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International audience; Theranostic translocations may be difficult to detect by routine techniques, especially when specimens are exiguous. We recently demonstrated in a series of translocated lung adenocarcinomas that LD-RT-PCR (ligation-dependent reverse transcription polymerase chain reaction) assay could identify ALK, ROS1 and RET rearrangements with 64% sensitivity and 100% specificity. Here, we report an upgraded version of this assay used in a routine prospective cohort of lung carcinomas. Newly diagnosed lung carcinomas referred to the Rouen molecular platform between 15/05/2018 and 15/05/2019 for ALK and ROS1 IHC, genotyping (SNaPshot© +/- high-throughput genotyping) and sometimes FISH (standard routine process) were tested prospectively in parallel with the LD-RT-PCR assay designed to detect at one go ALK, ROS1 and RET translocations and MET exon 14 skipping. 413 tumors from 396 patients were included. LD-RT-PCR had a global sensitivity of 91.43% (standard routine process: 80%), with a specificity of 100%. It detected 15/18 ALK and 4/4 ROS1 translocated tumors, but also 6/6 tumors with MET exon 14 skipping retrieved by genotyping. In addition, it retrieved 7 alterations missed by the routine process, then confirmed by other means: 5 MET exon 14 skipping and 2 RET translocated tumors. Finally, it allowed to deny an effect on MET exon 14 skipping for 8 mutations detected by routine genotyping. We successfully implemented LD-RT-PCR in routine analysis. This technique is cheap, fast, sensitive, specific, and easily upgradable (e.g., NTRK translocations), but still requires IHC to be performed in parallel. Owing to its advantages, we recommend considering it, in parallel with IHC and genotyping, as an excellent cost-effective alternative, for the systematic testing of lung adenocarcinoma, to FISH and to more expensive and complex assays such as RNA-seq.

High throughput RNA sequencing, also know as RNAseq, can easily be performed on the gold-standard technique of formalin-fixed paraffin-embedded tissue, which has long been successfully used in routine practice by pathologists. For this reason, RNAseq has been fully adopted in a very short period of time in most French molecular platforms of cancer genotyping, generating "high throughput" data, both qualitative (mutations, fusions) and quantitative (gene expression profiles). This technique opens new perspectives in oncology practice: from a diagnostic point of view (some gene fusions are specific of some diagnoses, some transcriptomic signatures suggest some types of cancer), but also from a prognostic point of view (gene expression profile of an aggressive tumor, or conversely of an indolent one), and above all from a predictive point of view, guiding the choice of potential targeted therapies (example of ALK, ROS1 or NTRK translocations). This technical approach has many advantages, first and foremost it detects, at one go, a plethora of molecular alterations which were previously analyzed sequentially using heterogenous assays (immunohistochemistry, DNA genotyping, fluorescent in situ hybridization, etc.). However, it also presents several drawbacks which may easily be overcome if certain pre-analytic parameters are correctly controlled, mainly aiming at the preservation of the quality of nucleic acids. In any event, the widespread use of RNAseq has had a profound impact on the algorithms of tumor tissue processing, shaping a new, holistic era in oncology.

Lung adenocarcinoma (LUAD) is the major subtype of non-small cell lung cancer, accounting for approximately 60% of cases. Molecular analysis of LUADs showed that the KRAS gene is mutated in up to 30% of cases; such cases were previously considered “undruggable”. The KRAS G12C mutation has become a hot topic of research after initial, promising, phase I and II trials with targeted inhibitors. We analyzed the morphological and genomic landscape of 202 KRAS G12C mutated LUADs using next-generation sequencing, and identified a specific subtype of patients that could show an improved response to KRAS G12C inhibitors. The main histological subtype was acinar in 29.7% of cases. Tumor-infiltrating lymphocytes (TILs) were highly or moderately abundant in more than 60% of cases. The immunohistochemical profile showed TTF1 positivity in 78.7% of cases and PD-L1 positivity in 44.1% of cases. The molecular profile showed an association between KRAS G12C and STK11 mutations in 25.2% of cases. This subgroup was associated with a statistically significant lower TTF1 (p = 0.0092) and PD-L1 (p < 0.0001) positivity. This type of combined morphological and molecular analysis can improve our understanding of tumor biology, and help us to identify specific patient subgroups that can achieve the best treatment response.

Background Testing for germline BRCA1/2 mutations has an established predictive role in breast and ovarian cancer risk assessment and EQA on germline mutation testing have been performed for a long time. With the European extension of indication for the PARP inhibitors, the screening of tumors first is increasingly important. However, tumoral BRCA1/2 testing is a different analytical process on formalin-fixed, paraffin-embedded (FFPE) material and with some challenging variants as large rearrangements. Validation of the test method and participation in external quality assessment programs are therefore required. Methods In the French national quality control programs (Gen&tiss) of 2017 and 2018, laboratories received 5 samples from ovarian cancer patients and 1 educational artificial sample with mutations present at different variant allelic frequencies in BRCA1/2 and a large rearrangement in BRCA1. Results The number of participants was 21 in 2017 and 26 in 2018. The number of labs with severe error was 3 in 2017 (14%) and 4 in 2018 (15%). For BRCA1, the average score remained stable between 2017 [9.6/10 (N = 21)] and 2018 [9.6/10 (N = 26)]. In both years, 2 laboratories made severe errors (false positive/false negative). In 2017 only 11 laboratories (N = 20) identified the educational variant present at 7% VAF. For BRCA2 the score slightly decreased from 9.8/10 (N = 22) to 9.4/10 (N = 26) and the number of laboratories with severe errors increased from 1 to 3. Half of the laboratories (N = 20) detected all three mutations present in the educational sample (VAF 10% to 30%) in the 2017 scheme. In 2018 a large deletion in BRCA1 present in the educational sample was only detected by 4 out of 20 laboratories. Three out of 26 laboratories detected the additional RAD51C variant and none the RAD51D variant. The main methods applied in France focus on BRCA1 and BRCA2 genes and amplification-based enrichment. Conclusions The genotype results were very similar between 2017 and 2018: acceptable but there are still more than 14% with severe errors. The limit of detection is a critical point. Few labs are ready to extend testing beyond BRCA1/2 genes. The question of large rearrangements is not yet solved for tumoral screening. EQA on tumoral BRCA1/2 testing is therefore essential to improve laboratory performance. Legal entity responsible for the study Gen&tiss - GFCO and AFAQAP. Funding AstraZeneca. Disclosure All authors have declared no conflicts of interest.

Background and objective Treatment optimization of non-squamous non-small-cell lung cancers (nonSq-NSCLC) relies on the molecular analysis of the tumour. We aimed to assess the predictive factors of molecular analysis feasibility (MAF) from samples of peripheral nonSq-NSCLC obtained by radial endobronchial ultrasound bronchoscopy (r-EBUS) and 1.5 mm microbiopsy forceps. Methods We reviewed data from consecutive peripheral lung nodules sampled with r-EBUS between January 2012 and July 2014 at a single French University Hospital. nonSq-NSCLC were systematically analysed for EGFR, KRAS, ALK, HER2, PI3K and BRAF throughout the study, and c-MET and ROS1 alterations for the last 10 months. Results Of 111 nonSq-NSCLC diagnosed by r-EBUS (113 procedures, mean nodule diameter 28 ± 15 mm), 88 were analysed for EGFR and ALK, 87 for KRAS, 86 for HER2, PI3K and BRAF and 14 for c-MET. Forty-one mutations were identified (23 KRAS, 10 EGFR, 2 BRAF, 1 HER2 and 5 ALK rearrangements). Four c-MET overexpressions were noted. MAF rose from 67% for the first 57 procedures to 89% for the last 56 procedures (P = 0.02) likely due to a higher number of biopsies performed (2 ± 1 vs 3 ± 2, P = 0.005). Upper or middle lobe location (OR 1.19, 95% CI: 1.02–1.38, P = 0.03), and at least three biopsies (OR 1.20, 95% CI: 1.04–1.40, P = 0.02) were predictive factors of MAF. Percentage of tumour cells, size of lesion and distance to the pleura did not correlate with MAF. Conclusion Multi-gene molecular analysis could be performed in nearly 80% of paraffin-embedded biopsies or smear specimens sampled by r-EBUS assisted bronchoscopy of peripheral tumoral lung nodules.

