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OBJECTIVES Epidermal growth factor receptor (mEGFR) and v-Ki-ras2 Kirsten rat sarcoma viral oncogen homologue (mKRAS) mutations are the two main oncogenic drivers in resected non-small-cell lung cancer (NSCLC). We aimed to evaluate the correlation between histopathological prognostic factors and these mutations in resected NSCLC. METHODS We retrospectively reviewed data from 841 patients who underwent a surgical resection with a curative intent for NSCLC between 2007 and 2012. RESULTS KRAS mutations were observed in 255 patients (32%) and mEGFR in 103 patients (12%). A correlation was observed between mKRAS patients and lymph node involvement [Cramer's V: 0.451, P < 0.001, OR: 7.5 (95% CI: 5.3-10.7), P < 0.001]. Otherwise, a correlation was observed between mKRAS and the risk of harbouring 2 N2 stations [Cramer's V: 0.235, P = 0.02, OR: 3.04 (95% CI: 1.5-6.3), P = 0.004]. High lymph node ratio and angioinvasion were also significantly more frequent in mKRAS [Cramer's V: 0.373, P < 0.001, OR: 6.37 (95% CI: 3.9-10.5), P < 0.001; and Cramer's V: 0.269, P < 0.001, OR: 3.25 (95% CI: 2.4-4.4), P < 0.001, respectively]. Skip N2 and microscopic N2 were significantly more frequent in mEGFR [Cramer's V: 0.459, P < 0.001, OR: 18 (95% CI: 5.6-57.8), P < 0.001; and (Cramer's V: 0.45, P < 0.001 OR: 21.14 (95% CI: 9.2-48.3), P < 0.001, respectively]. CONCLUSIONS We observed a correlation between mKRAS and negative histopathological prognostic factors and between mEGFR and positive prognostic factors. One can wonder whether histopathological prognostic factors are only clinical reflections of molecular alterations.

Background: We aimed to evaluate whether EGFR mutations (mEGFR) and KRAS amino acid substitutions can predict first site of recurrence or metastasis after non-small-cell lung cancer (NSCLC) surgery. Methods: Data were reviewed from 481 patients who underwent thoracic surgery for NSCLC between 2007 and 2012. Results: Patients with KRAS G12C developed significantly more bone metastases compared with the remainder of the cohort (59% vs 16%, P

Emerging data highlight different clinical behaviors according to KRAS amino acid substitutions (AASs) in patients with non-small-cell lung cancer (NSCLC). We aimed to evaluate whether different KRAS AASs were associated with different responses to chemotherapy.We retrospectively reviewed data from 1190 patients with KRAS mutations who underwent first-line platinum-based chemotherapy for stage IV NSCLC. The response to different chemotherapy regimens was evaluated using the Response Evaluation Criteria In Solid Tumors criteria (v 1.1). Overall survival and time to progression (TTP) were secondary endpoints.Taxane was associated with the best response in the entire cohort (odds ratio, 2.52; 95% confidence interval [CI], 1.82-3.48; P .001), especially in G12V patients (odds ratio, 2.15; 95% CI, 1.05-4.41; P = .036). Taxane was associated with improved TTP in the entire cohort (hazard ratio [HR], 0.31; 95% CI, 0.26-0.38; P .001), especially in G13D patients (HR, 0.47; 95% CI, 0.22-1.01; P = .054). Pemetrexed was associated with the worst TTP in the entire cohort, particularly in G12V patients, who had the worst response rates (HR, 0.55; 95% CI, 0.30-0.99; P = .049). No impact on overall survival was observed according to different chemotherapy regimens and AASs.KRAS-specific AAS appears to induce different responses to chemotherapy regimens after first-line platinum-based chemotherapy in advanced NSCLC.

