Specific KRAS amino acid substitutions and EGFR mutations predict site-specific recurrence and metastasis following non-small-cell lung cancer surgery.

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<0.0001). This was confirmed in multivariate analysis (MA) (odds ratio (OR): 0.113 (95% confidence interval (CI): 0.055-0.231), P<0.0001). Significantly, more patients with mEGFR developed liver and brain metastases compared with the remainder of the cohort (30% vs 10%, P=0.006; 59% vs 1%, P<0.0001, respectively). These were confirmed in MA (OR: 0.333 (95% CI: 0.095-0.998), P=0.05; OR: 0.032 (95% CI: 0.008-0.135), P<0.0001, respectively). Patients with KRAS G12V developed significantly more pleuro-pericardial metastases compared with the remainder of the cohort (94% vs 12%, P<0.0001). This was confirmed in MA (OR: 0.007 (95% CI: 0.001-0.031), P<0.0001). Wild-type patients developed significantly more lung metastases (35% vs 10%, P<0.0001). This was confirmed in MA (OR: 0.383 (95% CI: 0.193-0.762), P=0.006). CONCLUSION: Epidermal growth factor receptor mutation and KRAS amino acid substitutions seem to predict site-specific recurrence and metastasis after NSCLC surgery.

Impact of EGFR mutations and KRAS amino acid substitution on the response to radiotherapy for brain metastasis of non-small-cell lung cancer.

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.

Prognostic value of the KRAS G12V mutation in 841 surgically resected Caucasian lung adenocarcinoma cases.

BACKGROUND: Identifying patients who will experience lung cancer recurrence after surgery remains a challenge. We aimed to evaluate whether mutant forms of epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) (mEGFR and mKRAS) are useful biomarkers in resected non-small cell lung cancer (NSCLC). METHODS: We retrospectively reviewed data from 841 patients who underwent surgery and molecular testing for NSCLC between 2007 and 2012. RESULTS: mEGFR was observed in 103 patients (12.2%), and mKRAS in 265 (31.5%). The median overall survival (OS) and time to recurrence (TTR) were significantly lower for mKRAS (OS: 43 months; TTR: 19 months) compared with mEGFR (OS: 67 months; TTR: 24 months) and wild-type patients (OS: 55 months; disease-free survival (DFS): 24 months). Patients with KRAS G12V exhibited worse OS and TTR compared with the entire cohort (OS: KRAS G12V: 26 months vs COHORT: 60 months; DFS: KRAS G12V: 15 months vs COHORT: 24 months). These results were confirmed using multivariate analyses (non-G12V status, hazard ratio (HR): 0.43 (confidence interval: 0.28-0.65), P<0.0001 for OS; HR: 0.67 (0.48-0.92), P=0.01 for TTR). Risk of recurrence was significantly lower for non-KRAS G12V (HR: 0.01, (0.001-0.08), P<0.0001). CONCLUSIONS: mKRAS and mEGFR may predict survival and recurrence in early stages of NSCLC. Patients with KRAS G12V exhibited worse OS and higher recurrence incidences.

Outcome-based determination of optimal pyrosequencing assay for MGMT methylation detection in glioblastoma patients.

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 < 0.0001). The improvement compared to the predictive value of the test analyzing average methylation of CpG 74-78 (HR, 0.32; p < 0.0001) was however marginal. We recommend to test CpGs 74-78 when analyzing MGMT methylation status by PSQ because a commercial kit that has successfully been used in several studies is available, allowing reproducible and comparable results from one laboratory to another.

SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases.

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.

Methylation tolerance due to an O6-methylguanine DNA methyltransferase (MGMT) field defect in the colonic mucosa: an initiating step in the development of mismatch repair-deficient colorectal cancers.

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.

Rational design of peptides active against the gram positive bacteria Staphylococcus aureus.

In an attempt to increase the antimicrobial activity of the insect defensin from Anopheles gambiae, which is active against Staphylococcus aureus at low concentration, hybrid defensins were designed by combining conserved sequence regions and variable regions of insect defensins. Their activity against S. aureus strains sensitive and resistant to conventional antibiotics was evaluated, and the toxicity of the most active molecules was tested. The three-dimensional structure of Anopheles gambiae defensin and five hybrids were determined by NMR and molecular modelling. This strategy led to the design of two chimeric defensins with increased activity compared with the native molecule, but one of them appears to be toxic to mice at a rather low concentration. The structure of the CS alphabeta motif, which is a characteristic of insect defensin, is sensitive to sequence modifications, in particular in the N-terminal loop. The existence of the CS alphabeta is most probably a prerequisite for the stability and the activity of the molecule, but is not sufficient by itself since the hybrid displaying the best defined structure is not active against the tested strains. The analysis of the structure, in relation with the activity and the toxicity data, underlines the importance of turns and of the N-terminal loop. Residues located in the turns contributing to the preservation of positive electrostatic areas at the surface of the molecules seem particularly important for the activity of the molecule, while residues involved in the N-terminal loop are both involved in the modulation of the activity and the toxicity of the molecule.

KRAS-specific Amino Acid Substitutions are Associated With Different Responses to Chemotherapy in Advanced Non-small-cell Lung Cancer.

