Taiwan 2000PETACC 3 ASCO 2009

Molecular markers in colon cancer have a Molecular markers in colon cancer have a stage specific prognostic value. Results of stage specific prognostic value. Results of the translational study on the PETACC 3 - the translational study on the PETACC 3 -

EORTC 40993 -SAKK 60-00 trial.EORTC 40993 -SAKK 60-00 trial. A. D. Roth, S. Tejpar, P. Yan, R. Fiocca, D. Dietrich, M. Delorenzi, R. Labianca, D. Cunningham, E. Van

Cutsem, F. Bosman

PETACC 3 ASCO 2009

Rationale

• Need for new prognostic markers to guide treatment planning in colorectal cancer

• Lack of large data sets for the validation of potential markers

• PETACC 3: a large adjuvant trial in colon cancer where the pathological material of 1564 patients out of 3278 was prospectivelyprospectively collected– 20-25 slides/patient or block in bank => ~ half a ton of materialhalf a ton of material

(about 36,000 slides)(about 36,000 slides)– 669 blocks processed for tissue microarray banking

PETACC 3 ASCO 2009

Objectives

• To assess the frequency of molecular marker alterations: expression of p53, SMAD4, thymidylate synthetase (TS) and hTERT, and mutations of KRAS, BRAF, microsatellite instability (MSI) and 18qLOH in stage II and stage III colon cancer.

• To assess their prognostic relevance in the whole patient population using RFS as endpoint.

• To assess possible differences between stage II and stage III disease.

PETACC 3 ASCO 2009

Methods

• FFPE tissue blocks collected and cut in 5-20µ sections. • DNA extracted with phenol/chloroform from normal and

tumoral tissues after microdissection.

• Immunohistochemistry (IHC)Immunohistochemistry (IHC)

– P53, SMAD4, Thymidylate Synthetase (TS), Telomerase (HTERT)

• Molecular analysis:

– Microsatellite Instability (MSI): assessed on 10 markers (Cf. previous presentation)

– 18qLOH: multiple SNPs typing by pyrosequencing on Nor/Tu DNA

– KRAS exon 2 and BRAF exon 15: Allele specific real time PCR on Tu DNA

PETACC 3 ASCO 2009

Statistical Methods

• Prognostic value of the markers was analyzed per stage by Cox regression for RFS

• Interactions were tested using a likelihood ratio test

• p values of the multivariate analysis taken from the full model (unless indicated otherwise)

• Differences between the stages were assessed by a test of equal proportions

PETACC 3 ASCO 2009

Analysis success rate

Number of samples with results

Total analysed: -1530 patient slides analysed by IHC- DNA successfully extracted from 1404 patient slides (91.2%)

Marker Samples Success rate (%)

Telomerase 826 54% TS 1269 83% SMAD4 1443 94% P53 1447 95% MSI 1327 94% KRAS 1379 98% BRAF 1386 99% LOH in 18q - at least one snip ok

1220

87%

PETACC 3 ASCO 2009

Biomarker alteration observed

Alterations rate observed

Alteration rate reported (literature)

p53 overexpression* 35% 25-76%

SMAD4 loss** 21% 13-63%

TS*** 34% No consistent data

hTERT 46% No data available

MSI 15% 10-17%

18qLOH 68% 70%

KRAS 37% 32-40%

BRAF 8% 10%

* Intense expression, More than 45% cells positive. ** any loss.*** Positive = more than 25% cell positive.

PETACC 3 ASCO 2009

Marker Alteration Rate per stage

Marker Stage II (n=420)

Stage III (n=984)

p value*

MSI-H 22% 12% <0.0001

TS 43% 29% <0.0001

p53 30% 37% 0.01

SMAD4 18% 23% 0.03

18qLOH 63% 70% 0.04

hTERT 41% 48% 0.06

KRAS 35% 37% 0.81

BRAF 8% 8% 0.90

* Test of equal proportions

PETACC 3 ASCO 2009

Prognostic Value Univariate analysis

MarkerStage II (n=420) Stage III (n=984)