International audience; Detection of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 (ROS1), and rearranged during transfection (RET) gene rearrangements in lung adenocarcinoma is usually performed by immunohistochemistry (IHC) screening followed by fluorescence in situ hybridization (FISH), which is an expensive and difficult technique. Ligation-dependent reverse transcription polymerase chain reaction (RT-PCR) multiplex technique can detect gene rearrangements using probes specifically hybridized to either side of the break point. PCR products are then sequenced by pyrosequencing or high throughput sequencing in order to identify the two genes involved. The reagent cost is

Background The interpretation of testing results in molecular pathology is an essential phase. It is difficult to assesss this in external quality assessment on real samples. Here we propose a national measure of the concordance in the variant interpretation between laboratories participating in the national French quality control program Gen&tiss. Methods Laboratories were asked to interpret 5 variants each for melanoma, lung and colorectal cancers and 10 for ovarian cancer. These variants in KRAS, NRAS, BRAF, PIK3CA, PTEN, AKT1, TP53, EGFR, RAC1, KIT, BRCA1 and BRCA2 were not classical hotspots of mutation and 15 of these were annotated in OncoKB. A maximum of 25 variants was assessed per laboratories. Nine possible interpretations were proposed: from neutral to oncogenic and from no therapeutic impact to targeted by a drug. For each variant, a consensus score was determined based on public database as OncoKb and validated by 4 molecular experts with literature review. Three scores were possible: right answer (2 points), acceptable answer without clinical impact (1 point), wrong answer with clinical impact (0 points). Results 60 laboratories participated to the program for variant interpretation. The average scores on 10 were 5.9 for colorectal (N = 52), 7.0 for lung (N = 49), 6.0 for melanoma (N = 49) and 7.0 for ovarian cancer (N = 25). For BRCA genes in ovarian cancer, there was a pilot project in 2017, in which 5 identical variants were proposed to interpretation. 18 laboratories participated both in 2017 and 2018: 50% performed better, 28% performed worse, 17% had similar results. Conclusions As test results have will have a direct impact on patient management, ensuring a correct interpretation becomes more and more important. This EQA could help to validate the interpretation process used in the laboratories (SOP and databases) and could be extend to the individual expertise.The average score is acceptable, yet improvable and it really depends on the variants proposed. International consensus guidelines equivalent to the ACMG classification of constitutional variants would be helpful to improve reproducibility and inter-laboratory homogeneity. Legal entity responsible for the study Gen&tiss - GFCO and AFAQAP. Funding AstraZeneca. Disclosure All authors have declared no conflicts of interest.

The quick emerging of the several targeted therapies and the concept of personnalized medicine underlie the necessity to develop and to well organize a molecular biology (or molecular pathology) unit of high quality, dedicated to clinical care, in order to look for tissular and cellular theragnosis biomarkers. This new and sudden area of activity for a clinical pathologist is strongly linked to the knowledge of a new medical speciality in health care institutions. Thus, the molecular pathology (or molecular biology made from cellular or tissular samples) can nicely be implemented in a clinical pathology laboratory. This new mission for a pathologist has to be done in respect with a great quality assurance which should allow obtaining in a short-term an ISO 15189 accreditation to keep going to perform this activity. The present work aims to describe the main steps to be set up in the order to get an ISO 15189 accreditation in molecular pathology. The different chapters of this norm will not be described in their exhaustivity, but in their large lines. Finally, we will describe the potential difficulties and pitfalls to be avoided before getting this accreditation.

Background: Treatment optimization of non-squamous non-small-cell lung cancers (nonSq-NSCLC) relies on the molecular analysis of the tumour. Objective: We aimed to assess the predictive factors of molecular analysis feasibility (MAF) from samples of peripheral nonSq-NSCLC obtained by radial endobronchial ultrasound bronchoscopy (r-EBUS) and 1.5 mm microbiopsy forceps. Methods: we reviewed data from consecutive peripheral lung nodules sampled with r-EBUS between January 2012 and July 2014 at a single French University Hospital. nonSq-NSCLC were systematically analysed for EGFR , KRAS , ALK , HER 2, PI3K and BRAF throughout the study, and c- MET and ROS 1 alterations for the last 10 months. Results: Of 111 nonSq-NSCLC diagnosed by r-EBUS (113 procedures, mean nodule diameter 28 ± 15 mm), 88 were analysed for EGFR and ALK , 87 for KRAS , 86 for HER 2, PI3K and BRAF and 14 for c- MET . Forty-one mutations were identified (23 KRAS , 10 EGFR , 2 BRAF , 1 HER 2 and 5 ALK rearrangements). Four c- MET overexpressions were noted. MAF rose from 67% for the first 57 procedures to 89% for the last 56 procedures ( P = 0.02) likely due to a higher number of biopsies performed (2 ± 1 vs 3 ± 2, P = 0.005). Upper or middle lobe location (OR 1.19, 95% CI: 1.02–1.38, P = 0.03), and at least three biopsies (OR 1.20, 95% CI: 1.04–1.40, P = 0.02) were predictive factors of MAF. Percentage of tumour cells, size of lesion and distance to the pleura did not correlate with MAF. Conclusion: Multi-gene molecular analysis could be performed in nearly 80% of paraffin-embedded biopsies or smear specimens sampled by r-EBUS assisted bronchoscopy of peripheral tumoral lung nodules.

Contains fulltext : 171799.pdf (Publisher’s version ) (Closed access) Personalized medicine has gained increasing importance in clinical oncology, and several clinically important biomarkers are implemented in routine practice. In an effort to guarantee high quality of molecular testing in France, three subsequent external quality assessment rounds were organized at the initiative of the National Cancer Institute between 2012 and 2014. The schemes included clinically relevant biomarkers for metastatic colorectal (KRAS, NRAS, BRAF, PIK3CA, microsatellite instability) and non-small cell lung cancer (EGFR, KRAS, BRAF, PIK3CA, ERBB2), and they represent the first multigene/multicancer studies throughout Europe. In total, 56 laboratories coordinated by 28 regional molecular centers participated in the schemes. Laboratories received formalin-fixed, paraffin-embedded samples and were asked to use routine methods for molecular testing to predict patient response to targeted therapies. They were encouraged to return results within 14 calendar days after sample receipt. Both genotyping and reporting were evaluated separately. During the three external quality assessment rounds, mean genotype scores were all above the preset standard of 90% for all biomarkers. Participants were mainly challenged in case of rare insertions or deletions. Assessment of the written reports showed substantial progress between the external quality assessment schemes on multiple criteria. Several essential elements such as the clinical interpretation of test results and the reason for testing still require improvement by continued external quality assessment education.

