(CRC).

AIMAIM: To identify race-specific prognosticmolecular markers in CRCs, first we evaluatedmutational patterns of the p53 gene andcorrelated with patient outcomes based onrace.

Patient SelectionPatient Selection

CRC and corresponding normal tissues collected from373 patients (137 African-Americans patients & 236 non-Hispanic Caucasians patients).

Patients who underwent surgery with curative or palliativeintent at UAB Hospital during 1984-1995.

We selected only patients who have not received anypre- or –post surgery adjuvant therapy.

Patients with family or personal history were excluded. Patient demographics, pathologic, and clinical

information was extracted from medical records andphysicians charts

The follow-up information was collected from the UAB-Tumor Registry

Microsatellite Instability AnalysisMicrosatellite Instability Analysis

Microsatellite instability was analyzed at BAT25, BAT26, D2S123,D5S346 & D17S250 loci in 373 CRCs and marching normal tissues

BAT-25 by ABI

Bat 25 InstableBat 25 Stable

Bat 25 InstableBat 25 Stable

BAT-25 by Light Cycler

BAT-26 by Light Cycler BAT-26 by ABI

Microsatellite Instability & RaceMicrosatellite Instability & Race

Similar incidence of MSI-H has been observed betweenSimilar incidence of MSI-H has been observed betweenAfrican-American and Caucasian patientsAfrican-American and Caucasian patients

+1 Coding Region

Exons 1 to 11

5’ 3’

+ 1179+1

Analysis of Analysis of p53p53 in CRCs based on Race in CRCs based on Race

A] Sequence Analysis exon 4 through 9

B] Frequency of p53 mutations Codon wise by race

P53 mutations have been associated with poor survival of African-American(p= 0.001) and Caucasian (p= 0.028) patients with CRCs

African-American Patients Caucasian Patients

Survival Analysis based on p53 Status in CRCsby race

Codon 72 polymorphism of p53Codon 72 polymorphism of p53

SNP at codon 72 of p53 results in thesubstitution of arginine for proline

Susceptible for p53 mutations and Increaseddegradation of p53 protein

Increased tumorigenecity and oncogenic effect Uncontrolled malignant process and Increased

invasiveness and metastasis Risk of cancer progression and poor prognosis and Resistance to anticancer therapy

Analysis of genotype status at Codon 72 of p53By RFLP and Sequencing

M = Marker (100bp ladder)Lane 1 = G/G homozygous (Arg/Arg phenotype)

(160 and 119bp)Lane 2 = G/C heterozygous (Arg/Pro phenotype)

(279bp, 160 and 119bp)Lane 3 = C/C homozygous (Pro/Pro phenotype)

(279bp)

SequencingSequencing

PCR-RFLPPCR-RFLP

Genotype G/GHomozygous

Genotype G/CHeterozygous

Genotype C/CHomozygous

Codon 72 Phenotypes of p53 and Race &Tumor Characteristics

Pro/Pro phenotype is correlated with nodal metastasis tumor gradeand higher incidence of p53 mutations in African American patients

Hypothesis: The proline/proline phenotypes areassociated with aggressive tumor behavior.

Since higher frequency of proline/prolinephenotypes and these exhibited higher incidenceof p53 mutations, we analyzed their prognosticvalue in relation to other phenotypes(proline/arginine and arginine/arginine) based onrace .

Survival Analysis based on phenotypes codon 72polymorphism of p53 in CRCs by race

African-American Patients Caucasian Patients

Pro/Pro phenotype of P53 at codon 72 is associated with poor survival Only in African-American patients (p= 0.005), but not in Caucasian ( p= 0.886)patients with CRCs

Cox regression analysis to determine prognostic significance of p53 codon 72 phenotypes

Pro/Pro phenotype is an independent prognostic marker Only in AfricanAmerican patients with CRCs

Mutational spectra of p53 in African-American and Caucasian patients in relation to Pro/Pro phenotype

Pro/Pro phenotype of P53 at codon 72 is associated with higher frequency of P53 mutations particularly disruptive type only in in African-American patients[Differential mutational spectra in relation to Pro/Pro phenotype]

ConclusionsConclusions

p53 mutational pattern and prognostic value ofcodon 72 polymorphism differs with race incolorectal adenocarcinomas.

The incidence of Pro/Pro phenotype at codon 72of p53 is higher in African- American patients andassociated with higher incidence of p53mutations

Pro/Pro phenotype is associated with and poorclinical outcome of African-American but notCaucasian patients with CRCs.

Mentor: Upender Manne, Ph.D.Colleagues : Xu Jia, MD

Chandra Kumar , MD

Collaborators: Wen Wan, Ph.D.Sreelatha Meleth, Ph.D

Harvey Bumpers, MD,FACS. William E. Grizzle, MD, Ph.D.

Grant Support: RO1-CA98932-01 (NIH/NCI)2U54-CA118948-03 (NIH/NCI)

AcknowledgementsAcknowledgements