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Background: Small bowel adenocarcinoma is a rare disease. The genomic profiling tumours according to clinical characteristics and its impact on the prognosis remains unclear., Methods: A pooled analysis of clinical data, genomic profiling and MisMatch Repair (MMR) status from three databases was performed., Results: A total of 188 tumour samples were analysed. A predisposing disease was reported in 22.3%, mainly Lynch syndrome and Crohn's disease. The tumours were localized in 80.2% and metastatic in 18.8%. The most frequent mutations were KRAS (42.0%) among them 7/79 are G12C, TP53 (40.4%), APC (19.1%), PIK3CA (18.6%), SMAD4 (12.8%) and ERBB2 (9.6%). Mutation distribution differed according to predisposing disease for TP53, ERBB2, IDH1, FGFR3, FGFR1 and KDR. KRAS and SMAD4 mutations were more frequent in metastatic tumour, whereas ERBB2 mutations were absent in metastatic tumour. For localized tumour, APC mutation was independently associated with a poor overall survival (OS) (p = 0.0254). 31.8% of localized tumours and 11.3% of metastatic tumours were dMMR (29.8% of the entire cohort). A dMMR status was associated with a better OS (HR = 0.61 [0.39-0.96], p = 0.0316)., Conclusions: There is a different genomic profile according to the stage and predisposing disease. dMMR and APC mutation in localized tumour predict a better prognosis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: It is intuitively thought that early relapse is associated with poor survival after recurrence (SAR) in resected colon cancer (CC) patients, but this has never been formally studied., Methods: We pooled data from stage III patients treated with oxaliplatin-based adjuvant therapy in two phase III trials, to analyse time to recurrence (TTR) and its relationship with SAR. TTR and SAR were also studied according to molecular status (mismatch repair (MMR), RAS, and BRAF V600E ). Early relapsing patients were defined as patients having a TTR event within 12 months after starting adjuvant chemotherapy., Results: 4548 stage III CC patients were included in the present analysis. Deficient MMR (dMMR) CC patients experienced fewer recurrences than proficient (p)MMR CC patients (18.8% versus 27.6%) but had a significantly shorter median TTR (mTTR; 0.74 versus 1.40 years, p < 0.0001). In pMMR patients, BRAF and RAS mutations were also associated with earlier mTTR as compared to double wild-type (WT) patients (0.99 versus 1.38 versus 1.54 years, respectively, p < 0.0001). Early recurrence occurred in 397 patients and was associated with a median SAR (2.2 versus 3.3 years, p = 0.0007). However, this association was mainly due to pMMR/RAS and BRAF V600E mutated tumours and was not confirmed in dMMR and pMMR/double WT subgroups., Conclusion: In resected stage III CC treated with standard oxaliplatin-based adjuvant therapy, TTR varies between dMMR, pMMR/RAS, or BRAF V600E mutated and pMMR/double WT tumours. In addition, early relapse is associated with poor survival, mainly due to patients resected for a pMMR/RAS or BRAF V600E mutated tumour., Competing Interests: Declaration of Competing Interest JT has received honoraria as a speaker or in an advisory role from Sanofi, Roche, Merck Serono, Amgen, Servier, Pierre Fabre, Lilly, AstraZeneca, and MSD. PLP has received honoraria as a speaker or in an advisory role from ESMO, Amgen, Servier, Pierre Fabre, Biocartis, and stocks from Methys DX. SL has received honoraria as an invited speaker from Roche, Eli Lilly, Bristol Myers Squibb, Servier, Merck Serono, Pierre Fabre, GlaxoSmithKline, Amgen, and has participated in an advisory board for Amgen, Merck Serono, Eli Lilly, AstraZeneca, Incyte, Daiichi-Sankyo, Bristol Myers Squibb, Servier, and Merck Sharp & Dohme. TA has attended advisory board meetings and received consulting fees from AstraZeneca, Astellas, Aptitude Health, Bristol Myers Squibb, Gritstone Oncology, GamaMabs Pharma SA, Gilead, GlaxoSmithKline, Merck & Co. Inc., Nordic Oncology, Pierre Fabre, Seagen, Servier and Transgene; has received honoraria from AstraZeneca, Bristol Myers Squibb, GlaxoSmithKline, Merck & Co. Inc., Merck Serono, Pierre Fabre, Roche, Sanofi, Seagen and Servier; and has received support for meetings from Merck & Co. Inc., and Servier. CL was a consultant and had an advisory role for Novartis and AAA, and has received speaker honoraria from AMGEN, and Bayer. OB has received honoraria as a speaker and in an advisory role from Merck Serono, Amgen, Bayer, Servier, Pierre Fabre, Deciphera, Apmonia Therapeutics, and MSD. AL has received honoraria as a consultant and speaker from Advance Accelerator Applications, Amgen, Bayer, F. Hofmann-La Roche, Incyte Corporation, Ipsen Biopharmaceuticals Inc, Mylan Pharmaceuticals Inc, Novartis, Pierre Fabre Pharmaceuticals Inc, Sandoz, Sanofi Pasteur Inc and has received research grants from Bayer and Lilly Deutschland. CLO reports personal fees and non-financial support from Merck, Roche; personal fees from Servier; and personal fees from Amgen outside the submitted work. JBB has received personal fees from Amgen, Bayer, Bristol Myers Squibb, Merck Serono, Merck Sharp & Dohme, Pierre Fabre, Sanofi, Servier, and non-financial support from Amgen, Merck Serono, and Roche, outside the submitted work. JB is involved as an advisory board member for AstraZeneca, Bristol-Myers Squibb, MSD, Novartis, Ipsen, Servier, and Sanofi and has received honoraria from AstraZeneca, Bayer, Bristol Myers Squibb, Merck, MSD, Daiichi, Servier, Ipsen, Jansen, and Sanofi. CB has received honoraria as a consultant from Bayer, Molecular Partners, MSD, Pierre Fabre, and Servier, and has received research grants from Bayer, Roche, and Boehringer. All remaining authors have declared no conflicts of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: prognostic biomarkers could be useful to better select patients with borderline resectable (BR) or locally advanced (LA) pancreatic adenocarcinoma (PA) for chemoradiotherapy (CRT) and/or secondary resection., Aims: The main objective of this work was to study characteristics, received treatments and prognostic of patients with BR or LA PA according to their baseline circulating tumor DNA status and, for secondary objective, neutrophil-to-lymphocyte Ratio (NLR)., Methods: ctDNA status at baseline was determined using Next Generation Sequencing in a consecutive monocentric cohort of patients with a BR or LA PA., Results: 69 patients were included, 31 with BR PA and 38 with LA PA. 14 (20.3%) patients had baseline positive ctDNA. Five (7.8%) patients had NLR> 5. Patients with positive ctDNA had 3.7 months shorter progression free survival (p = 0.006). Patients with positive ctDNA had earlier progression after the beginning of CRT (4.4 vs 7.1 months; p = 0.068) and shorter relapse free survival after secondary resection (9.2 vs 22.9 months; p = 0.016)., Conclusions: positive ctDNA at baseline was associated with a worse prognosis in patients with BR or LA PA. These data are exploratory and must be confirmed in further prospective trials., Competing Interests: Declaration of Competing Interest JB Bachet has received personal fees from Amgen, AstraZeneca, Bayer, Merck Serono, Pierre Fabre, Roche, Sanofi, Servier, Shire, and non-financial support from Amgen, Merck Serono, and Roche. P Laurent-Puig declares consulting for or personal fees from Amgen, AstraZeneca, Biocartis, BMS, Boehringer-Ingelheim, Lilly, Merck Serono, MSD, Sanofi, Servier, Roche., (Copyright © 2022. Published by Elsevier Ltd.)