KRAS genotyping is mandatory before anti-epidermal growth factor receptor monoclonal antibody therapy in metastatic colorectal cancer, which is the second leading cause of cancer-related death in the United States and in Europe. Thus, large-scale KRAS mutation screening is needed for efficient patient management and in the future metastatic colorectal cancer genotyping might also include the detection of the BRAF V600E mutation, which is a very strong negative prognostic factor in colorectal cancer. We report our experience of routine KRAS/BRAF mutation screening practice performed on 1130 formalin-fixed paraffin-embedded tumor samples from 992 colorectal cancer patients. DNA was extracted from macrodissected tumor areas highlighted by a pathologist, KRAS codons 12/13 and BRAF V600E mutations were assessed in a single SNaPshot® multiplex assay and each mutation was confirmed by an independent analysis. KRAS and BRAF mutations were, respectively, present in 41.8 and 6.5% of the tumor samples. If KRAS and BRAF mutations were mutually exclusive, four samples presented two concomitant KRAS mutations. Genotyping of paired primary tumors and metastases from 44 patients indicated that 5 patients (11.4%) presented discordant KRAS mutational status. KRAS genotype heterogeneity was also observed within primary tumor sites in seven cases. Non-reproducible KRAS artefactual mutations were detected in 53 samples (4.7%). We found that the prominent mechanism leading to these artefactual mutations was the fragmentation of DNA occurring during tissue processing. Routine KRAS genotyping performed on formalin-fixed paraffin-embedded tissues requires, therefore, the development of quality control scheme for molecular pathology, especially because of DNA damages induced by formalin fixation. The tumor heterogeneity observed in some patients indicates that it should be more appropriate to perform KRAS genotyping on metastases if sample is available.

The advent of the targeted cancer therapies administered to patients, according to the results of molecular biology techniques (in particular, in situ hybridization, "polymerase chain reaction" amplification and sequencing), has modified the practice of the surgical pathology laboratories. The necessity to answer to the needs of physicians for optimizing the medical care for patients who develop cancer has led to a policy of national debate, spurred by the National Institute of Cancer (INCa), in order to implement new procedures in the pathology laboratories. Thus, in addition to the structuring of molecular biology platforms and their labeling by INCa, the upstream control of the steps present between resection of tumor samples and molecular analysis has proved to be crucial. Indeed, the quality of this upstream time, called "pre-analytical" phase, determines the reliability of the molecular biology results and therefore the therapeutic strategy. We describe here the main steps to be checked in the pre-analytical phase. The optimization of this pre-analytical phase within the surgical pathology laboratory aims to reduce or render insignificant the risk of errors of molecular biology tests. These errors can indeed lead to false negative or false positive results whose therapeutic consequences can be particularly harmful to patients with cancer.

Resume La pathologie moleculaire (PM) est une branche emergeante de l’anatomie et cytologie pathologiques. La mise en place des techniques de PM dans les laboratoires de pathologie a ete initialement facilitee par la proximite d’un laboratoire de genetique ou d’une unite de recherche. En effet, la reussite de ces transferts technologiques a ete en partie liee aux fortes interactions existantes entre les pathologistes et les geneticiens/biologistes moleculaires. La complementarite des pathologistes (maitrisant la conservation et l’identification des tissus) et des geneticiens (maitrisant les outils de BM) a permis ainsi de garantir la qualite des examens effectues a partir des tissus. Cependant, c’est avec le genotypage de KRAS effectue « a grande echelle » pour tous les carcinomes colorectaux metastatiques que la PM est apparu comme un outil indispensable a la pratique de la pathologie et egalement comme une evolution irremediable de notre discipline.

We describe a case of oligodendroglioma arising in an ovarian mature cystic teratoma associated with a loss of heterozygosity on the long arm of chromosomes 19 and 10. To our knowledge it is the second case reported in the literature at this site and the first one described in association with a characterized genetic alteration. The patient was 29 years old and presented with a history of 4 months of pelvic pain. Ultrasound examination showed a cystic mass arising in the left adnexa suggesting a teratoma. At laparotomy a cystic ovoid mass was found arising from the left adnexa, completely replacing the ovary. An ovariectomy was performed. Macroscopically a multilocular cyst containing hair, sebum, and a relatively well-defined solid zone of grayish-pink color strongly suggestive of a cerebral tissue, was observed. Microscopic analyses confirmed the teratomatous nature of the cyst. The solid area was composed of mature glial tissue in which was observed a proliferation of monotonous cells with round and homogenous nuclei, surrounded by a clear halo of cytoplasm ("honeycomb appearance") which immunohistochemically showed positivity for glial fibrilar acidic protein and for neurofilament protein. Ki-67 labeling index was about 3%. These findings were consistent with a low-grade oligodendroglioma arising in a mature ovarian cystic teratoma. Reverse transcription-polymerase chain reaction analysis showed a characterized loss of heterozygosity occurring in tumor DNA on chromosomes 10q and 19q13.

Purpose The antiepidermal growth factor receptor antibody cetuximab shows activity in irinotecan-refractory metastatic colorectal cancer (mCRC), mainly in wild-type KRAS tumors. Cetuximab may also exert antitumor effects through antibody-dependent cell-mediated cytotoxicity (ADCC) in which antibody Fc portion interacts with Fc receptors (FcγRs) expressed by immune cells. ADCC is influenced by FcγRIIa-H131R and FcγRIIIa-V158F polymorphisms that are clinically relevant in follicular lymphoma and metastatic breast cancer treated with rituximab and trastuzumab, respectively. We investigated the association of FcγR polymorphisms and KRAS mutation with the outcome of irinotecan-refractory mCRC patients treated with cetuximab plus irinotecan. Patients and Methods Tumor and normal tissues from 69 patients were screened for KRAS mutations using a sensitive multiplex assay and genotyped for FcγRIIa and FcγRIIIa polymorphisms by direct sequencing and multiplex allele-specific polymerase chain reaction, respectively. The results were correlated with response and progression-free survival (PFS). Results KRAS mutations were associated with lower response rate (4% v 27% in nonmutated patients; P = .021) and shorter PFS (3.0 v 5.3 months; P = .021). Patients with FcγRIIa-131H/H and/or FcγIIIa-158V/V genotypes had longer PFS than 131R and 158F carriers (5.5 v 3.0 months; P = .005). The difference remained significant for mutated-KRAS patients. By multivariate analysis, KRAS mutation and FcγR combined status were independent risk factors for PFS. Conclusion Combined FcγRIIa/FcγRIIIa polymorphisms are prognostic factors for disease progression in mCRC patients treated with cetuximab plus irinotecan. As these polymorphisms are also clinically relevant in mutated-KRAS mCRC, an important role of ADCC in cetuximab efficacy is presumed.

Background & Aims: Several quantitative genetic alterations have been suggested to have in colorectal cancer (CRC) either a prognostic or a therapeutic predictive value. Routine detection of these alterations is limited by the absence of simple methods. Methods: The somatic quantitative multiplex polymerase chain reaction of short fluorescent fragments (QMPSF) is based on the simultaneous amplification under quantitative conditions of several dye-labeled targets both from tumor and nonmalignant tissues. For each patient, the resulting QMPSF fluorescent profiles are superimposed, and quantitative changes are simply detected by an increase or decrease of the corresponding fluorescent peaks. Two assays were developed and applied to 57 CRC: a "bar code" exploring several loci with known prognostic value and a "kinogram" studying the copy number change of kinase genes, against which inhibitors have been developed. Results: The bar code revealed that the most frequent alterations were the gain of AURKA /20q13 (53%) and MYC /8q24 (39%) and heterozygous deletion of DCC /18q21.3 (39%) and TP53 /17p13 (23%). The kinogram detected a gene copy number increase for AURKA , PTK2 , MET , and EGFR in 53%, 37%, 33%, and 28% of the tumors, respectively. QMPSF results were validated by comparative genomic hybridization and multiplex real-time polymerase chain reaction on genomic DNA. Conclusions: The somatic QMPSF is a simple method able to detect simultaneously on a routine basis several quantitative changes in tumors. Its flexibility will allow the integration of clinically relevant genes. This high throughput method should be a valuable complementary tool of fluorescent in situ hybrization and comparative genomic hybridization.

