Chromosomal deletion as predictors Chromosomal deletion as predictors for recurrence of early stage CRCfor recurrence of early stage CRC

Wei Zhou, Ph.D.Wei Zhou, Ph.D.

Winship Cancer InstituteWinship Cancer InstituteOctober 2, 2005October 2, 2005

Falk Symposium 149: Highlights in GI Oncology

Colorectal Carcinoma

Early Stage Colon CancerEarly Stage Colon CancerSurgerySurgery

Colonoscopy with Colonoscopy with polypectomypolypectomySurgical resectionSurgical resection

Adjuvant TherapyAdjuvant Therapy55--FU/Leucovorin FU/Leucovorin IrinotecanIrinotecan (CPT(CPT--11), 11), OxaliplatinOxaliplatin, , C225C225Radiation therapyRadiation therapy

Adjuvant Therapy for Early Stage CRC?

Over 70Over 70--80% 580% 5--year survival with surgery alone year survival with surgery alone

Only benefit a small proportion of total Only benefit a small proportion of total populationpopulation

Risk, side effects and costs of adjuvant therapyRisk, side effects and costs of adjuvant therapy

Large number of Large number of noncancernoncancer related deathrelated death

Treatment Benefit in Dukes’ B Patients

No. of 5-year 5-yearPatients DFSsurvival

Surgery 509 73% 80%

5-FU+LV 507 76% 82%

FU: fluorouracil, LV: leucovorinBased on four National Surgical Adjuvant Breast and Bowel Project (NSABP) Clinical Trials (C-01, C-02, C-03 and C-04)Buyse & Piedbois, Seminars in Oncology, Vol 28, No.1 Suppl (Feb) 2001, pp20-24

How to Predict Patients at High Risk of Recurrence?

Colorectal Carcinoma

MIN and CIN Colorectal Cancers

MIN: Microsatellite Instability (MSI)MIN: Microsatellite Instability (MSI)Diploid Tumors (Near diploid tumors)Diploid Tumors (Near diploid tumors)~13% in colorectal, endometrial and gastric ~13% in colorectal, endometrial and gastric cancerscancers<2% in other cancer types<2% in other cancer types

CIN: Chromosome instabilityCIN: Chromosome instabilityAneuploidAneuploid TumorsTumors~85% in colorectal cancer~85% in colorectal cancer

Disease Prognosis in MIN CRC tumors

Better Overall survivalBetter Overall survivalHR=0.65; 95% CI, 0.59HR=0.65; 95% CI, 0.59--0.710.71

Tumors with MSI derived no benefit Tumors with MSI derived no benefit from adjuvant FUfrom adjuvant FU

HR=1.24; 95% CI 0.72 to 2.14HR=1.24; 95% CI 0.72 to 2.14

Journal of Clinical Oncology, 23,(3) 2005: pp. 609-618

LOH Analysis vs Other Methods

Underestimate the true extent of allele Underestimate the true extent of allele loss byloss by

KaryotypicKaryotypic studiesstudiesCGHCGHFISHFISH

Loss of a maternal or paternal allele in Loss of a maternal or paternal allele in a tumor are often accompanied by a a tumor are often accompanied by a gain of the opposite allelegain of the opposite allele

Early stage patients without 18q loss have better survival (I)

Jen. J et al, 1994, NEJM, 331: 213-221

Early stage patients without 18q loss have better survival (II)

Jen. J et al, 1994, NEJM, 331: 213-221

Follow-up studies are controversial

Study Overall survival Disease-free survivalSample size HR9 95% CI P -value HR 95% CI P -value

