Evaluation de la Recherche des Mutations du récepteur de l’EGF (EGF-R) et de K-RAS pour le Traitement par les Inhibiteurs de tyrosine kinase des Cancers bronchiques non à petite cellules. Apport des Techniques Sensibles pour la prédiction de la réponse et de la résistance aux Inhibiteurs de tyrosine kinase de l’EGFR.

Coordonnateur : Jacques Cadranel

Comité de Pilotage : Michèle Beau-Faller, Hélène Blons, Pascale Missy, Franck Morin

Gestionnaire de la Recherche : IFCT

Problems, hypothesis and objectives: Inhibitor of the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR), erlotinib, is approved for the treatment of patients with previously treated, advanced non-small cell lung cancer (NSCLC), based on evidence from the phase III BR.21 study. Since 2005, these results have led to an increase of erlotinib prescription. Clinical characteristics have been correlated to EGFR TKI response: female gender, adenocarcinoma, non smoking status, Asian ethnicity. Quite rapidly, sensitising mutations (EGFR exons 18, 19 and 21) and primary resistance mutations (K-Ras) have been described, and then secondary resistance mutations (T790M EGFR exon 20), leading to a better selection of patients potentially able to profit more (sensitising mutations) and not to profit (resistance mutations) from EGFR TKI treatment. The molecular data result usually from retrospective cohorts, with a limited number of mostly surgical and/or frozen samples, which is not reflecting routine diagnostic samples: small size paraffin embedded fixed samples, mostly bronchial biopsies, with a low percentage of tumor cells. Direct sequencing was considered to be the gold standard technique.

The ERMETIC STIC project funded in 2005 had two aims: 1) harmonizing EGFR and K-Ras mutations detection methods by direct sequencing in 15 French laboratories, on a retrospective cohort composed by 74 NSCLC, with paired frozen and paraffin-embedded surgical samples and their counterpart biopsies; 2) implementing these techniques on tumor samples of a prospective cohort of 530 stage IV NSCLC patients receiving an EGFR-TKI treatment (erlotinib) to define the best prescription strategy of these biomarkers. This cohort (end of inclusions 31/03/08) is representative because of its variety of tumor samples, including surgery and a majority of paraffin embedded biopsies, which may be poor in tumor cells (50% of samples with less than 50% of tumor cells). Thus, direct sequencing becomes unreliable. Techniques which are alternative to direct sequencing (fragment analysis, taqman, pyrosequencing, HRMA/DHPLC, PNA-clamped PCR, ARMS Scorpion, MASO PCR), more sensitive (detection down to 1% mutant DNA), faster and cheaper could be useful to optimize analysis of such routine samples. In this context, ERMETIC-2 project is a unique opportunity to perform in a blinded fashion, the detection of EGFR and K-Ras mutations by more sensitive techniques than direct sequencing, on a large number of routine tumor samples (at least 368 samples available from the 530 samples, according to last inventory) corresponding to clinically annotated tumors.

The main objective of this project is to evaluate the sensitivity of these more sensitive alternative  techniques compared to direct sequencing in an attempt to : i) limit “false-negative” and “unspecifiable“ results from specimens presenting a low percentage of tumor cells (<50%) or not amplifiable for direct sequencing;  ii) evaluate the clinical relevance of the detection of low abundance mutants i.e. sensitizing EGFR mutants as well as EGFR and K-Ras resistant mutants, in good quality samples considered as wild type by sequencing. Results will be collected on an eCRF elaborated by IFCT in collaboration with IGR, based on forms identical to the ones for sequencing in ERMETIC CRF and correlated to clinical and responses parameters, based on the same methodology as ERMETIC. We will compare the new techniques between them and with sequencing methods within each of the 3 set of mutations: EGFR hot-spot mutations predictive of sensitivity (exons 19 and 21), K-Ras mutations predictive of primary resistance, and low abundance secondary resistance mutation (EGFR exon 20 T790M).
Comparison of the new techniques within each of the three set of mutations: the new techniques will be compared between them and with the sequencing method within each of the three sets of mutations on the sensibility (minimal dilution with mutation detected), the feasibility rate (number of sample with results divided by the number of sample analysed), the rate of false positive results (on the normal samples) and the rate of positive results. The rates will be compared by chi² test or exact test (type Fisher test) if appropriate and presented with their 95% confidence interval. Clinical pertinence of the new techniques: The main endpoint to study the clinical pertinence of the new techniques is progression-free survival. Secondary endpoint will be tumour response at 3 months and overall survival.
ERMETIC-2 project will lead to completion of ERMETIC conclusions for detection of EGFR and K-Ras mutations using more sensitive alternative techniques, which are more suitable for routine clinical samples, for predicting patient response/resistance to EGFR TKI treatment. The regional expertise network certified and consolidated by ERMETIC, will also benefit from the technological development generated by this ERMETIC-2 project.

Financement : Laboratoire Roche

Lu 2481 fois Dernière modification le mardi, 02 décembre 2014 11:56