Introduction: Lung cancer is the leading cause of cancer deaths in the United States and worldwide. Biomarker testing is critical to personalized therapy in lung adenocarcinoma and has been extensively investigated in whites and Asians. However, little information addresses the underlying genetic changes among Caribbean and African Caribbean patients. In this study, we identified targetable biomarkers in Caribbean patients with lung adenocarcinoma. Methods: DNA extracted from lung adenocarcinoma specimens collected from 157 patients in whom primary lung adenocarcinoma was diagnosed from 2013 to 2015 in the University Hospital of Martinique was tested for mutation of the epidermal growth factor receptor gene (EGFR), Kirsten rat sarcoma viral oncogene homolog gene (KRAS), B-Raf proto-oncogene, serine/threonine kinase gene (BRAF), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA), ROS proto-oncogene 1, receptor tyrosine kinase gene (ROS), and MMNG HOS Transforming gene (MET). Clinical characteristics of our patients have been retrospectively gathered and correlated with mutational status. Results: Mutations in EGFR were identified in 57 cases (36%). Women accounted for 68% of patients with mutations versus 38% of those without mutations (p < 0.001). Eighteen percent of patients with mutations were smokers versus 62% of patients without mutations (p < 0.001). Sex, smoking habit, and age were significantly associated with differences in mutational status in univariate analysis, and the difference remained statistically significant in multivariate analysis (p = 0.0411, p = 0.001, and p = 0.0483, respectively). After the analysis was restricted to patients born in the French West Indies, the mutation rates reached 41%. Conclusion: Patients in Martinique, and specifically those of African descent, show very high levels of EGFR mutation as opposed towhat can be found inmainland France or in African Americans. These findings may be ascribed to low tobacco consumption aswell as togenetic factors. Systematic screening in patients of African Caribbean origin should be prescribed.

Background: When mutated, BRAF is an oncogenic driver of non-small cell lung cancer (NSCLC). Its prevalence in NSCLC is believed to be 1-4%. There is few published data on the clinical characteristics of BRAF mutated NSCLC patients. Aims: The aims of this study are to define BRAF mutation prevalence in a routinely tested French population and to describe clinical characteristics of patients. Methods: We conducted a retrospective study among non-squamous (NS) NSCLC diagnosed in Upper Normandy between sept 2011 and may 2014 which were systematically assessed in our institution for BRAF V600E and EGFR mutation. Results: From 2011 to 2014, 1923 NS-NSCLC were assessed for BRAF mutation. Prevalence of BRAF mutation was 32/1923 (1.6%). Sex ratio was 1.7. At the time of diagnosis, median age was 60, 22 patients (69%) were of PS 0-1, 8 patients (25%) were never-smokers. 27 patients (84%) had locally advanced or metastatic disease, with pleural, liver and bone being the most frequent metastatic sites.5 patients(15%) had a synchronous cancer (1 lymphoma, 1 rectal cancer, 1 colon and bladder cancer, 1 bladder cancer and 1 with 2 second primary lung cancers harboring each a different KRAS mutation). 1 other patient had concomitant KRAS mutation. Only 2 patients (6%) received a BRAF inhibitor (2 Vemurafenib, 1 Dabrafenib). Kaplan-Meyer estimated median survival was 19 months in metastatic patients and not reached for localized and locally advanced cancer, with median follow up of 4 months and 12 months respectively. Conclusion: In this series, the proportion of women and never-smokers and of synchronous cancer appears high. The series does not confirm the bad prognosis attached to BRAF mutated NSCLC.

The most common form of genomic instability observed in colorectal cancer is chromosomal instability (CIN), whose molecular bases remain to be determined. We have previously demonstrated that inactivation in human cells of several components of the Pes1-Bop1 complex (BOP1, GRWD1, PES1, ORC6L, and RPL3), involved in ribosome biogenesis, altered chromosome segregation. To determine the contribution to colorectal tumorigenesis of somatic alterations of genes involved in ribosome biogenesis, we screened 56 primary colorectal cancers, using quantitative multiplex PCR of short fluorescent fragments, a sensitive method for the detection of gene dosage alterations. We found that dosage increase of the BOP1 gene was a frequent event, being detected in 39% of the tumors, and we show that it is associated with an increase of BOP1 mRNA. Scanning of 8q24, on which BOP1 is located, revealed that in colorectal cancers, gene dosage increase of BOP1 can be independent from that of MYC and was more frequent than that affecting MYC. Finally, transient overexpression of BOP1 in human cells increased the percentage of multipolar spindles. Together with our previous results, the present study strongly suggests that deregulation of the BOP1 pathway contributes to colorectal tumorigenesis. © 2006 Wiley-Liss, Inc.

Gestational choriocarcinoma (CC) represents the most aggressive form of gestational tumours. In Europe and North America, gestational CC occurs in approximately 1/50 000 deliveries.1 We report the detection, in a gestational CC developed in a female partner of a patient with Li-Fraumeni syndrome (LFS) (MIM #151623), of the germline TP53 mutation initially detected in this LFS patient. In the French LFS series, we identified 78 fathers who were carriers of a germline TP53 mutation. Among the 213 corresponding pregnancies, we found two other cases of gestational CC in their partners. We estimate that gestational CC occurs in approximately 1% of the deliveries in female partners of TP53 mutation carriers. Gestational trophoblastic disease (GTD), which can occur after either abnormal or normal fertilisation, is characterised by the uncontrolled proliferation of trophoblastic cells normally producing the placenta. GTD includes premalignant (complete and partial hydatidiform moles) and malignant (invasive mole, gestational CC, placental-site trophoblastic and epithelioid trophoblastic tumours) lesions.1 We considered the diagnosis of LFS, a remarkable cancer predisposition characterised by the extent of tumour spectrum,2 in the family described in figure 1. The male index case had developed a cholangiocarcinoma at 37 years of age, …

To the Editor: Epidermal growth factor receptor is frequently overexpressed in colorectal cancer, and targeted therapies have been developed to block this receptor. Monoclonal antibodies such as cetuximab and panitumumab have demonstrated effectiveness in increasing the survival rates for some metastatic patients. Patients with mutations in codon 12 or 13 of the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) gene are not responsive to this therapy, however, and tumors from patients are systematically screened for somatic KRAS mutations before treatment. Several techniques have been developed to screen for KRAS mutations, and the differences in these techniques with respect to diagnostic sensitivity, diagnostic specificity, and efficiency for mutation detection have been described (1,–,4). The main potential weakness of direct sequencing is its low diagnostic sensitivity (approximately 20%). Other techniques, such as TaqMan® PCR–based assays and SNaPshot® (Applied Biosystems), have diagnostic sensitivities between 1% and 10%. In this study, we analyzed 1970 colorectal carcinoma tumors for KRAS mutations by direct sequencing. We detected 10 cases …

The aim of this study was to compare the detection of BRAF(V600E) by immunohistochemistry (IHC) using a mutation-specific antibody with molecular biology methods for evaluation of papillary thyroid carcinoma (PTC) patients.This study concerned 198 consecutive conventional PTC patients, of which the majority were women (133/198; 67%), with a mean age of 56 years (range 19-79 years). BRAF mutation analysis was performed using DNA-based (direct sequencing, pyrosequencing, and SNaPshot) and IHC (VE1 antibody) methods. The sensitivity and specificity of IHC for BRAF(V600E) was compared with the molecular biology data.A BRAF mutational result was obtained in 194 cases. A BRAF(V600E) mutation was detected in 153/194 (79%) cases of PTC when using at least one molecular method, and in 151/194 (78%) cases with IHC. No false positive results were noted using IHC to detect the BRAF(V600E) mutation. Besides this mutation, other rare BRAF mutations (BRAF(V600K) and BRAF(K601E)), used as negative controls, were consistently negative with IHC. The sensitivity and specificity of IHC for the detection of this mutation were 98.7% and 100% respectively. The IHC test demonstrated excellent performance at a level equivalent to two DNA-based counterparts (pyrosequencing and SNaPshot). Failure to achieve a result was more frequent with the direct sequencing method than with the three other methods.IHC using the VE1 antibody is a specific and sensitive method for the detection of the BRAF(V600E) mutation in PTC. IHC may be an alternative to molecular biology approaches for the routine detection of this mutation in PTC patients.