International audience; BACKGROUND: Pyrosequencing is recognized as a strong technique to analyze the MGMT status of glioblastoma patients. The most commonly used assay, quantifies the methylation levels of CpGs 74 to 78. A more recent CE-marked In Vitro Diagnostic Medical Device (CE-IVD) assay, Therascreen, analyzes CpGs 76-79.METHODS: We performed a comparison of these two assays to evaluate the potential impact of this shift in analyzed CpGs. Therascreen analysis was centrally performed for 102 glioblastoma patients, who were part of a prospective multicenter trial.RESULTS: A strong correlation was observed for the mean values of the 4 or 5 analyzed CpGs, with lower values recorded using the Therascreen assay, especially for values greater than 20%. When considering a classification in 3 categories (> 12%: methylated; ⩽ 8%: unmethylated; 9-12%: grey zone), 93% of patients were identically classified between the two assays. Using a binary classification, 95% and 97% of patients were identically classified with cut-offs of 8% and 12%, respectively. A strong prognostic significance was observed for both assays: median overall survival were 15.9 months and 34.9 months for respectively unmethylated and methylated patients with either test. CONCLUSION: The results demonstrate that these assays may be used interchangeably.

// Benoit Romain 1, 2 , Radhia Benbrika-Nehmar 1 , Laetitia Marisa 3 , Michele Legrain 4 , Viviane Lobstein 1 , Attila Oravecz 5 , Laetitia Poidevin 5 , Cyril Bour 1 , Jean-Noel Freund 6 , Isabelle Duluc 6 , Dominique Guenot 1 , Erwan Pencreach 1, 4, 7 1 Universite de Strasbourg, Progression Tumorale et Microenvironnement, Approches Translationnelles et Epidemiologie, Strasbourg, France 2 Hopitaux Universitaires de Strasbourg, Service de Chirurgie Generale et Digestive, Strasbourg, France 3 Cartes d’Identite des Tumeurs Program, Ligue Nationale Contre le Cancer, Paris, France 4 Hopitaux Universitaires de Strasbourg, Laboratoire de Biochimie et Biologie Moleculaire, Strasbourg, France 5 Universite de Strasbourg, CNRS, Department of Computer Science, ICube, Strasbourg, France 6 Universite de Strasbourg, INSERM Unit 1113, Strasbourg, France 7 Hopitaux Universitaires de Strasbourg, Centre de Ressources Biologiques, Departement de Pathologie, Strasbourg, France Correspondence to: Dominique Guenot, email: dominique.guenot@inserm.u-strasbg.fr Keywords: chemokine, valproate, butyrate, PCAF, acetylation Received: March 17, 2016 Accepted: February 27, 2017 Published: March 17, 2017 ABSTRACT CXCL12 has been shown to be involved in colon cancer metastasis, but its expression level and molecular mechanisms regulating its expression remain controversial. We thus evaluated CXCL12 expression in a large cohort of colon adenomas and carcinomas, investigated for an epigenetic mechanism controlling its expression and evaluated the impact of CXCL12 levels on cell migration and tumor growth. CXCL12 expression was measured in human colon adenomas and carcinomas with transcriptome array and RT-qPCR. The promoter methylation was analyzed with whole-genome DNA methylation chips and protein expression by immunohistochemistry. We confirm a reduced expression of CXCL12 in 75% of MSS carcinomas and show that the decrease is an early event as already present in adenomas. The methylome analysis shows that the CXCL12 promoter is methylated in only 30% of microsatellite-stable tumors. In vitro , treatments with HDAC inhibitors, butyrate and valproate restored CXCL12 expression in three colon cell lines, increased acetylation of histone H3 within the CXCL12 promoter and inhibited cell migration. In vivo , valproate diminished (65%) the number of intestinal tumors in APC mutant mice, slowed down xenograft tumor growth concomitant to restored CXCL12 expression. Finally we identified loss of PCAF expression in tumor samples and showed that forced expression of PCAF in colon cancer cell lines restored CXCL12 expression. Thus, reduced PCAF expression may participate to CXCL12 promoter hypoacetylation and its subsequent loss of expression. Our study is of potential clinical interest because agents that promote or maintain histone acetylation through HDAC inhibition and/or HAT stimulation, may help to lower colon adenoma/carcinoma incidence, especially in high-risk families, or could be included in therapeutic protocols to treat advanced colon cancer.

