Fabienne Escande

Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network

BACKGROUND There is scarce data available about epidermal growth factor receptor (EGFR) mutations other than common exon 19 deletions and exon 21 (L858R) mutations. PATIENTS AND METHODS EGFR exon 18 and/or exon 20 mutations were collected from 10 117 non-small-cell lung cancer (NSCLC) samples analysed at 15 French National Cancer Institute (INCa)-platforms of the ERMETIC-IFCT network. RESULTS Between 2008 and 2011, 1047 (10%) samples were EGFR-mutated, 102 (10%) with rare mutations: 41 (4%) in exon 18, 49 (5%) in exon 20, and 12 (1%) with other EGFR mutations. Exon 20 mutations were related to never-smoker status, when compared with exon 18 mutations (P < 0.001). Median overall survival (OS) of metastatic disease was 21 months [95% confidence interval (CI) 12-24], worse in smokers than in non-smoker patients with exon 20 mutations (12 versus 21 months; hazard ratio [HR] for death 0.27, 95% CI 0.08-0.87, P = 0.03). Under EGFR-tyrosine kinase inhibitors (TKIs), median OS was 14 months (95% CI 6-21); disease control rate was better for complex mutations (6 of 7, 86%) than for single mutations (16 of 40, 40%) (P = 0.03). CONCLUSIONS Rare EGFR-mutated NSCLCs are heterogeneous, with resistance of distal exon 20 insertions and better sensitivity of exon 18 or complex mutations to EGFR-TKIs, probably requiring individual assessment.

A Multicenter Blinded Study Evaluating EGFR and KRAS Mutation Testing Methods in the Clinical Non–Small Cell Lung Cancer Setting—IFCT/ERMETIC2 Project Part 1: Comparison of Testing Methods in 20 French Molecular Genetic National Cancer Institute Platforms

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors have limited use as first-line treatment for mutated EGFR metastatic non-small cell lung cancer. The French National Cancer Institute has installed molecular genetics platforms implementing EGFR and KRAS testing. However, there is considerable uncertainty as to which detection methods should be applied for routine diagnosis. This study aimed to compare the EGFR and KRAS genotyping methods developed by the IFCT/ERMETIC2 network platforms in two blind panels: 25 samples of serial dilutions of cell line DNA (20 centers) and 74 FFPE lung tumor samples (10 centers). The best threshold of mutation detection on cell lines was obtained using allele-specific amplification-based technologies. Nonamplifiable tissue samples were significantly less common when using alternative testing versus direct sequencing [15%; 95% confidence interval (CI), 14%-16% versus 40%; 95% CI, 39%-42%; P < 0.001]. Mutated cases increased from 42% (95% CI, 31%-54%) to 53% (95% CI, 41%-64%), with three supplementary EGFR mutations (p.G179A at exon 18 and p.L858R and p.L861Q at exon 21) and five supplementary KRAS mutations, when using alternative testing instead of direct sequencing. False-positive results were observed when using a PCR-based sizing assay, high-resolution melting, or pyrosequencing. Concordance analysis returned good kappa test scores for EGFR exon 19 and KRAS analysis when comparing sequencing with alternative methods and revealed no difference between alternative techniques themselves.

Clinical effect of molecular methods in sarcoma diagnosis (GENSARC): a prospective, multicentre, observational study