Background: To compare the genetic profile and phenotype of sinonasal Intestinal Type AdenoCarcinoma (ITAC) and colorectal adenocarcinoma. Methods: Between 1983 and 2001, 41 patients were treated for ethmoidal adenocarcinoma at Rouen University Hospital. All pathologic specimens were reassigned according to the new 2005 World Health Organization classification. Immunohistochemical study was carried out to evaluate EGFR and CDX2 expression. Thirty-eight out of the 41 tumor specimens had sufficient DNA for KRAS and EGFR mutation analysis. SNaPshot® multiplex system was used to determine presence of the most common mutations (located in exons 18, 19, 20 and 21 for EGFR and in exon 2 [codon 12 and 13] for KRAS). Results: Of the 41 patients, there were 37 men and four women. Mean age at presentation was 63.6 years (range: 40.7-86.4 years). Occupational exposure was documented for 32 patients, with 31 cases of wood exposure and one of leather exposure. Of the 38 tumors genotyped, 35 were ITAC (33 men and two women) and wood exposure was found in 29 (85%) of these patients. CDX2 expression was present in 31 out of the 35 ITAC (89%) and absent in the 3 non-intestinal adenocarcinomas. EGFR was expressed in 29 out of the 35 ITAC (83%) with different expression: 19 (56 %) 1+, 7 (21%) 2+ and 3 (6%) 3+ immunopositivity and the 3 non ITAC disclosed 1+ EGFR positivity. No EGFR mutation was found in the series. For KRAS, 5 out of the 35 ITAC (14%) disclosed KRAS exon 2 mutation and the 3 non-intestinal adenocarcinomas were KRAS wild type. Conclusion: CDX2 immunohistochemistry could be a useful tool for discriminating ITAC. Phenotype and genotype similarities between ITAC and colorectal carcinomas could lead to clinical trials using anti-EGFR therapy in patients with locally advanced or metastatic ITAC.

Context.—Current clinicopathologic assessment of malignant neoplastic diseases entails the analysis of specific genetic alterations that provide diagnostic, prognostic, or therapy-determining information.Objective.—To develop and validate a robust molecular method to detect clinically relevant mutations in various tissue types and anatomic pathology specimens.Design.—Genes of interest were amplified by multiplex polymerase chain reaction and sequence variants identified by liquid bead array cytometry. The BRAF assay was fully characterized by using plasmids and genomic DNA extracted from cell lines, metastatic colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissues, and thyroid nodule fine-needle aspirates.Results.—Qualitative multiplex assays for 22 different mutations in the BRAF, HRAS, KRAS, NRAS, or EGFR genes were established. The high signal-to-noise ratio of the technology enabled reproducible detection of BRAF c.1799T>A (p.V600E) at 0.5% mutant allele in 20 ng of genomic DNA. Precision studies with multiple operators and instruments showed very high repeatability and reproducibility with 100% (98.7%–100%) qualitative agreement among 292 individual measures in 38 runs. Evaluation of 1549 representative pathologic specimens in 2 laboratories relative to independent reference methods resulted in 99.0% (97.6%–99.6%) agreement for colorectal FFPE tissues (n = 416) and 98.9% (98.2%–99.4%) for thyroid fine-needle aspiration specimens (n = 1133) with an overall diagnostic odds ratio of 10 856 (2451–48 078).Conclusions.—The multiplex assay system is a sensitive and reliable method to detect BRAF c.1799T>A mutation in colorectal and thyroid lesions. This optimized technology platform is suitable for the rapid analysis of clinically actionable genetic alterations in cytologic and histologic specimens.

Background Prognosis of KRAS wild-type and mutant metastatic colorectal cancer (MCRC) patients (pts) treated with bevacizumab (BEV)-containing chemotherapy is not significantly different. Since specific KRAS mutations confer different aggressive behaviors, the prognostic role of prevalent KRAS mutations was retrospectively evaluated in MCRC pts treated with first line FIr-B/FOx, associating BEV to triplet chemotherapy. Methods Tumor samples were screened for KRAS codon 12, 13 and BRAF V600E mutations by SNaPshot and/or direct sequencing. MCRC pts 2 on days 1,2, 8, 9, 15, 16,22, 23), irinotecan plus BEV (160 mg/m2 and 5 mg/kg, respectively, on days 1,15); and oxaliplatin (80 mg/m2, on days 8,22). Pts were classified as liver-limited (L-L) and other/multiple metastatic (O/MM). Progression-free survival (PFS) and overall survival (OS) were compared using the log-rank test. Results Fifty-nine pts were evaluated at a median follow-up of 21.5 months. KRAS mutant pts: c.35 G > A, 15 (25.4%); c.35 G > T, 7 (11.8%); c.38 G > A, 3 (5%); other, 3 (5%). KRAS wild-type, 31 pts (52.7%). The objective response rate (ORR), PFS and OS were, respectively: c.35 G > A mutant, 71%, 9 months, 14 months; other than c.35 G > A mutants, 61%, 12 months, 39 months. OS was significantly worse in c.35 G > A pts compared to KRAS wild-type (P = 0.002), KRAS/BRAF wild-type (P = 0.03), other MCRC patients (P = 0.002), other than c.35 G > A (P = 0.05), other codon 12 (P = 0.03) mutant pts. OS was not significantly different compared to c.35 G > T KRAS mutant (P = 0.142). Conclusions KRAS c.35 G > A mutant status may be significantly associated with a worse prognosis of MCRC pts treated with first line FIr-B/FOx intensive regimen compared to KRAS/BRAF wild type and other than c.35 G > A mutant pts.

Introduction La mutation du gene BRAF ( v-Raf murine sarcoma viral oncogene homolog B ) est un driver oncogenique. Sa prevalence est faible, de l’ordre de 1 a 4 % des patients atteints de cancers bronchiques non a petites cellules (CBNPC). Il existe peu de publications concernant les caracteristiques cliniques et la survie des patients associees a ces mutations. Methodes Nous avons collecte, de maniere retrospective, les caracteristiques demographiques, cliniques et anatomopathologiques ainsi que les donnees de survie de patients porteurs de CBNPC mutes BRAFV600E ayant beneficie d’une recherche de biomarqueurs dans le laboratoire de biologie moleculaire de Rouen entre septembre 2011 et decembre 2015. Nous avons ensuite compare les donnees de survie de ces patients mutes BRAF a celles de patients non mutes EGFR, BRAF, KRAS, HER2, PIK3CA, ALK, ROS et MET. Resultats De septembre 2011 a decembre 2015, sur 2412 patients porteurs de CBNPC ayant beneficie d’une recherche de biomarqueurs, la mutation BRAF a ete identifiee chez 41 patients (1,7 %). Le sex-ratio hommes/femmes etait de 1,6. L’âge median au diagnostic etait de 63 ans. Il y avait 61 % de fumeurs essentiellement des hommes et 22 % de femmes non fumeuses. L’etat general etait bon (PS 0–1) pour 71 % des patients. Trente patients (73 %) etaient metastatiques au diagnostic. Un patient presentait une co-mutation KRAS. Chez 6 patients a ete diagnostique un cancer synchrone, pour l’un d’eux, il s’agissait d’un autre adenocarcinome bronchique mute KRAS. Six patients ont beneficie de therapies ciblees anti-BRAF avec une mediane de survie de 34,8 mois. La mediane de survie des patients mutes, tous stades confondus, etait de 11,5 mois, identique a celle des non-mutes. Conclusion Ce travail porte sur une grande serie de patients porteurs de CBNPC mutes BRAFV600E. Elle retrouve une proportion elevee d’hommes fumeurs. Les tumeurs BRAF presentaient frequemment un contingent micropapillaire. Cette serie ne confirme pas le pronostic pejoratif associe aux mutations BRAF avec des resultats encourageants chez les patients traites par inhibiteurs de BRAF.