Objectives The impact of skip N2 metastases (i.e. N2 lymph node metastases without N1) on survival in surgically resected non-small lung cancer remains an intriguing and rarely investigated topic. The goal of our study was to elucidate (i) skip N2 influence on overall survival (OS) and time to recurrence (TTR) in patients with resected lung adenocarcinoma and (ii) its link with epidermal growth factor receptor ( EGFR ) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog ( KRAS ) mutations. Methods A retrospective analysis of 279 consecutive patients with lung pN2 adenocarcinoma, operated in two institutions between 2003 and 2013, was conducted. OS and TTR were calculated using the Kaplan-Meier method. Crude and multivariable-adjusted comparisons by skip N2 for OS and TTR were performed using the Cox method with shared frailty (accounting for the within-centre correlation). Associations between skip N2 metastasis, clinicopathological characteristics and EGFR and KRAS mutations were investigated using the Fisher exact test and Cramer's V -test. Results The mean age at the time of surgery was 63 years (±12), and the median follow-up time was 36 months (min 3; max 101). Skip N2 was observed in 54 patients (19%). EGFR mutations were observed in 38 patients (14%); KRAS mutations were seen in 86 patients (31%). Patients with skip N2 metastasis were predominantly non-smokers ( P = 0.001), underwent segmentectomy or limited resections ( P = 0.004) and were not submitted to adjuvant therapy ( P = 0.022). Moreover, there was a correlation between EGFR mutations and skip N2 (Cramer's V : 0.25, P < 0.001). Indeed, EGFR mutations were significantly more frequent in skip N2 tumours (33%) compared with non-skip tumours (10%), P < 0.001. No correlation between skip N2 and KRAS mutations was observed (Cramer's V : 0.05, P = 0.46). The multivariable-adjusted model showed a significant skip N2 protective effect on OS (hazard ratio, HR 0.503; P = 0.014; 95% confidence interval, CI: 0.291-0.8704) but not on TTR (HR 0.788; P = 0.446; 95% CI: 0.427-1.454). Conclusions In our series, lung adenocarcinoma skip N2 metastasis demonstrated a favourable prognosis. The presence of EGFR mutations could have significance in the better survival and in the specific anatomic pathway of lymphatic metastases exhibited by skip N2 tumours.

// Stephane Renaud 1, 2, 3 , Francesco Guerrera 1, 4 , Joseph Seitlinger 1 , Lorena Costardi 4 , Mickael Schaeffer 5 , Benoit Romain 3, 6 , Claudio Mossetti 4 , Anne Claire-Voegeli 7 , Pier Luigi Filosso 4 , Michele Legrain 7 , Enrico Ruffini 4 , Pierre-Emmanuel Falcoz 1 , Alberto Oliaro 4 , Gilbert Massard 1 1 Department of Thoracic Surgery, Strasbourg University Hospital, Strasbourg, France 2 Department of Thoracic Surgery, Nancy University Hospital, Nancy, France 3 Research Unit EA3430, Tumoral Progression and Micro-Environment, Epidemiological and Translational Approaches, Strasbourg University, Strasbourg, France 4 Department of Thoracic Surgery, Azienda Ospedaliera Universitaria Citta della Salute e della Scienza di Torino, Torino, Italy 5 Department of Biostatistics, Strasbourg University Hospital, Strasbourg, France 6 Department of General and Digestive Surgery, Strasbourg University Hospital, Strasbourg, France 7 Department of Molecular Biology, Strasbourg University Hospital, Strasbourg, France Correspondence to: Stephane Renaud, email: sterenaud0@gmail.com Keywords: lung metastasectomy, colorectal cancer, KRAS, surgery, codon Received: August 25, 2016 Accepted: November 21, 2016 Published: November 29, 2016 ABSTRACT Introduction: The utilization of molecular markers as routinely used biomarkers is steadily increasing. We aimed to evaluate the potential different prognostic values of KRAS exon 2 codons 12 and 13 after lung metastasectomy in colorectal cancer (CRC). Results: KRAS codon 12 mutations were observed in 116 patients (77%), whereas codon 13 mutations were observed in 34 patients (23%). KRAS codon 13 mutations were associated with both longer time to pulmonary recurrence (TTPR) (median TTPR: 78 months (95% CI: 50.61–82.56) vs 56 months (95% CI: 68.71–127.51), P = 0.008) and improved overall survival (OS) (median OS: 82 months vs 54 months (95% CI: 48.93–59.07), P = 0.009). Multivariate analysis confirmed that codon 13 mutations were associated with better outcomes (TTPR: HR: 0.40 (95% CI: 0.17–0.93), P = 0.033); OS: HR: 0.39 (95% CI: 0.14–1.07), P = 0.07). Otherwise, no significant difference in OS ( P = 0.78) or TTPR ( P = 0.72) based on the type of amino-acid substitutions was observed among KRAS codon 12 mutations. Materials and Methods: We retrospectively reviewed data from 525 patients who underwent a lung metastasectomy for CRC in two departments of thoracic surgery from 1998 to 2015 and focused on 150 patients that had KRAS exon 2 codon 12/13 mutations. Conclusions: KRAS exon 2 codon 13 mutations, compared to codon 12 mutations, seem to be associated with better outcomes following lung metastasectomy in CRC. Prospective multicenter studies are necessary to fully understand the prognostic value of KRAS mutations in the lung metastases of CRC.