Background: Homozygous Recombination Deficiency (HRD) is associated with sensitivity to PARP-inhibitors (PARPi) in different cancer types. In pancreatic adenocarcinoma (PA) the main cause of HRD is BRCA1/2 germline mutation and patients with mutations in BRCA1/2 may benefit from PARPi. Recently other mechanisms leading to HRD were described in different cancer types, including gene mutations and epigenetic changes such as promoter hypermethylation. In PA, BRCA1 promoter hypermethylation, a known mechanism of gene silencing, was recently described. However, results are discordant between North American studies (0.7% of PA) and Asian ones (up to 60% of PA) and the association with HRD is not clear., Methods: Here, we developed 2 quantifications methods to explore BRCA1 and RAD51C promoter methylation in a series of 121 Formalin Fixed-Paraffin-Embedded (FFPE) specimens from resected PA without neoadjuvant treatment. The methylation-specific PCR was done with 2 different methods after DNA bisulfite conversion: a digital droplet PCR, and a PCR followed by capillary electrophoresis, to score the methylated / non methylated ratios in tumor samples. Methods were validated for specificity and sensibility using 100, 20, 10, 5 and 0% methylated commercial DNA for fragment analysis with a detection cutoff of 5-10%. Limit of blank was defined as 5 dropplets/20µL for RAD51C and 1 dropplet/20µL for BRCA1 for ddPCR. Samples were reviewed by a pathologist, macrodissected before DNA extraction to obtain 50-60% of tumoral cells. DNAs were treated for bisulfite conversion and analyzed using both methods in parallel to known positive and negative controls in each run., Results and Conclusion: No methylation at BRCA1 or RAD51C was found in this series of PA suggesting that HRD gene promoter methylation is a rare event in European patients., Competing Interests: Conflicts of Interest J.B. Bachet has received personal fees from Amgen, AstraZeneca, Bayer, Merck Serono, Pierre Fabre, Roche, Sanofi, Servier, Shire, and non-financial support from Amgen, Merck Serono, and Roche., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Purpose: Our team previously defined six quantitative transcriptomic components, and a classification in five subtypes by association of these components. In this study, we compared the robustness of quantitative components and qualitative classifications from different transcriptomic profiling techniques, investigated their clinical relevance, and proposed a new prognostic model., Experimental Design: A total of 210 patients from a multicentric cohort and 149 patients from a monocentric cohort were included in this study. RNA microarray profiles were obtained from 165 patients of the multicentric cohort. RNA sequencing (RNA-seq) profiles were obtained from all the patients., Results: For the patients with both RNA microarray and RNA-seq profiles, the concordance in subtype assignment was partial with an 82.4% coherence rate. The correlation between the two technique projections of the six components ranged from 0.85 to 0.95, demonstrating an advantage of robustness. On the basis of the Akaike information criterion, the RNA components showed more prognostic value in univariate or multivariate models than the subtypes. Using the monocentric cohort for training, we developed a multivariate Cox regression model using all six components and clinicopathologic characteristics (node invasion and resection margins) on disease-free survival (DFS). This prognostic model was highly associated with DFS ( P < 0.001). The evaluation of the model in the multicentric cohort showed significant association with DFS and overall survival ( P < 0.001)., Conclusions: We described the advantage of the prognostic value and robustness of the whole-tumor transcriptomic components than subtypes. We created and validated a new DFS-based multivariate Cox regression prognostic model, including six pancreatic adenocarcinoma transcriptomic component levels and pathologic characteristics., (©2021 American Association for Cancer Research.)

Gastric or gastro-oesophageal junction (GEJ) adenocarcinomas present poor overall survival (OS). First-line chemotherapy regimen for patients with HER2-negative tumours is based on a doublet or triplet of fluoropyrimidine plus platinum salt ± taxane. Second-line chemotherapy (Docetaxel or Irinotecan) improves OS which nonetheless remains poor (around 5 months). The first results of immune checkpoint inhibitors (anti-PD-1) combined with chemotherapy in metastatic gastric and GEJ cancers were discordant in recent phase III trials. Data on dual-blockade (anti-PD-L1 or anti-PD-1 plus anti-CTLA-4) plus chemotherapy are lacking. DURIGAST is a randomised, multicenter, non-comparative, phase II study, evaluating safety and efficacy of FOLFIRI plus Durvalumab (anti-PD-L1) versus FOLFIRI plus Durvalumab and Tremelimumab (anti-CTLA-4) as second-line treatment of advanced gastric and GEJ adenocarcinoma. The primary objective is the rate of patients alive and without progression at 4 months. The main inclusion criteria are: patients with advanced gastric or GEJ adenocarcinoma, pre-treated with fluoropyrimidine + platinum salt ± taxane. Due to a lack of data on FOLFIRI, Durvalumab and Tremelimumab combination, a 2-step safety run-in phase has been performed before the randomised phase II. The safety run-in phase did not show any safety issue and the randomised phase II starts in September 2020., Competing Interests: Conflict of interest None declared., (Copyright © 2020 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Purpose: Eryaspase is composed of l-asparaginase encapsulated in erythrocytes and has demonstrated significant efficacy in a randomized phase II trial. We assessed the prognostic and predictive value of circulating tumor DNA (ctDNA) in patients, plasma included in this trial., Experimental Design: Samples prospectively collected pretreatment were centrally analyzed by next-generation sequencing. Prognostic values of baseline ctDNA and ctDNA early changes between day 0 and 28 were assessed in both arms combined on objective response rate (ORR), progression-free survival (PFS), and overall survival (OS); three groups were defined: negative ctDNA (Neg), ctDNA responders (Resp), and ctDNA nonresponders (NResp). Predictive value of ctDNA for eryaspase efficacy was investigated., Results: ctDNA was positive at baseline in 77 patients of the 113 tested patients (68%). Detectable ctDNA was an independent negative prognostic factor for OS (4.6 vs. 8.8 months; P = 0.0025) and PFS (1.6 vs. 3.3 months; P = 0.00043). Early change in ctDNA levels was correlated with ORR (20%, 26%, 0%; P < 0.04), PFS (3.7, 3.4, 1.6 months; P < 0.0001), and OS (11.7, 6.5, 4.3 months; P < 0.0001) according to the three defined groups (Neg, Res, NResp, respectively). In patients with ctDNA detectable at baseline, eryaspase was associated with better PFS [HR = 0.53; 95% confidence interval (CI): 0.3-0.94)] and OS (HR = 0.52; 95% CI: 0.29-0.91)., Conclusions: We confirm from a prospective randomized trial that: (i) the presence of ctDNA at baseline is a major prognostic factor, (ii) the early change of ctDNA correlates with treatment outcome, and (iii) the ctDNA could be a predictive biomarker of eryaspase efficacy., (©2020 American Association for Cancer Research.)