Background Bevacizumab (BEV) plus triplet chemotherapy can increase efficacy of first-line treatment of metastatic colorectal cancer (MCRC), particularly integrated with secondary liver surgery in liver-limited (L-L) patients. The prognostic value of the KRAS genotype in L-L and other or multiple metastatic (O/MM) MCRC patients treated with the FIr-B/FOx regimen was retrospectively evaluated. Methods Tumoral and metastatic samples were screened for KRAS codon 12 and 13 and BRAF mutations by SNaPshot and/or direct sequencing. Fit MCRC patients 2, days 1, 2, 8, 9, 15, 16, 22 and 23; irinotecan (CPT-11) 160 mg/m2 plus BEV 5 mg/kg, days 1, 15; oxaliplatin (OXP) 80 mg/m2, days 8, 22; every 4 weeks. MCRC patients were classified as L-L and O/MM. Activity and efficacy were evaluated and compared using log-rank test. Results In all, 59 patients were evaluated: 31 KRAS wild-type (53%), 28 KRAS mutant (47%). At 21.5 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wild-type 90%, 14 months, 38 months; KRAS mutant 67%, 11 months, 20 months. PFS and OS were not significantly different. PFS and OS were significantly different in L-L compared to O/MM evaluable patients. In KRAS wild-type patients, clinical outcome of 12 L-L compared to 18 O/MM was significantly different: PFS 21 versus 12 months and OS 47 versus 28 months, respectively. In KRAS mutant patients, the clinical outcome of 13 L-L compared to 14 O/MM was not significantly different: PFS 11 months equivalently and OS 39 versus 19 months, respectively. Conclusions The KRAS genotype wild-type and mutant does not significantly affect different clinical outcomes for MCRC patients treated with the first-line FIr-B/FOx intensive regimen. KRAS wild-type patients with L-L disease may achieve a significantly prolonged clinical outcome due to integration with secondary liver surgery, with respect to KRAS mutant patients.

The quick emerging of the several targeted therapies and the concept of personnalized medicine underlie the necessity to develop and to well organize a molecular biology (or molecular pathology) unit of high quality, dedicated to clinical care, in order to look for tissular and cellular theragnosis biomarkers. This new and sudden area of activity for a clinical pathologist is strongly linked to the knowledge of a new medical speciality in health care institutions. Thus, the molecular pathology (or molecular biology made from cellular or tissular samples) can nicely be implemented in a clinical pathology laboratory. This new mission for a pathologist has to be done in respect with a great quality assurance which should allow obtaining in a short-term an ISO 15189 accreditation to keep going to perform this activity. The present work aims to describe the main steps to be set up in the order to get an ISO 15189 accreditation in molecular pathology. The different chapters of this norm will not be described in their exhaustivity, but in their large lines. Finally, we will describe the potential difficulties and pitfalls to be avoided before getting this accreditation.

The advent of the targeted cancer therapies administered to patients, according to the results of molecular biology techniques (in particular, in situ hybridization, "polymerase chain reaction" amplification and sequencing), has modified the practice of the surgical pathology laboratories. The necessity to answer to the needs of physicians for optimizing the medical care for patients who develop cancer has led to a policy of national debate, spurred by the National Institute of Cancer (INCa), in order to implement new procedures in the pathology laboratories. Thus, in addition to the structuring of molecular biology platforms and their labeling by INCa, the upstream control of the steps present between resection of tumor samples and molecular analysis has proved to be crucial. Indeed, the quality of this upstream time, called "pre-analytical" phase, determines the reliability of the molecular biology results and therefore the therapeutic strategy. We describe here the main steps to be checked in the pre-analytical phase. The optimization of this pre-analytical phase within the surgical pathology laboratory aims to reduce or render insignificant the risk of errors of molecular biology tests. These errors can indeed lead to false negative or false positive results whose therapeutic consequences can be particularly harmful to patients with cancer.

International audience; Recent studies have suggested that activation of the EGFR pathway leads to malignant transformation only if the p53 protein is inactivated. Therefore, we evaluated the impact of TP53 mutations on cetuximab-based chemotherapy (CT) sensitivity in combination with KRAS mutations that have been associated with cetuximab resistance. KRAS and TP53 status were assessed in tumours from 64 metastatic colorectal cancer patients treated with cetuximab-based CT and correlated to clinical response using the Fisher's exact test. Times to progression (TTPs) according to gene status were calculated using the Kaplan-Meier method and compared with log-rank test. TP53 mutations were found in 41 patients and were significantly associated with controlled disease (CD), as defined as complete response, partial response or stable disease (P=0.037) and higher TTP (20 vs 12 weeks, P=0.004). Remarkably, in the subgroup of 46 patients without KRAS mutation, but not in patients with KRAS mutation, TP53 mutations were also associated with CD (P=0.008) and higher TTP (24 vs 12 weeks, P=0.0007). This study suggests that TP53 mutations are predictive of cetuximab sensitivity, particularly in patients without KRAS mutation, and that TP53 genotyping could have a clinical interest to select patients who should benefit from cetuximab-based CT.

Introduction En Martinique, le cancer du poumon, bien que 5 e cause de cancer chez l’homme et 6 e chez la femme, reste respectivement la 2 e et 3 e cause de deces par cancer [1] . L’avenement des inhibiteurs de la tyrosine kinase a permis une amelioration de la survie des patients mutes EGFR [1] . Cette mutation est retrouvee chez 10,5 % des patients presentant un carcinome non a petites cellules non epidermoides. Nous avons recherche dans cette etude la frequence des mutations EGFR, dans les adenocarcinomes pulmonaires des patients diagnostiques en Martinique, population essentiellement afro-caribeenne peu tabagique, jusqu’ici non etudiee. Methodes Analyse retrospective des mutations EGFR retrouvees dans les carcinomes bronchiques non a petites cellules, non epidermoides, diagnostiques au CHU de Martinique entre janvier 2013 et mai 2015. Cette etude concerne les patients ayant beneficie d’une recherche de mutation theranostique. Resultats Quatre-vingt-onze cas d’adenocarcinome pulmonaire ont ete diagnostiques pendant cette periode et ont beneficie de la recherche de mutation EGFR. Trente-cinq (38 %) patients etaient porteurs d’une mutation de l’EGFR, contre 49 patients indemnes et 7 dont les prelevements n’etaient pas contributifs. Parmi les tumeurs mutees, 20 presentaient une deletion sur l’exon 19 et 11 autres presentaient une mutation ponctuelle l858r de l’exon 21. Soixante-deux pour cent des patients mutes etaient des femmes contre 33 % dans le groupe non mute ( p = 0,006). Il n’y a pas de difference d’âge entre les 2 groupes ( p > 0,05). Conclusion Notre population, principalement antillaise, presente, en particulier chez les femmes, un taux de mutation EGFR tres superieur a celui releve usuellement chez les caucasiens. Ces patients doivent beneficier d’une therapie ciblee par inhibiteurs de tyrosine kinase. Il importe de poursuivre l’analyse des facteurs cliniques associes a la mutation.