Interaction*HR§ p val** HR§ p val**

MSI (Hi vs Stable) 0.3 0.004 0.7 0.06 0.04

18qLOH 2.1 0.03 1 0.91 0.05

SMAD4 (any loss) 1.4 0.21 1.6 <0.0001 0.23

hTERT (High) 1.4 0.32 1.5 0.01 0.92

p53 (High) 1.0 0.98 1.3 0.03 0.37

TS (High) 0.5 0.03 0.7 0.02 0.30

KRAS (Mutated) 1.1 0.84 1.0 0.72 0.32

BRAF(Mutated) 0.9 0.90 1.2 0.28 0.38

* p value from Likelihood ratio test to assess differences between the stages** p values from the Wald test in a univariate Cox regression§ HR = hazard ratio

PETACC 3 ASCO 2009

Prognostic Value (RFS)Multivariate Analysis in whole population

MarkersStage II Stage III

HR§ p value* HR§ p value*

T Stage (T4 vs T3) 2.8 0.0001 1.6 0.0006

N Stage (N2 vs N1) N/A N/A 2.2 <0.0001

Histologic Grade (3-4 vs 1-2) 0.6 0.55 1.4 0.07

Age (>60 vs ≤60) 1.8 0.026 1.1 0.3

MSI (High vs Stable) 0.3 0.027 0.7 0.12

p53 (High) 0.7 0.27 1.3 0.015

SMAD4 (any loss) 1.0 0.9 1.6 0.0002

Treatment, Sex, Site, KRAS, BRAF,TS, 18qLOH (Stage II: HR 1.4, p=0.33), hTERT: not significant* p values from the Wald test in a multiivariate Cox regression§ HR = hazard ratio

PETACC 3 ASCO 2009

MSI and 18qLOH in a minimal multivariate model in stage II disease

Markers HR [95% CI] P value

T4 v. T3 2.34 [1.42 - 3.84] 0.00085

18qLOH 2.02 [1.03 - 3.96] 0.041

T4 v. T3 2.58 [1.56 - 4.28] 0.00024

MSI-H v. MSS 0.28 [0.10 - 0.72] 0.0089

18qLOH 1.37 [0.67 - 2.77] 0.38

PETACC 3 ASCO 2009

Graphical comparison between univariate and multivariate analysis

PETACC 3 ASCO 2009

E5202: A Pioneer study!

PETACC 3 ASCO 2009

Molecular predictors of survival after adjuvant chemotherapy for

colon cancer Watanabe T. et al NEJM 344: 1196-206 (2001)

“No marker had predictive value in the analysis of 121 patients with stage II cancer, possibly because

of the small sample”

PETACC 3 ASCO 2009

Really low risk?Really low risk?

PETACC 3 ASCO 2009

PETACC 3: RFS according to MSI or 18qLOH in stage II colon cancer

MSI 18qLOH

PETACC 3 ASCO 2009

PETACC 3: Additional prognostic value of 18qLOH on MSS and MSI-H tumors in stage II disease

• MSI-H population:– 18% 18qLOH (9/51)!– 18qLOH prognostic

value not assessable

P = 0.527

PETACC 3 ASCO 2009

Conclusions

• Stage II and III colon cancers express different biomarker alteration frequencies with different prognostic effects according to disease stage

• These data suggest that pooled analysis of stage II and III might be confounded by this heterogeneity

• The possibility that stage II and III are biologically different rather than sequential evolution steps in a pathological continuum needs to be considered

• Careful assessment of the relative weight of each prognostic marker in multivariate analysis according to disease stage is needed before translating them into clinic

PETACC 3 ASCO 2009

Thank You For All Your Efforts!!

Austria, Belgium, Bulgaria, Croatia, Czech Republic, Denmark, Egypt, Finland, France,

Germany, Greece & Cyprus, Hungary, Ireland, Iceland, Israel, Italy, Netherlands, Norway, Poland, Portugal,

Russia, South Africa, Slovakia, Slovenia, Spain, Sweden, Switzerland, Taiwan, Turkey & UK

We would also like to thank Pfizer for facilitating the execution and analysis of the PETACC3 study