Background: Recurrence patterns in stage III colon cancer (CC) patients according to molecular markers remain unclear. The objective of the study was to assess recurrence patterns according to microsatellite instability (MSI), RAS and BRAF V600E status in stage III CC patients., Methods: All stage III CC patients from the PETACC-8 randomized trial tested for MSI, RAS and BRAF V600E status were included. The site and characteristics of recurrence were analyzed according to molecular status. Survival after recurrence (SAR) was analyzed., Results: A total of 1650 patients were included. Recurrence occurred in 434 patients (26.3%). Microsatellite stable (MSS) patients had a significantly higher recurrence rate (27.2% vs. 18.7%, P = 0.02) with a trend to more pulmonary recurrence (28.8% vs. 12.9%, P = 0.06) when compared to MSI patients. MSI patients experienced more regional lymph nodes compared to MSS (12.9% vs. 4%, P = 0.046). In the MSS population, the recurrence rate was significantly higher in RAS (32.2%) or BRAF (32.3%) patients when compared to double wild-type patients (19.9%) (p < 0.001); no preferential site of recurrence was observed according to RAS and BRAF V600E mutations. Finally, decreased SAR was observed in the case of peritoneal recurrence or more than two recurrence sites., Conclusions: Microsatellite, RAS and BRAF V600E status influences recurrence rates in stage III CC patients. However, only microsatellite status seems to be associated with specific recurrence patterns. More than two recurrence sites and recurrence in the peritoneum were associated with poorer SAR.

Background: Epidermal growth factor receptor (EGFR) and hepatocyte growth factor (HGF)/mesenchymal-epithelial transition (MET) pathways, which promote tumour growth and proliferation, are often deregulated in advanced gastroesophageal adenocarcinomas. We assessed whether adding panitumumab (an EGFR inhibitor) or rilotumumab (a HGF inhibitor) to first-line fluoropyrimidine-based and platinum-based chemotherapy (modified oxaliplatin, leucovorin and fluorouracil [mFOLFOX6]) benefits to patients with advanced gastroesophageal adenocarcinoma., Patients and Methods: This phase II, open-label, randomised, three-arm study enrolled patients ≥18 years, with advanced gastroesophageal adenocarcinoma, Eastern Cooperative Oncology Group performance status 0-1 and no known HER2 overexpression. Patients were randomly assigned (1:1:1) mFOLFOX6 (oxaliplatin 85 mg/m 2 , leucovorin 400 mg/m 2 , 5-fluorouracil 400 mg/m 2 bolus then 2400 mg/m 2 over 46 h) alone or combined with panitumumab (6 mg/kg) or rilotumumab (10 mg/kg) every 2 weeks until limiting toxicity, patient's refusal or disease progression. The primary end-point was the 4-month progression-free survival (PFS) rate. Secondary end-points included overall survival (OS) and tolerance., Results: The study enrolled 162 patients in 29 French centres. The median follow-up was 23.6 months (interquartile range = 16.4-29.0). The 4-month PFS rate was 71% (95% confidence interval [CI] = 57-82) with chemotherapy alone, 57% (95% CI = 42-71) combined with panitumumab and 61% (95% CI = 47-74) combined with rilotumumab. Median OS was 13.1 months (95% CI = 8.7-16.9) with chemotherapy alone, 8.3 months (95% CI = 6.2-13.2) combined with panitumumab and 11.5 months (95% CI = 7.9-17.1) combined with rilotumumab. Adverse events grade ≥III occurred less frequently with chemotherapy alone (62%) than with panitumumab (83%) and rilotumumab (89%)., Conclusions: We found no benefit in adding panitumumab or rilotumumab to mFOLFOX6 first-line chemotherapy to treat advanced gastroesophageal adenocarcinoma patients., Trial Registration: European Clinical Trials Database, number 2009-012797-12., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Multiple nodules in the lung are being diagnosed with an increasing frequency thanks to high-quality computed tomography imaging. In patients with lung cancer, this situation represents up to 10% of patients who have an operation. For clinical management, it is important to classify the disease as intrapulmonary metastasis or multiple primary lung carcinoma to define TNM classification and optimize therapeutic options. In the present study, we evaluated the respective and combined input of histological and molecular classification to propose a classification algorithm for multiple nodules., Methods: We studied consecutive patients undergoing an operation with curative intent for lung adenocarcinoma (N = 120) and harboring two tumors (N = 240). Histological diagnosis according to the WHO 2015 classification and molecular profiling using next-generation sequencing targeting 22 hotspot genes allowed classification of samples as multiple primary lung adenocarcinomas or as intrapulmonary metastasis., Results: Next-generation sequencing identified molecular mutations in 91% of tumor pairs (109 of 120). Genomic and histological classification showed a fair agreement when the κ test was used (κ = 0.43). Discordant cases (30 of 109 [27%]) were reclassified by using a combined histomolecular algorithm. EGFR mutations (p = 0.03) and node involvement (p = 0.03) were significantly associated with intrapulmonary metastasis, whereas KRAS mutations (p = 0.00005) were significantly associated with multiple primary lung adenocarcinomas. EGFR mutations (p = 0.02) and node involvement (p = 0.004) were the only independent prognostic factors., Conclusion: We showed that combined histomolecular algorithm represents a relevant tool to classify multifocal lung cancers, which could guide adjuvant treatment decisions. Survival analysis underlined the good prognosis of EGFR-mutated adenocarcinoma in patients with intrapulmonary metastasis., (Copyright © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.)

Multimodal primary treatment of localised adenocarcinoma of the stomach, the oesophagus and the oesophagogastric junction (AEG) was reviewed by a multidisciplinary expert panel in a moderated consensus session. Here, we report the key points of the discussion and the resulting recommendations. The exact definition of the tumour location and extent by white light endoscopy in conjunction with computed tomography scans is the backbone for any treatment decision. Their value is limited with respect to the infiltration depth, lymph node involvement and peritoneal involvement. Additional endoscopic ultrasound was recommended mainly for tumours of the lower oesophagogastric junction (i.e. AEG type II and III according to Siewert) and in early cancers before endoscopic resection. Laparoscopy to diagnose peritoneal involvement was thought to be necessary before the start of neoadjuvant treatment in all gastric cancers and in AEG type II and III. In general, perioperative multimodal treatment was suggested for all locally advanced oesophageal tumours and for gastric cancers with a clinical stage above T1N0. There was consensus that the combination of fluorouracil, folinic acid, oxaliplatin and docetaxel is now a new standard chemotherapy (CTx) regimen for fit patients. In contrast, the optimal choice of perioperative CTx versus neoadjuvant radiochemotherapy (neoRCTx), especially for AEG, was identified as an open question. Expert treatment recommendations depend on the tumour location, biology, the risk of incomplete (R1) resection, response to treatment, local or systemic recurrence risks, the predicted perioperative morbidity and patients' comorbidities. In summary, any treatment decision requires an interdisciplinary discussion in a comprehensive multidisciplinary setting., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Ampullary adenocarcinoma (AA) originates from either intestinal (INT) or pancreaticobiliary (PB) epithelium. Different prognostic factors of recurrence have been identified in previous studies., Methods: In 91 AA patients of the AGEO retrospective multicentre cohort, we evaluated the centrally reviewed morphological classification, panel markers of Ang et al. including CK7, CK20, MUC1, MUC2 and CDX2, the 50-gene panel mutational analysis, and the clinicopathological AGEO prognostic score., Results: Forty-three (47%) of the 91 tumours were Ang-INT, 29 (32%) were Ang-PB, 18 (20%) were ambiguous (Ang-AMB) and one could not be classified. Among these 90 tumours, 68.7% of INT tumours were Ang-INT and 78.2% of PB tumours were Ang-PB. MUC5AC expression was detected in 32.5% of the 86 evaluable cases. Among 71 tumours, KRAS, TP53, APC and PIK3CA were the most frequently mutated genes. The KRAS mutation was significantly more frequent in the PB subtype. In multivariate analysis, only AGEO prognostic score and tumour subtype were associated with relapse-free survival. Only AGEO prognostic score was associated with overall survival., Conclusions: Mutational analysis and MUC5AC expression provide no additional value in the prognostic evaluation of AA patients. Ang et al. classification and the AGEO prognostic score were confirmed as a strong prognosticator for disease recurrence.