Contexte L’EBUS radiale par minisonde a montre son interet pour le diagnostic histologique des nodules pulmonaires peripheriques (NPP). Cependant les prelevements obtenus sont de petite taille, et il n’y a pas de donnees disponibles concernant l’analyse moleculaire des adenocarcinomes (ADK) diagnostiques sur ces petits prelevements. Objectif Determiner la rentabilite des prelevements realises par EBUS radiale pour l’analyse moleculaire des ADK. Methode Tous les patients ayant beneficie d’une EBUS radiale pour l’exploration d’un NPP de janvier 2010 a juillet 2014 au CHU de Rouen ont ete selectionnes pour cette etude. L’analyse moleculaire (EGFR, Kras, Alk, Her2, PI3 K, Braf) etait realisee selon les techniques de routine par le laboratoire de genetique somatique des tumeurs du CHU de Rouen (plateforme INCA) a partir des etalements cytologiques ou des coupes en paraffine des ADK. Resultats Cinq cent seize EBUS radiales ont ete realisees dans la periode de l’etude. La sensibilite de l’examen pour les diagnostics de cancers etait de 78 %. Le diagnostic d’ADK a ete obtenu chez 193 patients (37 %). 118/193 ADK (61 %) ont pu faire l’objet d’une analyse moleculaire, identifiant 46 anomalies (Mutations Kras, Egfr, Her2 et Braf : 29, 10, 1 et 1 respectivement, translocation ALK : 5). Le taux de faisabilite de l’analyse moleculaire des ADK passait de 45 % en 2010–2011 a 70 % en 2012–2013 et 89 % depuis debut 2014. Conclusion L’analyse moleculaire des ADK est faisable sur une majorite des prelevements realises par EBUS. L’optimisation de la gestion des prelevements se traduit par une augmentation de la rentabilite de l’analyse moleculaire.

Among the identified factors involved in malignant transformation, abnormal methylation of the CDKN2A/p16(INK4a) gene promoter has been described as an early event, particularly in bronchial cell cancerization. Precancerous bronchial lesions (n = 70) prospectively sampled during fluorescence endoscopy in a series of 37 patients at high risk for lung cancer were studied with respect to the methylation status of the CDKN2A gene. Methylation-specific polymerase chain reaction was performed on DNA extracted from pure bronchial cell populations derived from biopsies and detection of p16 protein was studied by immunohistochemistry on contiguous parallel biopsies. Aberrant methylation of the CDKN2A gene promoter was found in 19% of preinvasive lesions and its frequency increased with the histologic grade of the lesions. Methylation in at least 1 bronchial site was significantly more frequent in patients with cancer history, although there was no difference in the outcome of patients with or without methylation in bronchial epithelium. The other risk factors studied (tobacco and asbestos exposure) did not influence the methylation status. There was no relationship between CDKN2A methylation and the evolutionary character of the lesions. Our results confirm that abnormal methylation of the CDKN2A gene promoter is an early event in bronchial cell cancerization, which can persist for several years after carcinogen exposure cessation, and show that this epigenetic alteration cannot predict the evolution of precancerous lesions within a 2-year follow-up.

e14596 Background: Prognosis of MCRC patients (pts) treated with bevacizumab (BEV) and chemotherapy (CT) is not significantly different according to KRAS genotype. Specificmutations confer different aggressiveness. Prognostic relevance of the prevalent c.35 G > A KRAS mutation was retrospectively evaluated in pts treated with first line FIr-B/FOx, and post-progression. Methods: KRAS codon 12/13 and BRAF mutations were screened by SNaPshot and/or sequencing. FIr-B/FOx: weekly 2 days/12h-timed-flat-infusion/5-fluorouracil 900 mg/m2, weekly alternating irinotecan 160 mg/m2/BEV 5 mg/kg, or oxaliplatin 80 mg/m2. Second-line treatment was selected according to fitness, KRAS genotype, previous efficacy and safety. Efficacy was evaluated and compared according to treatment or KRAS genotype, using log-rank. Results: At 21.5 months, 59 pts were evaluated. KRAS mutant pts: c.35 G > A, 15 (25.4%); c.35 G > T, 7 (11.8%); c.38 G > A, 3 (5%); other, 3 (5%). KRAS wild-type, 31 pts (52.7%). ORR, PFS, OS were, respectively: c.35 G > A, 71%, 9 months, 14 months; other mutants, 61%, 12 months, 39 months. OS was significantly worse in c.35 G > A pts compared to KRAS wild-type (p = 0.002), KRAS/BRAFwild-type (p = 0.03), other pts (p = 0.002), other mutants (p = 0.05), other codon 12 (p = 0.03) mutant pts; PFS was not significantly different. Post-progression, at 14 months follow-up, PFS and OS were significantly worse in c.35 G > A compared to wild-type and/or other mutant pts. ORR of second line treatments was 38%, metastasectomies 12.5%, PFS 10 months, OS 14 months. PFS and OS were significantly favourable in pts treated with triplet CT plus targeted agent compared to triplet, respectively: PFS 13 months versus 8 months; OS not reached versus 11 months. Conclusions: KRAS c.35 G > A mutation may significantly affect worse OS of MCRC pts. It does not significantly affect worse PFS of intensive first line FIr-B/FOx, while after progression significantly affect worse efficacy of second line treatments. Re-challenge of intensive regimens may affect significantly favourable PFS and OS.

Background Bevacizumab (BEV) plus triplet chemotherapy can increase efficacy of first-line treatment of MCRC (Bruera G et al, BMC Cancer 2010, 10:567), particularly if integrated with secondary liver surgery in liver-limited (L-L) patients (pts) (Bruera G et al, Clin Colorectal Cancer 2012). Clinical outcome of FIr-B/FOx regimen was evaluated according to KRAS genotype in L-L and other MCRC pts. Methods Tumoral and metastatic samples were screened for KRAS codon 12 and 13, and BRAF mutations by SNaPshot and/or direct sequencing. MCRC pts were classified as L-L and other or multiple metastatic (O/MM). Activity and efficacy were evaluated and compared using log-rank test. Results Fifty-nine pts were evaluated: 31 KRAS wild-type, 53%; 28 KRAS mutant, 47%. At 21.5 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wild-type 90%, 14 months, 38 months; KRAS mutant 67%, 11 months, 20 months. PFS and OS were, respectively: overall in 25 L-L compared to 32 O/MM evaluable pts, 17 and 12 months, 47 and 21 months, significantly different; in KRAS wild-type, 12 L-L compared to 18 O/MM, 21 and 12 months, 47 and 28 months, significantly different; in KRAS mutant, 13 L-L compared to 14 O/MMS, 11 months equivalently, 39 and 19 months, not significantly different. Conclusion First line FIr-B/FOx regimen can increase activity and efficacy of KRAS wild-type and mutant MCRC pts; integration with secondary liver surgery significantly discriminates increased clinical outcome in KRAS wild-type L-L compared to O/MM pts while not in KRAS mutant pts. Disclosure All authors have declared no conflicts of interest.

e14010 Background: Bevacizumab (BEV) plus triplet chemotherapy can increase efficacy of first-line treatment of MCRC, particularly if integrated with secondary liver surgery in liver-limited (L-L) patients (pts). Clinical outcome of FIr-B/FOx regimen was evaluated according to KRAS genotype in L-L and other MCRC pts. Methods: Tumoral and metastatic samples were screened for KRAS codon 12 and 13, and BRAF mutations by SNaPshot and/or direct sequencing. MCRC pts were classified as L-L and other or multiple metastatic (O/MM). Activity and efficacy were evaluated and compared using log-rank test. Results: Fifty-nine pts were evaluated: 31 KRAS wild-type, 53%; 28 KRAS mutant, 47%. At 21.5 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wild-type 90%, 14 months, 38 months; KRAS mutant 67%, 11 months, 20 months. PFS and OS were, respectively: overall in 25 L-L compared to 32 O/MM evaluable pts, 17 and 12 months, 47 and 21 months, significantly different; in KRAS wild-type, 12 L-L compared to 18 O/MM, 21 and 12 months, 47 and 28 months, significantly different; in KRAS mutant, 13 L-L compared to 14 O/MMS, 11 months equivalently, 39 and 19 months, not significantly different. Conclusions: First line FIr-B/FOx regimen can increase activity and efficacy of KRAS wild-type and mutant MCRC pts; integration with secondary liver surgery significantly discriminates increased clinical outcome in KRAS wild-type L-L compared to O/MM pts while not in KRAS mutant pts.

