Tissue biomarkers (BIOM) in colon cancer (COC): The translational study on the randomized phase III trial comparing infused irinotecan / 5-fluorouracil (5-FU)/folinic acid (FA) to 5-FU/FA in stage II-III COC patients (pts) (PETACC 3 - EORTC

40993 -SAKK 60-00)

A.D. Roth1, S. Tejpar2, P. Yan3, R. Fiocca4, D. Dietrich5, G. Bodoky6, R. Labianca7, D. Cunningham8, E. Van Cutsem2, F.

Bosman3

1Oncosurgery, Geneva University Hospital, Geneva, Switzerland, 2Digestive Oncology Unit, University Hospital Gasthuisberg, Leuven, Belgium, 3Dpt of Pathology, Lausanne University, Lausanne, Switzerland, 4Dpt of Surgical and Morphological Sciences, University of Genova, Genova, Italy, 5Swiss Group of Clinical Cancer Research, Bern, Switzerland, 6Oncology, St Lazlo Hospital, Budapest, Hungary, 7Unit of Medical Oncology, Ospedali Riuniti, Bergamo, Italy, 8Medical Oncology, The Royal Marsden Hospital, Sutton, United Kingdom.

Rationale

• New prognostic markers are eagerly needed to better select patients with colon cancer (COC) who need adjuvant chemotherapy

• The value of tissue biomarkers in COC in adjuvant setting is still a matter of debate because of lack of validation in large data sets

• We took advantage of PETACC 3, a large adjuvant trial with 3005 patients,to assess in a large cohort the prognostic and predictive value and the individual interactions of tissue biomarkers selected on the basis of their prognostic potential according to the results of previous smaller studies

Objectives

• To assess the feasibility of biomarker analysis on formalin fixed paraffin embedded (FFPE) routine material prospectively collected in a large international study with 386 centers

• To evaluate or confirm the prognostic relevance of selected biological markers in the whole patient population using DFS and OS as endpoints

• To assess the predictive effect of some of them on the impact of a chemotherapeutic regimen including irinotecan in addition to 5-FU on disease free survival (DFS) and overall survival (OS).

Methods (1)

• FFPE tissue blocks prospectively collected and cut in 5-20µ sections

• Immunohistochemistry (IHC)– P53: mouse mAb clone D07, ABC Basic DAB Detection (Ventana

medical system)– SMAD4: mouse mAb clone B8 (IgG1, Santa Cruz Biotechnology).

Novocastra polymer detection kit– Thymidylate Synthetase (TS): Monoclonal antibody TS 106/4H4B1

(IgG1, Zymed). DAKO EnVision detection system– Telomerase (HTERT): Monoclonal antibody NCL-hTERT (IgG2,

Novocastra). DAKO EnVision detection system

Methods (2)

• DNA was extracted with phenol/chloroform from normal (Nor) and tumoral (Tu) tissues after microdissection of FFPE sections.

• Molecular analysis:– Microsatellite Instability (MSI): assessed on 10 markers (BAT-25,

BAT-26, D5S346, D2S123, D17S250, BAT-40, TGF-ß RII, D18S58, D18S69, D17S787)

– 18q and 8p LOH: multiple SNPs typing by pyrosequencing on Nor/Tu DNA

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

– UGT1A1 7/7 genotype: PCR and fragment sizing on Nor DNA

Statistics

• Determination of cutpointsFor markers that have no established cutpoints, potential cutpoints are selected based on the Martingale residuals from a Cox regression of disease-free survival time

• Associations between two categorical variables were examined by chi-squared or Fisher’s exact test

• Disease-free survival times of different groups were estimated by Kaplan-Meier method and examined by log-rank test

• The associations between specific toxicities (diarrhea, febrile neutropenia, neutropenia) and UGT1A1 genotype are examined by logistic regression

Statistical constraints

3-year DFS

Proportion of marker Positive specimens (M+)

M+ M- HR

10% 20% 30% 40% 50% 60% 70% 80% 90% 70% 60% 0.70 901 476 340 278 249 241 255 310 508 70% 55% 0.60 494 255 178 141 123 116 118 139 218

Required number of events for a power of 80% (significant level 5%)

Number of patients and events available for this preliminary analysis

UICC stage II UICC stage III UICC stage II+III Marker

Patients Events Patients Events Patients Events Telomerase 243 31 544 187 787 218 TS 383 54 826 280 1209 334 SMAD4 416 63 962 340 1378 403 P53 414 62 968 342 1382 404 MSI 394 58 857 299 1251 357 KRAS 404 59 892 308 1296 367 BRAF 406 59 898 311 1304 370 LOH in 18q - at least one snip ok

368

55

807

285

1175

340

UGT1A1 391 - 868 - 1259 - Total 419 63 982 346 1401 409

Analysis success rate

Number of samples with results

Total analysed: -1530 patient slides analysed by IHC- DNA successfully extracted from 1401 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%

UGT1A1 1335 95%

Biomarker alteration (mutation, expression or deletion) observed

Alterations rate observed

Alteration rate reported (literature)

P53 overexpression* 37% 25-76%

SMAD4 loss** 15% 13-63%

TS*** 48% No consistent data available

HTERT 48% No data available

MSI 15% 10-17%

18q 65% 70%

KRAS 37% 32-40%

BRAF 8% 10%

UGT1A1 (7/7 genotype) 12% 10-15%

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

Patterns of SMAD-4 expression in colon cancer: (a) complete loss of expression in tumor glands as compared with normal crypts (arrow); (b) non-homogeneous expression: loss of expression in the lower part of the field contrasts with marked expression in the upper part.

SMAD4: Preliminary results (stage III)

Cutpoint % patients Estimated 3-year DFS1 Log-rank ≤ cutpoint ≤ cutpoint > cutpoint p-value 0 15% 53% 68% <0.001

0 500100015000.40.50.60.70.80.91.0

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Day:

Proportion disease freeno expressionexpression present

At risk:145 95 57 2817 663 449 38

MSI: Preliminary results (stage III)

0 500100015000.50.60.70.80.91.0/

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Day:

Proportion disease freestable/lowhigh

At risk:753 595 396 33104 89 61 3 % patients Estimated 3-year DFS1 Log-rank

high stable/low high p-value 12% 65% 74% 0.04

Discussion

• The high success rate of the analysis (>80%) shows for most biomarkers that it is possible to conduct large translational studies on routine FFPE material

• The confirmed correlation between the biomarker alteration rates found with those published confirm the methodological reliability of our study

• Preliminary results show an improved prognosis in MSI high tumours and an impaired prognosis for tumours not expressing SMAD4 (a marker associated with 18q deletion), results which are in accordance with the literature

• Because of statistical constraints final analysis cannot be performed before additional follow up data are available

Conclusions

• Preliminary results show that this translational study which is so far the largest conducted prospectively in stage II-III colon cancer will be able to add significant information regarding the prognostic and predictive strength of the biomarkers tested

• A complete analysis including a multivariate analysis shall be conducted when updated data on DFS and OS shall be available from the clinical study