Mutational heterogeneity could explain different metastatic patterns among IIIA-N2 lung cancer and influence prognosis. The identification of subclonal mutations using deep sequencing to evaluate the degree of molecular heterogeneity may improve IIIA-N2 classification. The aim of this prospective study was to assess mutational and immunohistochemical characteristics in primary tumours and involved lymph nodes (LN) in operated patients. Four patients operated for primary lung carcinoma and unisite N2 mediastinal involvement were consecutively selected. Samples (tumour and paired LN) were analysed for PD1, PD-L1 and CD8 immunostaining. Somatic mutation testing was performed by deep targeted next generation sequencing (NGS), with the AmpliSeq™ Colon and Lung Cancer Panel (LifeTechnology). A total of 9 primary lung cancer samples and 10 LN stations were analysed. For each cancer, we found 2 mutations, with allelic ratios from 3% to 72%. Mutational patterns were heterogeneous for 2 primary tumours. In 3 cases, mutations observed in the primary tumour were not found in LN metastases (ALK, FGFR3, MET). Inversely, in 1 case, a KRAS mutation was found in LN but not in the primary tumour. All primary tumours were found PD-L1 positive while CD8+ T cells infiltrate varied. In the different examined LN samples, PD-L1 expression, CD8+ and PD1+ T cells infiltrate were not similar to the primary tumour. This preliminary prospective study shows the diversity of intra-tumour and LN mutations using routinely-used targeted NGS, concerning both mutated gene and allelic ratio. Further studies are needed to evaluate its prognostic impact.

Background & Aims: Genomic studies have revealed subtypes of pancreatic ductal adenocarcinoma (PDA) based on their molecular features, but different studies have reported different classification systems. It is a challenge to obtain high-quality, freshly frozen tissue for clinical analysis and determination of PDA subtypes. We aimed to redefine subtypes of PDA using a large number of formalin-fixed and paraffin-embedded PDA samples, which are more amenable to routine clinical evaluation., Methods: We collected PDA samples from 309 consecutive patients who underwent surgery from September 1996 through December 2010 at 4 academic hospitals in Europe; nontumor tissue samples were not included. Samples were formalin fixed and paraffin embedded. DNA and RNA were isolated; gene expression, targeted DNA sequencing, and immunohistochemical analyses were performed. We used independent component analysis to deconvolute normal, tumor, and microenvironment transcriptome patterns in samples. We devised classification systems from an unsupervised analysis using a consensus clustering approach of our data set after removing normal contamination components. We associated subtypes with overall survival and disease-free survival of patients using Cox proportional hazards regression with estimation of hazard ratios and 95% confidence interval. We used The Cancer Genome Consortium and International Cancer Genome Consortium PDA data sets as validation cohorts., Results: We validated the previously reported basal-like and classical tumor-specific subtypes of PDAs. We identified features of the PDA, including microenvironment gene expression patterns, that allowed tumors to be categorized into 5 subtypes, called pure basal like, stroma activated, desmoplastic, pure classical, and immune classical. These PDA subtypes have features of cancer cells and immune cells that could be targeted by pharmacologic agents. Tumor subtypes were associated with patient outcomes, based on analysis of our data set and the International Cancer Genome Consortium and The Cancer Genome Consortium PDA data sets. We also observed an exocrine signal associated with acinar cell contamination (from pancreatic tissue)., Conclusions: We identified a classification system based on gene expression analysis of formalin-fixed PDA samples. We identified 5 PDA subtypes, based on features of cancer cells and the tumor microenvironment. This system might be used to select therapies and predict patient outcomes. We found evidence that the previously reported exocrine-like (called ADEX) tumor subtype resulted from contamination with pancreatic acinar cells. ArrayExpress accession number: E-MTAB-6134., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Introduction: In advanced gastric cancer, doublet regimen including platinum salts and fluoropyrimidine is considered as a standard first-line treatment. The addition of docetaxel (75 mg/m 2  q3w) to cisplatin (75 mg/m 2  q3w) and 5-fluorouracil has been shown to improve efficacy. However, this regimen (DCF) was associated with frequent severe toxicities (including more complicated neutropenia), limiting its use in clinical practice. Interesting alternative docetaxel-based regimens have been developed that need to be validated., Aim: GASTFOX study is a randomized phase III trial comparing FOLFOX alone or with docetaxel at 50 mg/m 2 (TFOX regimen) in first-line treatment for advanced gastric cancer. In both arms, cycle is repeated every 2 weeks until disease progression or unacceptable toxicity., Materials and Methods: Main eligibility criteria: histologically proven locally advanced or metastatic gastric or esogastric junction adenocarcinoma, HER negative status, measurable disease, ECOG performance status 0 or 1, and adequate renal, hepatic and bone marrow functions., Results: The primary endpoint is radiological/clinical progression-free survival (PFS). A difference of 2 months for the median PFS in favor of TFOX is expected (HR = 0.73) Based on a two-sided α risk of 5% and a power of 90%, 454 events are required to show this difference. Secondary endpoints included overall survival, overall response rate, safety, quality of life and the therapeutic index., Conclusion: This study is planned to include 506 patients to demonstrate the superiority of TFOX over FOLFOX in first-line advanced gastric cancer treatment (NCT03006432)., (Copyright © 2018 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Importance: The prognostic impact of DNA mismatch repair (MMR) status in stage III colon cancer patients receiving FOLFOX (folinic acid, fluorouracil, and oxaliplatin) adjuvant chemotherapy remains controversial., Objective: To determine the association of MMR status with disease-free survival (DFS) in patients with stage III colon cancer treated with FOLFOX., Design, Setting, and Participants: The evaluated biomarkers for MMR status were determined from prospectively collected tumor blocks from patients treated with FOLFOX in 2 open-label, phase 3 randomized clinical trials: NCCTG N0147 and PETACC8. The studies were conducted in general community practices, private practices, and institutional practices in the United States and Europe. All participants had stage III colon adenocarcinoma. They were enrolled in NCCTG N0147 from February 2004 to November 2009 and in PETACC8 from December 2005 to November 2009., Interventions: Patients in the clinical trials were randomly assigned to receive 6 months of chemotherapy with FOLFOX or FOLFOX plus cetuximab. Only those patients treated with FOLFOX alone were included in the present study., Main Outcomes and Measures: Association of MMR status with DFS was analyzed using a stratified Cox proportional hazards model. Multivariable models were adjusted for age, sex, tumor grade, pT/pN stage, tumor location, ECOG (Eastern Cooperative Oncology Group) performance status, and BRAF V600E mutational status., Results: Among 2636 patients with stage III colon cancer treated with FOLFOX, MMR status was available for 2501. Of these, 252 (10.1%) showed deficient MMR status (dMMR; 134 women, 118 men; median age, 59 years), while 2249 (89.9%) showed proficient MMR status (pMMR; 1020 women, 1229 men; median age, 59 years). The 3-year DFS rates in the dMMR and pMMR groups were 75.6% and 74.4%, respectively. By multivariate analysis, patients with dMMR phenotype had significantly longer DFS than those with pMMR (adjusted hazard ratio, 0.73; 95% CI, 0.54-0.97; P = .03)., Conclusions and Relevance: The deficient MMR phenotype remains a favorable prognostic factor in patients with stage III colon cancer receiving FOLFOX adjuvant chemotherapy., Trial Registration: clinicaltrials.gov Identifier: NCT00079274 for the NCCTG N0147 trial and EudraCT identifier: 2005-003463-23 for the PETACC8 trial.