BACKGROUND Advances in molecular genetics of sarcoma have enabled the identification of type-specific aberrations. We aimed to assess the clinical effect of systematic implementation of molecular assays to improve sarcoma misdiagnosis. METHODS In this multicentre, observational study, we recruited patients from 32 centres of the French Sarcoma Group/Reference Network in Pathology of Sarcomas. Eligibility criteria included: biopsy or surgical resection; suspicion of: dermatofibrosarcoma protuberans (cohort 1), dedifferentiated liposarcoma (cohort 2), Ewings sarcoma family of tumours (cohort 3), synovial sarcoma (cohort 4), alveolar rhabdomyosarcoma (cohort 5), and myxoid or round cell liposarcoma (cohort 6); review by one sarcoma-expert pathologist; availability of frozen material (except for cohort 1 of patients with dermatofibrosarcoma protuberans because anti-CD34 immunohistochemistry is performed on paraffin-embedded tissue); and patient information. For each case, the pathologist made one primary diagnosis followed by up to two differential diagnoses, based on histological characteristics only. Each diagnosis was classified as certain, probable, or possible. For each case to determine the molecular classification, we did fluorescence in-situ hybridisation on paraffin-embedded samples. We also did comparative genomic hybridisation and quantitative PCR (cohort 2) or reverse transcriptase PCR (cohorts 3-6) on frozen and paraffin-embedded samples. We made a final diagnosis based on the molecular results. The clinical effect of diagnosis correction was assessed by a board of experts. FINDING Between June 22, 2009, and Oct 30, 2012, 395 patients were enrolled in the study, of which 384 were eligible for inclusion. The diagnosis was eventually modified by molecular genetics for 53 patients: eight (16%) of 50 patients with dermatofibrosarcoma (cohort 1), seven (23%) of 30 patients with dedifferentiated liposarcoma (cohort 2), 13 (12%) of 112 with Ewings sarcoma family of tumours (cohort 3), 16 (16%) of 97 patients with synovial sarcoma (cohort 4), seven (15%) of 46 patients with alveolar rhabdomyosarcoma (cohort 5), and two (4%) of 49 patients with myxoid or round cell liposarcoma (cohort 6), with an effect on primary management or prognosis assessment in 45 cases. INTERPRETATION Molecular genetic testing should be mandatory for diagnostic accuracy of sarcoma and appropriate clinical management, even when histological diagnosis is made by pathologist experts in this field. FUNDING French National Cancer Institute and Nice University Hospital.

A quality control program for mutation detection in KIT and PDGFRA in gastrointestinal stromal tumours

BackgroundAlthough most gastrointestinal stromal tumours (GIST) carry oncogenic mutations in KIT exons 9, 11, 13 and 17, or in platelet-derived growth factor receptor alpha (PDGFRA) exons 12, 14 and 18, around 10% of GIST are free of these mutations. Genotyping and accurate detection of KIT/PDGFRA mutations in GIST are becoming increasingly useful for clinicians in the management of the disease.MethodTo evaluate and improve laboratory practice in GIST mutation detection, we developed a mutational screening quality control program. Eleven laboratories were enrolled in this program and 50 DNA samples were analysed, each of them by four different laboratories, giving 200 mutational reports.ResultsIn total, eight mutations were not detected by at least one laboratory. One false positive result was reported in one sample. Thus, the mean global rate of error with clinical implication based on 200 reports was 4.5%. Concerning specific polymorphisms detection, the rate varied from 0 to 100%, depending on the laboratory. The way mutations were reported was very heterogeneous, and some errors were detected.ConclusionThis study demonstrated that such a program was necessary for laboratories to improve the quality of the analysis, because an error rate of 4.5% may have clinical consequences for the patient.

Validation of the high-performance of pyrosequencing for clinical MGMT testing on a cohort of glioblastoma patients from a prospective dedicated multicentric trial

Background The goal of this prospective multicentric trial was to validate a technique that allowed for MGMT promoter methylation analysis in routine clinical practice. Methods The MGMT status of 139 glioblastoma patients, whom had received standard first line treatment, was determined using pyrosequencing (PSQ) and a semi-quantitative Methylation-specific PCR (sqMS-PCR) method, using both frozen and formalin-fixed paraffin-embedded FFPE samples. Eight participating centers locally performed the analysis, including external quality controls. Results There was a strong correlation between results from FFPE and frozen samples. With cut-offs of 12% and 13%, 98% and 91% of samples were identically classified with PSQ and sqMS-PCR respectively. In 12% of cases frozen samples were excluded because they had a low percentage of tumor cells. In 5-6% of cases the analysis was not feasible on FFPE samples. The optimized risk cut-offs were higher in both techniques when using FFPE samples, in comparison to frozen samples. For sqMS-PCR, we validated a cut-off between 13-15% to dichotomize patients. For PSQ, patients with a low level of methylation (<= 8%) had a median progression-free survival under 9 months, as compared with more than 15.5 months for those with a level above 12%. For intermediate values (9-12%), more discordant results between FFPE and frozen samples were observed and there was not a clear benefit of temozolomide treatment, which indicated a “grey zone”. Conclusions MGMT status can reliably be investigated in local laboratories. PSQ is the ideal choice as proven by strong interlaboratory reproducibility, along with threshold agreements across independent studies.