International audience; 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 (

OBJECTIVES The role of perioperative chemotherapy (POC) and targeted therapies in lung metastasectomy for colorectal cancer (CRC) is still subject to debate. We aimed to evaluate whether POC and targeted therapies were associated with different outcomes according to the mutational status. METHODS We reviewed data from 223 patients who underwent pulmonary metastasectomy for CRC from 1998 to 2015 and for whom the V-Ki-ras2 Kirsten sarcoma viral oncogene homologue (KRAS) and V-raf Murine sarcoma viral oncogene homologue B1 (BRAF) mutational statuses were known. RESULTS A total of 167 patients (74%) underwent POC: 62 (37%) received neoadjuvant therapy, 59 (35%) were in the adjuvant setting and 46 (28%) were in both the neoadjuvant and adjuvant settings. POC did not significantly influence either the loco-regional recurrence free survival (LRRFS) (P = 0.21) or the overall survival (OS) (P = 0.29). Furthermore, in cases of adjuvant chemotherapy, outcomes were not significantly different in cases of neoadjuvant chemotherapy or both neoadjuvant and adjuvant treatment (P = 0.26 for OS, P = 0.14 for LRRFS). For patients with KRAS mutation, perioperative bevacizumab was associated with a significant improvement in both LRRFS [70 months (41.58–98.42) vs 24 months (1.15–46.86), P = 0.001] and OS [101 vs 55 months (49.77–60.23), P = 0.004]. However, this benefit was only significant in cases of KRAS exon 2 codon 12 mutations [median OS: 101 months (83.97–118.02) vs 60 months (53–66.99), P

Colon carcinogenesis encompasses the stepwise accumulation of genomic aberrations correlated with the transition of aberrant crypt-adenoma-carcinoma. Recent data have revealed that, in addition to the microsatellite-instable phenotype, the chromosome instability pathway, representing four fifth of the colon carcinoma, could be involved in heterogeneous molecular alterations. Our project was aimed at determining the existence of distinct molecular subtypes in 159 non-microsatellite-instable colon polyps and their correlation with histology and dysplasia, using allelotyping, MGMT promoter gene methylation status, and K-RAS mutation analyses. Allelic imbalance, MGMT methylation, and K-RAS mutations arise in 62%, 39%, and 32% of polyps, respectively. Only 14% of polyps had no alterations. A 2-way hierarchical clustering analysis of the allelic imbalances identified subgroups of polyps according to their allelic imbalance frequency and distribution. Not only tubulovillous adenoma but also high-grade adenomas were correlated with high global allelic imbalance frequency (P = .005 and P = .003), with allelic imbalance at microsatellites targeting chromosomes 1, 6, and 9. In conclusion, the data presented in this study show that a large heterogeneity exists in the molecular patterns of alterations in precancerous colon lesions, favoring different modes of tumor initiation. Therefore, molecular alterations correlated with tubulovillous-type and high-grade dysplasia could represent targets identifying predictive factors of progression.