Background: RAS mutations have been shown to confer resistance to anti- epidermal growth factor receptor (EGFR) treatment. We analysed the results of the PETACC8 trial (cetuximab + FOLFOX vs FOLFOX) in full RAS and BRAF wildtype (WT) patients (pts) with resected stage III colon cancer., Patients and Methods: Exons 2, 3 and 4 of KRAS and NRAS, and BRAF exons 11 and 15, were sequenced using the Ampliseq colon-lung cancer panel version 2, in PETACC8 trial pts who consented to translational research. The impact of cetuximab on time to recurrence (TTR), disease-free survival (DFS) and overall survival (OS) was investigated in pts with tumours harbouring RAS and BRAF WT, and RAS mutations. The prognostic value of each individual mutation was also tested., Results: Among the 2559 pts analysed, 745 pts (29%) were known to have KRAS exon 2 mutations and 163 pts (6.4%) the BRAF V600E mutation. Of the remaining 1651 pts, 1054 were assessed by NGS, showing that a further 227 pts (21%) had KRAS exon 2, 3, 4 or NRAS exon 2, 3, 4 mutations, and that 46 pts (4.4%) had a newly diagnosed BRAF mutation. Cetuximab added to FOLFOX did not significantly improve TTR, DFS or OS in pts with RAS WT or RAS and BRAF WT tumours (HR 0.77-1.03, all P > 0.05). Cetuximab addition was not either significantly deleterious in RAS mutant pts or in pts with rare RAS or BRAF mutations. In the overall trial population, NRAS and KRAS codon 61 mutations were the only rare mutations with the same pejorative prognostic value as KRAS exon 2 or BRAF V600E mutations., Conclusion: Though not significant, the clinically relevant 0.76 adjusted HR observed for DFS in favour of adding cetuximab to FOLFOX, in full RAS and BRAF WT stage III colon cancer pts, may justify a new randomized controlled trial testing EGFR inhibitors in this setting., Clinical Trial Number: This is an ancillary study of the PETACC8 trial: EUDRACT 2005-003463-23., (© The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: The prognostic value of BRAF and KRAS mutations within microsatellite-unstable (MSI) and microsatellite-stable (MSS) subgroups of resected colon carcinoma patients remains controversial. We examined this question in prospectively collected biospecimens from stage III colon cancer with separate analysis of MSI and MSS tumors from patients receiving adjuvant FOLFOX +/- cetuximab in two adjuvant therapy trials., Methods: Three groups were defined: BRAF Mutant, KRAS Mutant, and double wild-type. The analytic strategy involved estimation of study-specific effects, assessment of homogeneity of results, and then analysis of pooled data as no differences in patient outcome were found between treatment arms in both trials. Associations of mutations with patient outcome were analyzed, and multivariable models were adjusted for treatment and relevant factors., Results: Four thousand four hundred eleven tumors were evaluable for BRAF and KRAS mutations and mismatch repair status; 3934 were MSS and 477 were MSI. In MSS patients, all BRAF V600E mutations (hazard ratio [HR] = 1.54, 95% confidence interval [CI] = 1.23 to 1.92, P < .001), KRAS codon 12 alterations, and p.G13D mutations (HR = 1.60, 95% CI = 1.40 to 1.83, P < .001) were associated with shorter time to recurrence (TTR) and shorter survival after relapse (SAR; HR = 3.02 , 95% CI = 2.32 to 3.93, P < .001, and HR = 1.20, 95% CI = 1.01 to 1.44, P = .04, respectively). Overall survival (OS) in MSS patients was poorer for BRAF-mutant patients (HR = 2.01, 95% CI = 1.56 to 2.57, P < .001) and KRAS-mutant patients (HR = 1.62, 95% CI = 1.38 to 1.91, P < .001) vs wild-type. No prognostic role of KRAS or BRAF mutations was seen in MSI patients. Furthermore, no interaction was found between treatment arm (with or without cetuximab) and KRAS and BRAF mutations for TTR or OS in MSS patients., Conclusions: In a pooled analysis of resected stage III colon cancer patients receiving adjuvant FOLFOX, BRAF or KRAS mutations are independently associated with shorter TTR, SAR, and OS in patients with MSS, but not MSI, tumors. Future clinical trials in the adjuvant setting should consider these mutations as important stratification factors., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Rationale: Tumor-infiltrating immune cells affect lung cancer outcome. However, the factors that influence the composition and function of the tumor immune environment remain poorly defined and need investigation, particularly in the era of immunotherapy., Objectives: To determine whether the tumoral immune environment is related to lung adenocarcinoma mutations., Methods: This retrospective cohort included 316 consecutive patients with lung adenocarcinoma (225 men; 258 smokers) studied from 2001 to 2005 in a single center. We investigated the association of densities of intratumoral mature dendritic cells (mDCs), CD8 + T cells, neutrophils, and macrophages with clinical and pathological variables and tumor cell mutation profiles obtained by next-generation sequencing., Measurements and Main Results: In 282 tumors, we found 460 mutations, mainly in TP53 (59%), KRAS (40%), STK11 (24%), and EGFR (14%). Intratumoral CD8 + T-cell density was high in smokers (P = 0.02) and TP53-mutated tumors (P = 0.02) and low in BRAF-mutated tumors (P = 0.005). Intratumoral mDC density was high with low pathological tumor stage (P = 0.01) and low with STK11 mutation (P = 0.004). Intratumoral neutrophil density was high and low with BRAF mutation (P = 0.04) and EGFR mutation (P = 0.02), respectively. Intratumoral macrophage density was low with EGFR mutation (P = 0.01). Intratumoral CD8 + T-cell and mDC densities remained strong independent markers of overall survival (P = 0.001 and P = 0.02, respectively)., Conclusions: Intratumoral immune cell densities (mDCs, CD8 + T cells, neutrophils, macrophages) were significantly associated with molecular alterations in adenocarcinoma underlying the interactions between cancer cells and their microenvironment.