EGFR tyrosine kinase inhibitors versus chemotherapy in EGFR wild-type pre-treated advanced nonsmall cell lung cancer in daily practice.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are approved for second-line treatment of EGFR wild-type (EGFR-wt) nonsmall cell lung cancer (NSCLC). However, results from randomised trials performed to compare EGFR-TKIs with chemotherapy in this population did not show any survival benefit. In the era of immunotherapy, many drugs are approved for second-line treatment of EGFR-wt NSCLC and there is a need to reassess the role of EGFR-TKIs in this setting. The Biomarkers France study is a large nationwide cohort of NSCLC patients tested for EGFR mutations. We used this database to collect clinical, biological, treatment and outcome data on EGFR-wt patients who received second-line treatment with either EGFR-TKIs or chemotherapy. Among 1278 patients, 868 received chemotherapy and 410 received an EGFR-TKI. Median overall survival and progression-free survival were longer with chemotherapy than with an EGFR-TKI. Overall survival was 8.38 versus 4.99 months, respectively (hazard ratio 0.70, 95% CI 0.59–0.83; p<0.0001) and progression-free survival was 4.30 versus 2.83 months, respectively (hazard ratio 0.66, 95% CI 0.57–0.77; p<0.0001). This study is helpful to guide a multiline treatment strategy for EGFR-wt NSCLC patients. Immunotherapy is approved for second-line treatment. For third-line treatment, chemotherapy results in longer overall survival and progression-free survival, and should be preferred to EGFR-TKIs. Biomarkers France study: second-line chemotherapy gave longer PFS and OS than TKI in NSCLC EGFR-wt patients http://ow.ly/rEXk30b3tak

1309PERMETIC-2 PROJECT : IMPACT OF SYSTEMATIC EGFR AND KRAS MUTATION EVALUATION BY ALTERNATIVE TESTING METHODS ON PROGRESSION-FREE (PFS) AND OVERALL SURVIVAL (OS) IN PATIENTS WITH ADVANCED NON–SMALL-CELL LUNG CANCER (NSCLC) TREATED BY ERLOTINIB (E) IN THE IFCT ERMETIC COHORT

ABSTRACT Aim: ERMETIC is a French (IFCT) prospective study of EGFR and KRAS mutation evaluation by sequencing in patients with advanced NSCLC treated by E. The aim of the ERMETIC-2 project was to determine the prognostic impact of using alternative molecular techniques in the available tumor samples of the ERMETIC cohort. Methods: 239 patients (pts) with tumors were available for re-analyze by previously described alternative techniques (J Mol Diag 2014). Multivariate Cox model with performance status, histology, smoking status and initial number of metastatic sites was used for prognostic analysis. Results: Population included 59% of adenocarcinoma, 37% of women and 19% of non-smokers. Overall mutation rate is 46%: 31 EGFR mutations (13%) and 78 KRAS mutations (33%); 40 new mutations compared to previous study were found: 9 EGFR and 31 KRAS. In the ERMETIC 2 cohort, OS and PFS remained significantly (global test p Conclusions: 1. KRAS but not EGFR mutations were detected in higher proportion by alternative molecular techniques compared to sequencing. 2. KRAS mutations identified by more sensitive technique did not identified a group of patients with a different prognostic. Disclosure: All authors have declared no conflicts of interest.

A quality control program for mutation detection in KIT and PDGFRA in gastrointestinal stromal tumours.

BackgroundAlthough most gastrointestinal stromal tumours (GIST) carry oncogenic mutations in KIT exons 9, 11, 13 and 17, or in platelet-derived growth factor receptor alpha (PDGFRA) exons 12, 14 and 18, around 10% of GIST are free of these mutations. Genotyping and accurate detection of KIT/PDGFRA mutations in GIST are becoming increasingly useful for clinicians in the management of the disease.MethodTo evaluate and improve laboratory practice in GIST mutation detection, we developed a mutational screening quality control program. Eleven laboratories were enrolled in this program and 50 DNA samples were analysed, each of them by four different laboratories, giving 200 mutational reports.ResultsIn total, eight mutations were not detected by at least one laboratory. One false positive result was reported in one sample. Thus, the mean global rate of error with clinical implication based on 200 reports was 4.5%. Concerning specific polymorphisms detection, the rate varied from 0 to 100%, depending on the laboratory. The way mutations were reported was very heterogeneous, and some errors were detected.ConclusionThis study demonstrated that such a program was necessary for laboratories to improve the quality of the analysis, because an error rate of 4.5% may have clinical consequences for the patient.