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Tissue biomarkers (BIOM) in colon cancer (COC): The translational study on the randomized phase III trial comparing infused irinotecan / 5-fluorouracil.

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Presentation on theme: "Tissue biomarkers (BIOM) in colon cancer (COC): The translational study on the randomized phase III trial comparing infused irinotecan / 5-fluorouracil."— Presentation transcript:

1 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. Roth 1, S. Tejpar 2, P. Yan 3, R. Fiocca 4, D. Dietrich 5, G. Bodoky 6, R. Labianca 7, D. Cunningham 8, E. Van Cutsem 2, F. Bosman 3 1 Oncosurgery, Geneva University Hospital, Geneva, Switzerland, 2 Digestive Oncology Unit, University Hospital Gasthuisberg, Leuven, Belgium, 3 Dpt of Pathology, Lausanne University, Lausanne, Switzerland, 4 Dpt of Surgical and Morphological Sciences, University of Genova, Genova, Italy, 5 Swiss Group of Clinical Cancer Research, Bern, Switzerland, 6 Oncology, St Lazlo Hospital, Budapest, Hungary, 7 Unit of Medical Oncology, Ospedali Riuniti, Bergamo, Italy, 8 Medical Oncology, The Royal Marsden Hospital, Sutton, United Kingdom.

2 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

3 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).

4 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

5 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

6 Statistics Determination of cutpoints For 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

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

8 Number of patients and events available for this preliminary analysis

9 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%)

10 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 HTERT48%No data available MSI15%10-17% 18q65%70% KRAS37%32-40% BRAF8%10% UGT1A1 (7/7 genotype)12%10-15% * Intense expression, More than 45% cells positive ** Any loss *** Positive = more than 25% cell positive

11 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.

12 SMAD4: Preliminary results (stage III)

13 MSI: Preliminary results (stage III)

14 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

15 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

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