Small bowel cancer represents less than 5% of all gastrointestinal cancers, while small bowel adenocarcinoma (SBA) accounts for about one third of all cancers of the small bowel. Although SBA frequently appears sporadically, some diseases are risk factors, such as Crohn's disease and some genetic predispositions to cancer. Progress in the identification of molecular alterations suggests some similarities in carcinogenesis between SBA and colorectal cancer. Evidence levels for the treatment and prognosis of these tumors are insufficient because of the scarcity of this disease and the absence of randomized trials. Chemotherapy based on fluoropyrimidine plus a platinum salt appears to be the most effective treatment regimen in non-randomized prospective trials for advanced SBA. Targeted therapy, against the angiogenic pathway or the epidermal growth factor receptor (EGFR) pathway, for example, is not yet established, but seems promising given the over-expression of vascular epithelial growth factor (VEGF)-A or EGFR observed in SBA. Phase I and II studies are currently evaluating the safety and efficacy of these targeted therapies in SBA treatment. The low incidence of SBA should promote the development of international collaborations to improve our knowledge of the biological mechanisms underlying these tumors and to set up therapeutic trials., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Ten years after the discovery of the predictive value of KRAS status for anti-EGFR antibodies, other genes involved in oncogenesis and therapeutic responses were identified and are now systematically sought. Molecular diagnosis often requires invasive procedures, sometimes iatrogenic, and is limited by feasibility problems, quantity and quality of samples. Identifying these mutations from blood biomarkers would reduce costs and diagnostic delay. The circulating tumor DNA (ctDNA) is one of the most promising blood biomarkers. In this review, we report and discuss the latest results obtained with ctDNA in colorectal cancer, non-small cell lung cancer, and adenocarcinoma of the pancreas. If the methods highlighting appear very heterogeneous, the correlation between mutations found in tumor and those identified in the blood exceeds 95 % specificity in numerous studies. The detection sensitivity is in turn strongly related to tumor stage patients. The presence of ctDNA appears as a prognostic factor for progression-free survival and overall survival. Finally, recent studies have shown that the changing rate ctDNA during systemic treatments had a predictive value for therapeutic efficacy. These results allow to consider the use of ctDNA in monitoring patients to identify early recurrence or progression., (Copyright © 2015 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Background: Histologic classification of lung adenocarcinoma subtype has a prognostic value in most studies. However, lung adenocarcinoma characteristics differ across countries. Here, we aimed at validating the prognostic value of this classification in a large French series of lung adenocarcinoma., Methods: We reviewed 407 consecutive lung adenocarcinomas operated on between 2001 and 2005 and reclassified them according to the International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) classification and subsequently graded them into low, intermediate, and high grade. We analyzed the relevance of this classification according to clinical, pathologic, and molecular analysis., Results: Patients (median age, 61 years; 288 men) underwent lobectomy (n = 378) or pneumonectomy (n = 29). Patients' overall survival at 5 and 10 years was 53.2% and 32.6%, respectively. Union for International Cancer Control stage distribution was 189 stage I, 104 stage II, 107 stage III, and seven stage IV. Low-grade tumor was found in one patient, intermediate grade in 275 patients, and high grade in 131 patients. KRAS and EGFR mutations were detected in 34% and 9.6%, respectively. Histologic grade was significantly correlated with extent of resection (P = .01), thyroid transcriptional factor-1 expression (P = .00000001), vascular emboli (P = .03), and EGFR mutations (P = .01). Mucinous adenocarcinomas were associated with KRAS mutations (P = .003). At univariate analysis, age, extent of resection, histologic grade, pleural invasion, vascular emboli, pathologic T and N, and stage were predictive of survival. At multivariate analysis, age (P = .0001), histologic grade (P = .03), and stage (P = .000003) were independent prognostic factors., Conclusions: IASLC/ATS/ERS classification of lung adenocarcinomas predicts survival in French population. Histologic grade correlates with clinical, pathologic and molecular parameters suggesting different oncogenic pathways.

Background: Since the 1990s, fluorouracil-based adjuvant chemotherapy has significantly reduced the risk of tumour recurrence in patients with stage III colon cancer. We aimed to assess whether the addition of cetuximab to standard adjuvant oxaliplatin, fluorouracil, and leucovorin chemotherapy (FOLFOX4) in patients with stage III colon cancer improved disease-free survival (DFS)., Methods: For this open-label, randomised phase 3 study done in nine European countries, we enrolled patients through an interactive voice response system to the central randomisation centre, with a central stratified permuted block randomisation procedure. We randomly assigned patients with resected (R0) stage III disease (1:1) to receive 12 cycles of FOLFOX4 twice a week with or without cetuximab. Patients were stratified by N-status (N1 vs N2), T-status (T1-3 vs T4), and obstruction or perforation status (no obstruction and no perforation vs obstruction or perforation or both). A protocol amendment (applied in June, 2008, after 2096 patients had been randomly assigned to treatment-restricted enrolment to patients with tumours wild-type at codons 12 and 13 in exon 2 of the KRAS gene (KRAS exon 2 wild-type). The primary endpoint was DFS. Analysis was intention to treat in all patients with KRAS exon 2 wild-type tumours. The study is registered at EudraCT, number 2005-003463-23., Findings: Between Dec 22, 2005, and Nov 5, 2009, 2559 patients from 340 sites in Europe were randomly assigned. Of these patients, 1602 had KRAS exon 2 wild-type tumours (intention-to-treat population), 791 in the FOLFOX4 plus cetuximab group and 811 in the FOLFOX4 group. Median follow-up was 3·3 years (IQR 3·2-3·4). In the experimental and control groups, DFS was similar in the intention-to-treat population (hazard ratio [HR] 1·05; 95% CI 0·85-1·29; p=0·66), and in patients with KRAS exon 2/BRAF wild-type (n=984, HR 0·99; 95% CI 0·76-1·28) or KRAS exon 2-mutated tumours (n=742, HR 1·06; 95% CI 0·82-1·37). We noted heterogeneous responses to the addition of cetuximab in preplanned subgroup analyses. Grade 3 or 4 acne-like rash (in 209 of 785 patients [27%] vs four of 805 [<1%]), diarrhoea (113 [14%] vs 70 [9%]), mucositis (63 [8%] vs 10 [1%]), and infusion-related reactions (55 [7%] vs 30 [4%]) were more frequent in patients treated with FOLFOX4 plus cetuximab than in those patients who received FOLFOX4 alone., Interpretation: The addition of cetuximab to FOLFOX4 did not improve DFS compared with FOLFOX4 alone in patients with KRAS exon 2 wild-type resected stage III colon cancer. This trial cannot conclude on the benefit of cetuximab in the studied population, but the heterogeneity of response suggests that further investigation of the role of FOLFOX4 plus cetuximab in specific patient subgroups is warranted., Funding: Fédération Francophone de Cancérologie Digestive (FFCD), Merck KGaA, and Sanofi-Aventis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aim of the Study: Small bowel adenocarcinoma (SBA) is a rare and aggressive tumour with poor outcomes. Because of its low incidence, the number prospective studies remains insufficient leading to poor knowledge and absence of standard of care. Aiming to better understand small bowel carcinogenesis we investigated the frequency of somatic mutations in a large data set of patients in more than 740 mutational hotspots among 46 genes., Methods: In total, 83 SBA cases were selected from two European databases. The sequencing was performed using the Ion 316 Chip. Additionally we looked into ERBB2 expression and microsatellite instability (MSI) status., Results: The tumours most frequently were duodenal (47%) and stage ⩾3 (63%). Eight genes were mutated with a frequency >5%: KRAS, TP53, APC, SMAD4, PIK3CA, ERBB2, BRAF and FBXW7. ERBB2 alterations are present in 10 patients (12%) through mutations (7 cases) or amplifications (3 cases). ERBB2 mutations were significantly associated with duodenal tumour location (P=0.04). In this group, there was a positive association with dMMR status (P=0.006) and APC mutation (P=0.02) but negative association with p53 mutations (P=0.038)., Conclusions: This study describes the first large screening of somatic mutations in SBA using next generation sequencing. The ERBB2 mutation was revealed to be one of the most frequent alterations in SBA with a distribution dependent on tumour location. In most cases ERBB2 mutation was identical (p.L755S). In clinical practice, this may suggest that more than 10% of the patients with SBA could be treated using an anti-ERBB2-targeted agent., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: To test the prognostic value of tumour protein and genetic markers in colorectal cancer (CRC) and examine whether deficient mismatch repair (dMMR) tumours had a distinct profile relative to proficient mismatch repair (pMMR) tumours., Methods: This prospective multicentric study involved 251 stage I-III CRC patients. Analysed biomarkers were EGFR (binding assay), VEGFA, thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) expressions, MMR status, mutations of KRAS (codons 12-13), BRAF (V600E), PIK3CA (exons 9 and 20), APC (exon 15) and P53 (exons 4-9), CpG island methylation phenotype status, ploidy, S-phase, LOH., Results: The only significant predictor of relapse-free survival (RFS) was tumour staging. Analyses restricted to stage III showed a trend towards a shorter RFS in KRAS-mutated (P=0.005), BRAF wt (P=0.009) and pMMR tumours (P=0.036). Deficient mismatch repair tumours significantly demonstrated higher TS (median 3.1 vs 1.4) and TP (median 5.8 vs 3.5) expression relative to pMMR (P<0.001) and show higher DPD expression (median 14.9 vs 7.9, P=0.027) and EGFR content (median 69 vs 38, P=0.037) relative to pMMR., Conclusions: Present data suggesting that both TS and DPD are overexpressed in dMMR tumours as compared with pMMR tumours provide a strong rationale that may explain the resistance of dMMR tumours to 5FU-based therapy.