5565 Background: Sinonasal carcinomas are very rare tumors which are sometimes not eligible for curative surgery. Alternative therapies (i.e. conventional chemotherapies/radiotherapy) are used but with poor results. Therefore, the need for a more efficient treatment is mandatory. Aims of our study: Comparing EGFR and KRAS genetic profiles, EGFR and CDX2 phenotypes of sinonasal intestinal type adenocarcinomas (ITAC) with colorectal adenocarcinomas (CRC). Methods: 41 patients were treated in our institution between 1983 and 2007. All pathological specimens were reclassified according to the 2005 WHO classification. An immunohistochemical (IHC) study was carried out for EGFR and CDX2 expression. We were able to analyze 38 of the 41 specimens for KRAS and EGFR mutations. SNaPshot multiplex system was used to determine the presence of the most common mutations which are located in exon 18, 20 and 21 for EGFR and in exon 2 (codon 12 and13) for KRAS. Fragment analysis method was used for EGFR exon 19 deletions. Results: Thirty five of the 38 patients were classified as ITAC (33 men and 2 women). The mean age was 64.5 years. Exposure to wood work was found in 29 cases (85%). CDX2 expression was present in 31 (89%) cases of ITAC and absent in all non intestinal adenocarcinomas (3 cases). EGFR was expressed in 29 ITACs (83%) with various degrees of IHC expression: 19 (56 %) 1+, 7 (21%) 2+, and 3 with 3+ immunopositivity. No EGFR mutation was found in the whole population; 5 ITAC patients (14%) disclosed KRAS mutations. Conclusions: Histological, phenotype and genetic profiles of ITAC are very similar to those of colorectal adenocarcinoma. These results suggest that ITACs with wt KRAS could respond to anti MoAb anti-EGFR therapy in the same way as metastatic CRC. We propose that all sinonasal tumors should undergo: firstly, CDX2 IHC in order to confirm the ITAC histological subtype and then KRAS genotyping to select the wt population which could benefit from such anti EGFR targeted therapy.

Introduction Most of sporadic or hereditary colorectal cancers (CRC) with microsatellite instability (MSI) have a peculiar histopathologic pattern, notably with a frequent inflammatory lymphocytic component. MSI CRC are associated with a better outcome than microsatellite stable (MSS) CCR, probably in relation with a more effective immune response. The aim of our work was to highlight the inflammatory mechanisms occurring in MSI CRC, by analyzing the cytokines and the in situ immune response encountered in this tumor subtype. Methods A panel of 48 cytokines was measured from 48 MSI CRC and 62 MSS CRC using the bioplex cytokine multiplexed assay (Biorad, Hercules, CA, USA). Immunohistochemistry was performed on a tissue microarray constructed from the same tumors, with the following markers: CD3 (T lymphocytes), CD8 (cytotoxic T lymphocytes), CD45RO (memory T lymphocytes), T-bet (Th1 CD4 cells) and FoxP3 (regulatory T-cells). After immunostaining of each marker, T-cell density was assessed by image analysis. Results Analysis of the 48 cytokines allowed us to assess a higher level of IL1β, IL1-Rα, IL16, Gro-a, IL-8, Ip-10, Mig, Mip-1α, Mip-1β, Rantes and IFN-γ in MSI CRC than in MSS CRC (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 317. doi:1538-7445.AM2012-317

235 Background: Pancreatic adenocarcinomas account for 90% of pancreatic malignancies. It is often diagnosed at an unresectable stage (at a metastatic and / or at a locoregional invasion stage). Histological diagnosis is mandatory to establish palliative chemotherapy. Endoscopic ultrasound-guided fine needle aspiration (EUGA) of pancreatic tumors is a difficult gesture, not without risk. Furthermore, it frequently provides pauci-cellular, hemorrhagic or necrotic material unsuitable for precise histologic diagnosis. Techniques for detecting circulating tumor cells (CTC) have been recently developed to highlight the presence of malignant cells circulating in peripheral blood. Screencells technology is based on the separation of CTCs from other circulating cells by blood filtration through a polycarbonate membrane with 7m pores. The aims of this pilot study were 1) to compare the results of detections CTC to EUGA, 2) to assess the feasibility and the contribution of this innovative technique in the care of a limited series of patients. Methods: A sample of peripheral venous blood was performed in 15 patients with a suspicious CT scan of a pancreatic head tumoral lesion, before the completion of an EUGA. 4 ml of blood were filtered using ScreencellsCyto columns. After Giemsa staining, a pathologist analyzed each filter. Cells visualized on the filter under light microscopy were considered as tumoral if they met the following morphological criteria: anisocytosis, anisokaryosis, nuclear diameter>7m, nuclear membrane irregularitie, large nucleolus. Results: The malignant feature of the pancreatic lesion could be histologically assessed after liver biopsy for two patients. EUGA showed malignancy in 10 cases. CTC presence was identified in 7 patients with tumoral histological diagnosis. Conclusions: This study demonstrates the feasibility of CTC detection in patients with pancreatic head tumor. The identification of CTCs could facilitate patient care, thus avoiding difficult and potentially dangerous EUGA. A prospective trial is needed to confirm the positive predictive value of this biomarker and to evaluate its contribution in the diagnosis procedure of such tumors.

521 Background: Bevacizumab (BEV) addition to doublet chemotherapy significantly increases efficacy without differentially affecting prognosis in KRAS wild-type (wt) and mutant (m) MCRC. Present study evaluates clinical outcome of BEV added to triplet chemotherapy, FIr-B/FOx (Bruera G et al, BMC Cancer 2010, 10:567), according to KRAS genotype. Methods: MCRC patients (pts) were treated with first line FIr-B/FOx regimen including BEV (5 mg/kg, days 1,15) and triplet chemotherapy, weekly alternating irinotecan (160 mg/m2 days 1,15) or oxaliplatin (80 mg/m2, days 8, 22) associated to fluorouracil (900 mg/m2, days 1-2, 8-9, 15-16, 22-23); every 4 weeks. Tumoral samples were simultaneously screened for KRAS codon 12 and 13, and BRAF c.1799 T > A mutations by SNaPshot multiplex assay (Di Fiore F, Br J Cancer 2007;96(8):1166-1169) and/or direct sequencing. Activity and efficacy were evaluated and compared using log-rank test. Results: Forty-five MCRC pts were evaluated: 25 KRAS wt (56%), 20 KRAS m (44%). After 30 months median follow-up, objective response rate (ORR), progression-free survival (PFS) and overall survival (OS) were, respectively: KRAS wt 88% (C.I. ± 14), 14 months, 38 months; KRAS m 80% (C.I. ± 19), 12 months, 21 months. PFS and OS were not significantly different. Among KRAS m pts, ORR, PFS and OS at 19 months median follow-up in c.35 G > A (13 pts) were 69% (C.I. ± 26), 9 months, 11 months, respectively. OS of c.35 G > A KRAS m was significantly worse compared to KRAS wt (p = 0.003) and to other than c.35 G > A mutant pts (p = 0.022). Conclusions: First line FIr-B/FOx intensive regimen adding BEV to triplet chemotherapy can further increase activity and efficacy without significantly affecting different clinical outcome in KRAS wt and m MCRC pts. A significant interaction between the most prevalent c.35 G > A KRAS mutation and worse prognosis was observed, compared to KRAS wt pts and to other than c.35 G > A KRAS exon 2 mutations, depending from different biological aggressiveness and sensitivity to BEV addition to triplet chemotherapy.