Among pediatric malignancies, medulloblastoma (MB) is one of the most common malignant tumors of the CNS. In the past few years, thanks to a multidisciplinary approach including surgery, chemo- and radiation therapy, survival has significantly improved. Despite that, a third of patients still have a low chance of being cured and long-term survivors experience severe treatment-related sequelae. MBs are usually classified according to a clinical risk stratification, based on histological features, age at diagnosis, extent of tumor resection and presence or absence of metastases. However, these clinical variables have recently been reported to be poor for defining risk-related disease. Retrospective studies have identified histological or biological factors that have distinct roles in prognosis. As several pathways have been discovered to be involved in MB pathogenesis, they should be taken into account to more accurately stratify patients and their treatment and to develop innovative therapies.

Introduction Recent clinical success of epidermal growth factor (EGFR)-tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) have raised hopes that targeting other deregulated growth factor signaling, such as the hepatocyte growth factor/MET pathway, will lead to new therapeutic options for NSCLC. Furthermore, NSCLC present secondary EGFR-TKIs resistance related to exons 20 and 19 EGFR mutations or more recently to MET amplification. The aim of this study was to determine MET copy number related to EGFR copy number and K-Ras mutations in a targeted TKI naive NSCLC cohort. Methods We investigated 106 frozen tumors from surgically resected NSCLC patients. Genes copy number of MET and EGFR were assessed by quantitative relative real-time polymerase chain reaction and K-Ras mutations by sequencing. Results MET is amplified in 22 cases (21%) and deleted in nine cases (8.5%). EGFR is amplified in 31 cases (29%). K-Ras is mutated in 11 cases (10.5%). As observed for EGFR amplification, MET amplification is never associated with K-Ras mutation. MET amplification could be associated with EGFR amplification. MET amplification is not related to clinical and pathologic features. MET amplification and EGFR amplification showed a trend toward poor prognosis in adenocarcinomas. Conclusion In EGFR-TKIs naive NSCLC patients, MET amplification is a frequent event, which could be associated with EGFR amplification, but not with K-Ras mutation. MET amplification may identify a subset of NSCLC for new targeted therapy. It will also be important to evaluate MET copy number to properly interpret future clinical trials.

Background: Our study aimed to evaluate response rate (RR) to brain metastasis radiotherapy (RT), depending on the genomic status of non-small-cell lung cancer. Material & methods: We retrospectively reviewed 1971 non-small-cell lung cancer files of patients with EGFR and KRAS testing and focused on 157 patients who had undergone RT for brain metastasis. Results: A total of 16 patients (10.2%) harbored EGFR mutations (mEGFR) and 45 patients (28.7%) KRAS (mKRAS). In univariate analysis, RR was significantly higher for mEGFR compared with wild-type EGFR/KRAS (odds ratio [OR]: 4.96; p = 0.05) or mKRAS (OR: 1.81; p = 0.03). In multivariate analysis, KRAS G12V or G12C status was associated with both poor RR (OR: 0.1; p < 0.0001) and overall survival (OR: 3.41; p < 0.0001). Conclusion: mEGFR are associated with higher RR to brain RT than wild-type EGFR/RAS or mKRAS.

Biobanking or collection and storage of specimens for future research purposes have become an essential tool in many fields of biomedical research and aims to provide a better understanding of disease mechanisms as well as the identification of disease-specific biomarkers that can navigate in complex diseases. In this study, we assessed the use of Flinders Technology Associates (FTA) cards as a long-term storage device for cervical specimens with suspected human papillomavirus (HPV) infections. HPV detection and genotyping results in liquid-based transport media were compared to HPV results from FTA cards. The overall agreement for the presence of any HPV infection between liquid-based medium and FTA cards stored for 1 year at ambient temperature was 100%. Reproducibility analysis of HPV detection and genotyping from FTA cards demonstrated that FTA cards are a reliable medium to store and preserve viral nucleic acids. Biobanking of cervical cells on FTA cards may provide a key resource for epidemiological and retrospective HPV studies.