Small bowel adenocarcinomas are rare tumours, but their incidence is increasing. Their most common primary location is the duodenum. The few studies that have collected data regarding small bowel adenocarcinoma are not homogeneous and are widely spread over time. Even though these tumours are most often sporadic, some predisposing diseases have been identified, among which Crohn's disease and genetic syndromes. Early diagnosis of small bowel adenocarcinoma remains difficult despite significant radiological and endoscopic progress. After surgical resection the main prognostic factor is node invasion; in this case, adjuvant chemotherapy can be expected to be beneficial, although this has not been established by randomised trials. For metastatic disease, platinum-based chemotherapy seems to be the most effective treatment. Targeted therapies have not yet been evaluated in this type of cancer., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Small bowel adenocarcinoma (SBA) is a rare tumour with a poor prognosis. Molecular biology data on SBA carcinogenesis are lacking., Methods: Expression of HER2, β-catenin, p53 and mismatch repair (MMR) protein was assessed by immunohistochemistry. KRAS, V600E BRAF mutations and microsatellite instability were investigated., Results: We obtained samples from 63 SBA patients (tumour stages: I-II: 30%; III: 35%; IV: 32%; locally advanced: 3%). HER2 overexpression (3+) was observed in 2 out of 62 patients, overexpression of p53 in 26 out of 62, abnormal expression of β-catenin in 12 out of 61, KRAS mutation in 21 out of 49, BRAF V600E mutation in 1 out of 40 patients, MMR deficiency (dMMR) in 14 out of 61 and was consistent with Lynch syndrome in 9 out of 14 patients. All of the dMMR tumours were in the duodenum or jejunum and only one was stage IV. Median overall survival (OS) was 36.6 months (95% CI, 26.9-72.2). For all patients, in univariate analysis, stages I-II (P<0.001), WHO PS 0-1 (P=0.01) and dMMR phenotype (P=0.02) were significantly associated with longer OS. In multivariate analysis, disease stage (P=0.01) and WHO PS 0-1 (P=0.001) independently predicted longer OS. For stage IV patients, median OS was 20.5 months (95% CI: 14.6; 36.6 months). In multivariate analysis, WHO PS 0-1 (P=0.0001) and mutated KRAS status (P=0.02) independently predicted longer OS., Conclusion: This large study suggests that molecular alterations in SBA are closer to those in colorectal cancer (CRC) than those in gastric cancer, with low levels of HER 2 overexpression and high frequencies of KRAS mutations. The seemingly higher frequency of dMMR than in CRC may be explained by the higher frequency of Lynch syndrome in SBA patients. A dMMR phenotype was significantly associated with a non-metastatic tumour (P=0.02). A trend for a good prognosis and a duodenum or jejunum primary site was associated with dMMR.

EML4-ALK adenocarcinomas constitute a new molecular subgroup of lung tumours that respond very well to crizotinib, an ALK inhibitor. However, the diagnosis of ALK rearrangement in lung cancer is challenging. The aim of this study was to compare the diagnostic accuracy of five different methods in a series of 20 EGFR(wt/wt) lung adenocarcinomas from non- or light- smokers. Multiplex RT-PCR was considered as gold standard and identified four ALK-rearranged tumours among the 20 tested tumours. qRT-PCR got an interpretability rate of 100% and accurately typed all 20 tumours. qRT-PCR from corresponding formalin-fixed paraffin-embedded (FFPE) specimens got an interpretability rate of 65%. Out of the four previously identified ALK-rearranged cases, three were interpretable and two were retrieved using FFPE qRT-PCR. ALK break-apart FISH got an interpretability rate of 60% and accurately typed all of the twelve remaining cases. Anti-ALK immunohistochemistry (IHC) accurately typed all twenty tumours using a cut-off value of strong staining of 100% tumour cells. The 16 non ALK-rearranged tumours got no/light staining in 13 cases, and a moderate staining of 80-100% tumour cells in 3 cases. We then analysed four solid signet-ring lung adenocarcinomas. FFPE qRT-PCR, FISH and immunohistochemistry were concordant in three cases, with positive and negative results in respectively one and two cases. The fourth case, which was positive by FISH and immunohistochemistry but negative by RT-PCR, was shown to have a non-EML4-ALK ALK-rearrangement. As various factors such as RNA quality, fixation quality and type of ALK rearrangement may impede ALK screening, we propose a combined FISH/molecular biology diagnostic algorithm in which anti-ALK immunohistochemistry is used as a pre-screening step., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Metastasis is a multistep process and the main cause of mortality in lung cancer patients. We previously showed that EGFR mutations were associated with a copy number gain at a locus encompassing the TWIST1 gene on chromosome 7. TWIST1 is a highly conserved developmental gene involved in embryogenesis that may be reactivated in cancers promoting both malignant conversion and cancer progression through an epithelial to mesenchymal transition (EMT). The aim of this study was to investigate the possible implication of TWIST1 reactivation on the acquisition of a mesenchymal phenotype in EGFR mutated lung cancer. We studied a series of consecutive lung adenocarcinoma from Caucasian non-smokers for which surgical frozen samples were available (n = 33) and showed that TWIST1 expression was linked to EGFR mutations (P<0.001), to low CDH1 expression (P<0.05) and low disease free survival (P = 0.044). To validate that TWIST1 is a driver of EMT in EGFR mutated lung cancer, we used five human lung cancer cell lines and demonstrated that EMT and the associated cell mobility were dependent upon TWIST1 expression in cells with EGFR mutation. Moreover a decrease of EGFR pathway stimulation through EGF retrieval or an inhibition of TWIST1 expression by small RNA technology reversed the phenomenon. Collectively, our in vivo and in vitro findings support that TWIST1 collaborates with the EGF pathway in promoting EMT in EGFR mutated lung adenocarcinoma and that large series of EGFR mutated lung cancer patients are needed to further define the prognostic role of TWIST1 reactivation in this subgroup.

Butyrate, a short-chain fatty acid produced by the colonic bacterial fermentation is able to induce cell growth inhibition and differentiation in colon cancer cells at least partially through its capacity to inhibit histone deacetylases. Since butyrate is expected to impact cellular metabolic pathways in colon cancer cells, we hypothesize that it could exert its antiproliferative properties by altering cellular metabolism. We show that although Caco2 colon cancer cells oxidized both butyrate and glucose into CO(2) , they displayed a higher oxidation rate with butyrate as substrate than with glucose. Furthermore, butyrate pretreatment led to an increase cell capacity to oxidize butyrate and a decreased capacity to oxidize glucose, suggesting that colon cancer cells, which are initially highly glycolytic, can switch to a butyrate utilizing phenotype, and preferentially oxidize butyrate instead of glucose as energy source to produce acetyl coA. Butyrate pretreated cells displayed a modulation of glutamine metabolism characterized by an increased incorporation of carbons derived from glutamine into lipids and a reduced lactate production. The butyrate-stimulated glutamine utilization is linked to pyruvate dehydrogenase complex since dichloroacetate reverses this effect. Furthermore, butyrate positively regulates gene expression of pyruvate dehydrogenase kinases and this effect involves a hyperacetylation of histones at PDK4 gene promoter level. Our data suggest that butyrate exerts two distinct effects to ensure the regulation of glutamine metabolism: it provides acetyl coA needed for fatty acid synthesis, and it also plays a role in the control of the expression of genes involved in glucose utilization leading to the inactivation of PDC., (Copyright © 2010 UICC.)