Jen et al. [1994] 145 2.83 1.32-6.08 0.008Watanabe et al. [2001] 221 2.04 1.3-3.2 0.002Barratt et al. [2002] 108 0.86 0.57-1.28 0.45Aschele et al. [2004] 42 2.16 1.04-4.49 0.04Carethers et al. [1998] 70 1.22 0.32-4.72 0.84Diep et al. [2003] 184 2.7 1.33-5.48 0.006Halling et al. [1999] 386 1.2 0.81-1.76 0.37 1.25 0.81-1.91 0.32Lanza et al. [1998] 112 7.13 2.1-23.9 <0.001 5.76 2.2-15 <0.001Ogunbuyi et al. [1996] 126 2 1.26-3.86 0.003Zauber et al. [2004] 93 0.91 not reported 0.86

Popat & Houlston, Euro J. Cancer, 41, pp2060-2070

Detection of LOH by Microsatellite Analysis

Jen. J et al, 1994, NEJM, 331: 213-221

N T

LOH Analysis in Primary Tumors is NOT Reliable

Normal tissue contaminationNormal tissue contaminationDNA from nonDNA from non--neoplastic cells can easily mask neoplastic cells can easily mask LOHLOH

Tumor DNA degradationTumor DNA degradationLiu, J. Liu, J. et al.et al. Loss of heterozygosity in tumor cells Loss of heterozygosity in tumor cells requires rerequires re--evaluation: the data are biased by the sizeevaluation: the data are biased by the size--dependent differential sensitivity of allele detection. dependent differential sensitivity of allele detection. FEBS FEBS LettLett.. 462462, 121, 121--8 (1999).8 (1999).

Low DNA yield with microLow DNA yield with micro--dissection dissection techniquestechniques

Sampling errorsSampling errors

N T

Counting Alleles: Fault-Tolerant LOH Analysis

ParaffinParaffin--embedded samplesembedded samplesNot affected by PCRNot affected by PCR--bias, DNA bias, DNA degradation and normal tissue degradation and normal tissue contaminationcontamination

SingleSingle--step reaction setupstep reaction setup

QuantitativeQuantitativeRigorous mathematical toolsRigorous mathematical toolsTolerate up to 50% normal tissue Tolerate up to 50% normal tissue contamination contamination

Counting allelesC T

+A

G

AT

GC Single molecule

amplification

Molecular beacon detection

Statistical Analysis

Tolerate upto 50% contamination

CRC Patient Information

4 cohorts, 196 cases4 cohorts, 196 casesScotland, Italy, New York and Scotland, Italy, New York and BaltimoreBaltimore

DukeDuke’’s Stage A or Bs Stage A or B

Exclude HNPCC and FAPExclude HNPCC and FAP

Chromosome Markers

8p8pLost during late stage of CRC developmentLost during late stage of CRC development9 SNP markers9 SNP markers

1818Commonly lost during transition between Commonly lost during transition between early adenomas to late early adenomas to late adeonmasadeonmas11 SNP markers11 SNP markers

Data Analysis

Ch. 8p (9) & Ch. 18 (11)Ch. 8p (9) & Ch. 18 (11)

L tumors:L tumors: lost both lost both R/L tumors:R/L tumors: lost eitherlost eitherR tumors:R tumors: retain bothretain both

Allele Status and Recurrence

TypesTypes PrognosisPrognosis RecurrenceRecurrence

R tumorsR tumors ExcellentExcellent 0%0%

R/L tumorsR/L tumors intermediateintermediate 27%27%

L tumorsL tumors PoorPoor 46%46%

Allele status and Prognosis

P<0.001

L patients with Duke’s A have a worse prognosisthan R patients with Duke’s B disease

R R R/LR/L LL

DukesDukes’’ AA 0%0% 14%14% 38%38%

DukesDukes’’ BB 0%0% 30%30% 48%48%

Recurrence Rate

(P<0.002)

Why are L type tumors more likely to recur

AneuploidyAneuploidyL types tumors lose both 8p and 18L types tumors lose both 8p and 18More advance disease stageMore advance disease stageA general feature for all solid tumors???A general feature for all solid tumors???