High grade gliomas (HGG) are usually associated with a very dismal prognosis, which was moderately improving in the last decade with the introduction of the alkylating agent temozolomide in their treatment. The methylation status of MGMT (O6 methylguanine DNA-methyltransferase) promoter is one of the strongest predictive and prognostic factors for the patient chemoresponse. For instance, the molecular method of assessment for MGMT promoter status is not standardized. In this background, we developed a fluorescent capillary gel electrophoresis-based methylation specific-PCR. This technique allowed a semi-quantitative estimate of the relative ratio between methylated and unmethylated alleles. The efficacy and accuracy of the technique was assessed in a retrospective cohort of 178 newly diagnosed adult HGGs, who were homogeneously treated. First, we analyzed the impact on survival of different cut-off points in the MGMT promoter methylation and, to go further, we correlated these different rates to other well-known prognostic molecular factors involved in adult HGGs. This strategy allowed to validate our technique as a very sensitive technique (detection of a low methylation percentage, < 5%), which was feasible in fresh-frozen as well as in FFPE samples and had the propensity to detect intra-tumor heterogeneity. This technique identified a new sub-group of anaplastic oligodendrogliomas or oligoastrocytomas defined by a minor methylation and a worse outcome and, therefore, will help to substratify accurately into more homogeneous subgroups of methylated tumors.

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. The online version of this article (doi:10.​1007/​s11060-013-1332-y) contains supplementary material, which is available to authorized users.; International audience; The methylation of O(6)-methylguanine DNA methyltransferase (MGMT) gene promoter is a key biological marker in clinical neuro-oncology. Nevertheless, there is no consensus concerning the best technique for its assessment. In a recent study comparing five methods to analyze MGMT status, we found that the best prediction of survival was obtained with a pyrosequencing (PSQ) test assessing methylation of 5 CpGs (CpGs 74-78). In the present study we extended our PSQ analysis to 16 CpGs (CpGs 74-89) identified as critical for transcriptional control of the gene. The predictive value of the methylation levels at each CpG, as well as the mean methylation levels of selected sets of consecutive CpGs was tested in a cohort of 89 de novo glioblastoma patients who had received standard of care treatment (Stupp protocol). Using an optimal risk cut-off, each CpG or combination of CpGs, was associated with overall survival (OS) and progression free survival. The best predictive models for OS after stratification on performance score and age were obtained with CpG 89, CpG 84 and mean methylation of CpG 84-88 (Hazard ratio (HR), 0.31; p

International audience; 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 \textless 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.

Background Until now, FISH has been the gold standard technique to identify HER2 amplification status in ambiguous cases of breast cancer. Alternative techniques have been developed to increase the capacities of investigating HER2 amplification status. The aims of this multicenter study in a large series of breast cancer patients were to prospectively compare the level of performance of CISH, SISH, and qPCR alternative techniques on paraffin-embedded core biopsies with “gold standard FISH” for evaluation of HER2 amplification status. Methods This study was performed on 840 cases scored by immunohistochemistry (IHC): 0=317 (38%), 1+=183 (22%), 2+=109 (13%), 3+=231 (27%). Each of the 15 French centers participating in the study analyzed 56 breast carcinoma cases diagnosed on fixed paraffin-embedded core biopsies. HER2 amplification status was determined by commercially available FISH used as the reference technique with determination of the HER2/CEN17 ratio or HER2 copy number status. The alternative techniques performed on the same cases were commercially available SISH or CISH and a common qPCR method especially designed for the study including a set of 10 primer pairs: 2 for HER2 (exons 8 and 26), 5 to evaluate chromosome 17 polysomy TAOK1, UTP6, MRM1, MKS1, SSTR2 and 3 for diploidy control TSN, LAP3 and ADAMTS16. Results The concordance between IHC and FISH was 96% to 95% based on the HER2/CEN17 ratio (n=766) or HER2 copy number (n=840), respectively. The concordance of the alternative techniques with FISH was excellent: 97% and 98% for SISH (498 and 587 cases), 98% and 75% for CISH (108 and 204 cases) and 95% and 93% (699 and 773 cases) for qPCR based on the HER2/CEN17 ratio or HER2 copy number, respectively. Similarly, sensitivity ranged from 99% to 95% for SISH, 100% to 99% for CISH and 89% to 80% for qPCR. The concordance with FISH (ratio) in the 2+ cases was 89% for SISH, 100% for CISH and 93% for qPCR. Conclusion These alternative techniques showed an excellent concordance with FISH in core biopsies allowing their use in routine clinical practice. This newly designed qPCR on paraffin-embedded core biopsies deserves special attention, as it is reliable, easy to perform and less expensive than ISH tests.