BACKGROUND: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSION: KRAS-specific AAS appears to induce different responses to chemotherapy regimens after first-line platinum-based chemotherapy in advanced NSCLC.

SNAI2 and TWIST1 in lymph node progression in early stages of NSCLC patients.

Lymph node metastasis is an important prognosis factor in non-small cell lung cancer (NSCLC) patients. The aim of this study was to investigate the role of epithelial to mesenchymal transition (EMT) in lymph node progression in the early stages of NSCLC. We studied a retrospective cohort of 160 consecutive surgically treated NSCLC patients with available frozen tumor samples for expression of EMT markers (CDH1, CTNNB1, CDH2, and VIMENTIN), inducers (TGFB1, c-MET, and CAIX), and transcription factors (EMT-TF: SNAI1, SNAI2, ZEB1, TWIST1, and TWIST2). Partial EMT was more frequent in N1-2 (N+) vs N0 patients (P < .01). TGFB1 (P = .02) as well as SNAI2 (P < .01) and TWIST1 (P = .04) were the most differentially expressed genes in N+ tumors. In this group, ZEB1 was correlated with all EMT inducers and other EMT-TFs were overexpressed depending on the inducers. CAIX was an independent prognostic factor for overall survival (IC 95% HR: 1.10-5.14, P = .03). Partial EMT is involved in lymph node progression of NSCLC patients and depends on the TGFß pathway. EMT-TFs are differentially expressed depending on EMT inducers. CAIX might be a relevant prognostic marker in early stage NSCLC.

Histone hypoacetylation contributes to CXCL12 downregulation in colon cancer: impact on tumor growth and cell migration.

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.

Perioperative bevacizumab improves survival following lung metastasectomy for colorectal cancer in patients harbouring v-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue exon 2 codon 12 mutations†.

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 (41.58­98.42) vs 24 months (1.15­46.86), P = 0.001] and OS [101 vs 55 months (49.77­60.23), P = 0.004]. However, this benefit was only significant in cases of KRAS exon 2 codon 12 mutations [median OS: 101 months (83.97­118.02) vs 60 months (53­66.99), P < 0.001; median LRRFS: 76 months (64.62­87.38) vs 44 months (35.27­52.73), P < 0.001]. CONCLUSION: Perioperative bevacizumab appears to be beneficial in patients with exon 2 codon 12 KRAS mutations who have undergone lung metastasectomy for CRC.

KRAS exon 2 codon 13 mutation is associated with a better prognosis than codon 12 mutation following lung metastasectomy in colorectal cancer.

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.

Epidermal growth factor receptor mutations are linked to skip N2 lymph node metastasis in resected non-small-cell lung cancer adenocarcinomas.

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 Cramér'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 (Cramér'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 (Cramér'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.

Clinical validation of the CE-IVD marked Therascreen MGMT kit in a cohort of glioblastoma patients.

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.

Long-term storage and safe retrieval of human papillomavirus DNA using FTA elute cards.

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.

Epidermal growth factor receptor and v-Ki-ras2 Kirsten rat sarcoma viral oncogen homologue-specific amino acid substitutions are associated with different histopathological prognostic factors in resected non-small-cell lung cancer.

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.

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

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

An Innovative Fluorescent Semi-quantitative Methylation-specific PCR Method for the Determination of MGMT Promoter Methylation is Reflecting Intra-tumor Heterogeneity.

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.

Lead optimization of antifungal peptides with 3D NMR structures analysis.

Antimicrobial peptides are key components of the innate immune response in most multicellular organisms. These molecules are considered as one of the most innovative class of anti-infective agents that have been discovered over the last two decades, and therefore, as a source of inspiration for novel drug design. Insect cystine-rich antimicrobial peptides with the CS alpha beta scaffold (an alpha-helix linked to a beta-sheet by two disulfide bridges) represent particularly attractive templates for the development of systemic agents owing to their remarkable resistance to protease degradation. We have selected heliomicin, a broad spectrum antifungal CS alpha beta peptide from Lepidoptera as the starting point of a lead optimization program based on phylogenic exploration and fine tuned mutagenesis. We report here the characterization, biological activity, and 3D structure of heliomicin improved analogs, namely the peptides ARD1, ETD-135, and ETD-151. The ARD1 peptide was initially purified from the immune hemolymph of the caterpillars of Archeoprepona demophoon. Although it differs from heliomicin by only two residues, it was found to be more active against the human pathogens Aspergillus fumigatus and Candida albicans. The peptides ETD-135 and ETD-151 were engineered by site-directed mutagenesis of ARD1 in either cationic or hydrophobic regions. ETD-135 and ETD-151 demonstrated an improved antifungal activity over the native peptides, heliomicin and ARD1. A comparative analysis of the 3D structure of the four molecules highlighted the direct impact of the modification of the amphipathic properties on the molecule potency. In addition, it allowed to characterize an optimal organization of cationic and hydrophobic regions to achieve best antifungal activity.

Sporadic early-onset colorectal cancer is a specific sub-type of cancer: a morphological, molecular and genetics study.

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.