Inactivation of TSG on 8p and 18qInactivation of TSG on 8p and 18q8p: ???8p: ???18q: DCC or DPC4/SMAD418q: DCC or DPC4/SMAD4

Disease Progression of L types tumors in other organ sites (I)

Allelic statuses of 8p and 18 does not Allelic statuses of 8p and 18 does not predict disease recurrence in stage II predict disease recurrence in stage II and III prostate tumorsand III prostate tumors

Zhou et al, The Prostate, 2004, 15 (61), 81-91

Disease Progression of L types tumors in other organ sites (II)

Morikawa et al 2005, in review

VariableNumber of

patients OR p Value 95% CI

Allelic typesR 8pR&18qR 0R/L 8pR&18qL 11 referentR/L 8pL&18qR 19 9.0 0.049* (1.17, 69.47)L type 8pL&18qL 24 6.0 0.12* (0.76, 47.22)

Based on chi-square test and Fisher's exact test* , approximate 95% confidence intervalsAI = allelic imbalance, R = retention/ no allelic imbalance, OR = odds ratio, CI = confidence intervals

No Distant Relapse Distant Relapse

n/a n/a

9

101015

19

Allelic status distribution in relation to distant relpase in IDC

Why are L type tumors more likely to recur

AneuploidyAneuploidyL types tumors lose both 8p and 18L types tumors lose both 8p and 18More advance disease stageMore advance disease stageA general feature for all solid tumors???A general feature for all solid tumors???

Inactivation of TSG on 8p and 18qInactivation of TSG on 8p and 18q8p: ???8p: ???18q: DCC or DPC4/SMAD418q: DCC or DPC4/SMAD4

Counting alleles in CRC

18q loss associates with vascular 18q loss associates with vascular invasion in early stage colorectal cancerinvasion in early stage colorectal cancer

Zhou W, et al. Nat Zhou W, et al. Nat BiotechnolBiotechnol 2001;19(1):782001;19(1):78--81.81.

Conclusions

Counting alleles provides a sensitive and Counting alleles provides a sensitive and reliable approach for assessing LOH in clinical reliable approach for assessing LOH in clinical samples.samples.

Quantified assessment of AI of 8p and 18 can Quantified assessment of AI of 8p and 18 can identify subgroups of patients in whom tumors identify subgroups of patients in whom tumors are likely or not likely to recurare likely or not likely to recur

AI of 8p and 18 are probably associated with AI of 8p and 18 are probably associated with inactivation of tumor suppressor genes not a inactivation of tumor suppressor genes not a simple reflection of simple reflection of aneuploidyaneuploidy

Improving the “counting alleles” method

Emulsion PCREmulsion PCR

Dressman, et al, PNAS, 100 (15): 8817-8822

High Density SNP arraysThe The GeneChipGeneChip®® Human Mapping 100K SetHuman Mapping 100K Set

Two arraysTwo arraysExtensive genomic coverage (26 Kb/marker)Extensive genomic coverage (26 Kb/marker)One primer setOne primer set

ApplicationsApplicationsLOH analysisLOH analysisCopy number analysisCopy number analysis

ChallengeChallengeTissue DegradationTissue DegradationTissue contaminationTissue contamination

School of Public HealthRobert LylesMichael GoodmanJack Mandel

Johns HopkinsBert VogelsteinKenneth W. KinzlerSteven N. GoodmanElizabeth A. MontgomeryIeMing ShihKatharine E. RomansMichael A. Choti

Second University of Naples, School of Medicine, ItalyGennaro GaliziaEva LietoFrancesca Ferraraccio

University of EdinburghMedical School, UKColin A. PurdieJuan PirisRobert MorrisDavid J. Harrison

Memorial Sloan-KetteringPhilip B. PatyAl Culliford

University Hospital AntwerpCecile Colpaert

Funding SupportACS, DoD, Georgia Cancer Coalition

AcknowledgementsZhou LabDiansheng ZhongAki MorikawaMichael GuoXiuju LiuTanisha Williams

PathologyMahul AminMilton DattaSharon Lim

UrologyJohn PetrosKyle RusthovenKathy Lachenmyer

Atlanta VAVijay VarmaTheresa Gillespie