International audience; BACKGROUND AND AIMS: O(6)-Methylguanine-DNA methyltransferase (MGMT) removes methyl adducts from O(6)-guanine. Known as methylation tolerance, selection for mismatch repair (MMR)-deficient cells that are unable to initiate lethal processing of O(6)-methylguanine-induced mismatches in DNA is observed in vitro as a consequence of MGMT deficiency. It was therefore hypothesised that an MGMT field defect may constitute a preneoplastic event for the development of MMR-deficient tumours displaying microsatellite instability (MSI). METHODS: MGMT expression was investigated by immunohistochemistry and the methylation status of the gene promoter by PCR in neoplastic, adjacent and distant mucosal tissues of patients with MSI or non-MSI (MSS) colorectal cancer (CRC). The cancers were familial (42 MSI, 13 MSS) or sporadic (40 MSI, 49 MSS) in origin, or arose in the context of inflammatory bowel disease (IBD; 13 MSI, 36 MSS). Colonic mucosa from patients with diverticulitis (n=20) or IBD (n=39 in 27 patients) without cancer served as controls. RESULTS: Loss of MGMT expression was more frequent in MSI than MSS CRC (p=0.047). In comparison with MSS tumours, MSI CRC occurred more frequently adjacent to patches of mucosa that lacked MGMT expression (p=0.002). Overall, loss of MGMT expression was associated with MGMT gene promoter methylation (p=0.03). CONCLUSION: MGMT field defects are more frequently associated with MSI than MSS CRC. These findings indicate that methylation tolerance may be a crucial initiating step prior to MMR deficiency in the development of MSI CRC in familial, sporadic and IBD settings.

International audience; Non-small cell lung cancers (NSCLC), in particular adenocarcinoma, are often mixed with normal cells. Therefore, low sensitivity of direct sequencing used for K-Ras mutation analysis could be inadequate in some cases. Our study focused on the possibility to increase the detection of K-Ras mutations in cases of low tumour cellularity. Besides direct sequencing, we used wild-type hybridisation probes and peptide-nucleic-acid (PNA)-mediated PCR clamping to detect mutations at codons 12 and 13, in 114 routine consecutive NSCLC frozen surgical tumours untreated by targeted drugs. The sensitivity of the analysis without or with PNA was 10 and 1% of tumour DNA, respectively. Direct sequencing revealed K-Ras mutations in 11 out of 114 tumours (10%). Using PNA-mediated PCR clamping, 10 additional cases of K-Ras mutations were detected (21 out of 114, 18%, P

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib improves survival of lung cancer as second- or third-line therapy. However, after an initial response, most patients will recur, particularly within the central nervous system. The present study reports the case of a 27-yr-old nonsmoking male presenting with a metastatic lung adenocarcinoma with EGFR exon 19 deletion, associated with sensitivity to EGFR-TKI. Gefitinib, followed by chemotherapy and finally erlotinib resulted in prolonged disease control, until multiple liver metastases were detected. After stopping EGFR-TKI, brain metastases with carcinomatous meningitis were diagnosed. A secondary T790M mutation, associated with resistance to EGFR-TKI, was found on the liver biopsy but not in the cerebrospinal fluid. Erlotinib was reintroduced and allowed a quick neurological improvement, even though the extra-cranial disease remained resistant to erlotinib. The present report underscores the interest of molecular monitoring in lung cancer. Persistent cerebral tyrosine kinase inhibitor sensitivity should be considered in patients presenting with an early central nervous system relapse after stopping epidermal growth factor receptor tyrosine kinase inhibitor, even with a T790M-resistant mutation in noncerebral metastases. Questions remain concerning the selection of sub-clones during epidermal growth factor receptor tyrosine kinase inhibitor therapy, which could differ according to metastatic sites, especially in the central nervous system.

Cytogenetically detectable elongation of the 15q proximal region can be associated with Prader–Willi/Angelman critical region interstitial duplications or with inherited juxtacentromeric euchromatic variants. The first category has been reported in association with developmental delay and autistic disorders. These pathogenic recurrent duplications are more frequently of maternal origin and originate from unequal meiotic crossovers between chromosome 15 low-copy repeats. 15q juxtacentromeric euchromatic variants reflect polymorphic copy number variations of segments containing pseudogenes and usually segregate without apparent phenotypic consequence. Pathogenic relevant 15q11–q13 duplications are not distinguishable from the innocuous euchromatic variants with conventional cytogenetic methods. We report cytogenetic and molecular studies of a patient with hypotonia, developmental delay and epilepsy, carrying, on the same chromosome 15, both a de novo 15q11–q13 interstitial duplication and an inherited 15q juxtacentromeric amplification from maternal origin. The duplication, initially suspected by fluorescent in situ hybridization (FISH), has been confirmed by molecular studies. The 15q juxtacentromeric region amplification, which segregates in the family for at least three generations, has been confirmed by FISH using BAC probes overlapping the NF1 and GABRA5 pseudogenes. This report emphasizes the importance to distinguish proximal 15q polymorphic variants from clinically significant duplications. In any patient with inherited 15q proximal variant but unexplained developmental delay suggesting 15q11–q13 pathology, a pathogenic rearrangement has to be searched with adapted strategies, in order to detect deletions as well as duplications of this region.

Sporadic early onset colorectal carcinoma (EOCRC) which has by definition no identified hereditary predisposition is a growing problem that remains poorly understood. Molecular analysis could improve identification of distinct sub-types of colorectal cancers (CRC) with therapeutic implications and thus can help establish that sporadic EOCRC is a distinct entity. From 954 patients resected for CRC at our institution, 98 patients were selected. Patients aged 45-60 years were excluded to help define "young" and "old" groups. Thirty-nine cases of sporadic EOCRC (patients ≤ 45 years with microsatellite stable tumors) were compared to both microsatellite stable tumors from older patients (36 cases, patients>60 years) and to groups of patients with microsatellite instability. Each group was tested for TP53, KRAS, BRAF, PIK3CA mutations and the presence of a methylator phenotype. Gene expression profiles were also used for pathway analysis. Compared to microsatellite stable CRC from old patients, sporadic EOCRC were characterized by distal location, frequent synchronous metastases and infrequent synchronous adenomas but did not have specific morphological characteristics. A familial history of CRC was more common in sporadic EOCRC patients despite a lack of identified hereditary conditions (p = 0.013). Genetic studies also showed the absence of BRAF mutations (p = 0.022) and the methylator phenotype (p = 0.005) in sporadic EOCRC compared to older patients. Gene expression analysis implicated key pathways such as Wnt/beta catenin, MAP Kinase, growth factor signaling (EGFR, HGF, PDGF) and the TNFR1 pathway in sporadic EOCRC. Wnt/beta catenin signaling activation was confirmed by aberrant nuclear beta catenin immunostaining (p = 0.01). This study strongly suggests that sporadic EOCRC is a distinct clinico-molecular entity presenting as a distal and aggressive disease associated with chromosome instability. Furthermore, several signaling pathways including the TNFR1 pathway have been identified as potential biomarkers for both the diagnosis and treatment of this disease.