The CpG island methylator phenotype (CIMP) is a distinct phenotype in colorectal cancer, associated with specific clinical, pathologic, and molecular features. However, most of the studies stratified methylation according to two subgroups (CIMP-High versus No-CIMP/CIMP-Low). In our study, we defined three different subgroups of methylation (No-CIMP, CIMP-Low, and CIMP-High) and evaluated the prognostic significance of methylation status on a population-based series of sporadic colon cancers. A total of 582 colon adenocarcinomas were evaluated using methylation-specific PCR for 5 markers (hMLH1, P16, MINT1, MINT2, and MINT31). No-CIMP status was defined as no methylated locus, CIMP-Low status as one to three methylated loci, and CIMP-High status as four or five methylated loci. Clinicopathologic and molecular characteristics were correlated to the methylation status. Crude and relative survival was compared according to methylation status. In the microsatellite-stable (MSS) group, CIMP-High was significantly associated with proximal location (P = 0.011) and BRAF mutation (P < 0.001). KRAS mutations were more associated with CIMP-High and CIMP-Low status (P = 0.008). A shorter 5-year survival was observed in MSS cancer patients with CIMP-Low or CIMP-High status. These results remained significant in multivariate analysis adjusted for age, stage, and BRAF and KRAS mutational status [CIMP-Low: hazard ratio (HR), 1.85; 95% confidence interval (95% CI), 1.37-2.51; CIMP-High, HR, 2.90; 95% CI, 1.53-5.49 compared with No-CIMP]. Within the high-level microsatellite instability group, no difference in survival was observed between the different CIMP groups. Our results show the interest of defining three subgroups of patients according to their methylation status (No-CIMP/CIMP-Low/CIMP-High). Methylation is an independent prognostic factor in MSS colon cancer.

The RAS-MAPK, PI (3)K signaling pathways form a network that play a central role in tumorigenesis. The BRAF, KRAS and PI3KCA genes code 3 partners of this network and have been found to be activated by mutation in colorectal cancer; these mutations lead to unrestricted cell growth. We evaluated the clinicopathological features and the prognosis of patients with activated-network colon cancers in a population-based study. A total of 586 colon adenocarcinomas were evaluated using sequencing for mutations of KRAS and PI3KCA, and allelic discrimination for mutation of BRAF. Clinicopathological characteristics were correlated to the risk of bearing a mutation of the network using logistic regression. Three-year survival rates were compared with the Log rank test. A multivariate survival analysis using the Cox model was performed. After adjustment for age and microsatellite instability, activation of the network by mutation of at least 1 of the 3 genes was significantly associated with female sex (p = 0.02) and proximal location (p < 0.001). Lower levels of 3-year survival were associated with activation of the network by mutation of at least 1 of the 3 genes (59.4 and 69.4%, respectively; p = 0.009). These results remained significant in a multivariate analysis adjusted for sex, age, location, stage and microsatellite instability (HR = 1.48; CI CI(95%) = [1.07-2.04]). Our study is the first report to underline the potential role of RAS-MAPK, PI (3)K network mutations on survival in colon cancers. Because of the role of this signaling network on anticancer agents, the evaluation of its mutations could have clinical implications., ((c) 2008 Wiley-Liss, Inc.)

Background: The HNPCC syndrome (hereditary nonpolyposis colon cancer) is an inherited condition defined by clinical and genealogical information, known as Amsterdam criteria. In about 70% of cases, HNPCC syndrome is caused by germline mutations in MMR genes, leading to microsatellite instability of tumor DNA (MSI phenotype). Patients affected by the disease are at high risk for colorectal and endometrial carcinomas, but also for small intestine, urothelial, ovary, stomach and biliary tract carcinomas. HNPCC syndrome is responsible for 5% of colorectal cancers. Identification and management of this disease are part of a multidisciplinary procedure., Methods: Twelve experts have been mandated by the French Health Ministry to analyze and synthesize their consensus position, and the resulting document has been reviewed by an additional group of 4 independent experts., Main Recommendations: The lack of sensitivity of Amsterdam criteria in recognizing patients carrying a MMR germline mutation led to an enlargement of these criteria for the recruitment of possible HNPCC patients, and to a 2-steps strategy, asking first for a tumor characterization according to MSI phenotype, especially in case of early-onset sporadic cases. The identification of germline MMR mutations has no major consequence on the cancer treatments, but influences markedly the long-term follow-up and the management of at-risk relatives. Gene carriers will enter a follow-up program regarding their colorectal and endometrial cancer risks, but other organs being at low lifetime risk, no specific surveillance will be proposed.

Cancer is a DNA disease characterized by uncontrolled cell proliferation due to the accumulation of genetic alterations. Recent progress in molecular biology allowed the identification of markers potentially usefull for patients management through the identification of these genetic alterations and a best understanding of chemotherapy molecular targets. Several examples in digestive oncology underline the relevance of molecular biology in clinical research. If almost all colorectal cancers (CRC) correspond to the same histopathological type (adenocarcinoma), molecular biology allowed the identification of two different molecular mechanisms of colorectal carcinogenesis: chromosomal instability characterized by recurrent allelic losses on chromosomes 17, 5, 18, 8 and 22 that contribute to the inactivation of tumor suppressor genes, and genetic instability characterized by the instability of microsatellite loci due to an alteration of DNA mismatch repair leading to the accumulation of mutations in genes involved in the control of cell cycle and apoptosis. These data are potentially interesting for the management of CRC patients. Indeed, microsatellite instability seems not only to be a good prognostic factor but also a molecular factor that can predict response to adjuvant 5-fluorouracil based chemotherapy. Therapeutic clinical trials taking into account these molecular parameters are still going on. DNA microarray-based gene expression profiling technology that allows the simultaneous analysis of thousand of tumor genes represents also an interesting approach in oncology with the recent identification of a "genetic signature" as a risk factor of tumor recurrence in stage II CRC, a setting in which the benefit of adjuvant chemotherapy remains on debate. At last, a best understanding of chemotherapy molecular targets allowed the identification of genetic markers that can predict the response and/or the toxicity of anti-cancer drugs used in gastrointestinal cancers, which could be helpful in the future to propose for each patient a personalized treatment. Mutations that can predict the response of new target therapies such as the inhibitors of the c-KIT tyrosine kinase activity in gastrointestinal stromal tumors have also been found and will allow the selection of patients who can have benefit from these new therapeutic drugs.

After the primary diagnosis of an adenocarcinoma, diagnosis of the origin of a second adenocarcinoma is a problem (primitive or metastatic). Although the clinical evolution or location sometimes makes it possible to determine the origin of the new lesion, in some cases the diagnosis cannot be confirmed. Although a pathological examination is essential for diagnosis, it may still be inconclusive, making management difficult. The use of molecular biology can help solve this problem. We report the case of a patient who presented with an ovarian adenocarcinoma of an undetermined origin one year after an adenocarcinoma of the colon. This clinical case illustrates the contribution of molecular biology in the diagnosis of the origin of an ovarian adenocarcinoma by characterizing allelic losses in 5 chromosome segments using microsatellite markers genotyping in the two lesions. The comparative analysis suggested the primitive origin of the ovarian lesion.

Background: A novel member of the Wnt signalling pathway, Chibby, was recently identified. This protein inhibits Wnt/beta-catenin mediated transcriptional activation by competing with Lef-1 (the transcription factor and target of beta-catenin) to bind to beta-catenin. This suggests that Chibby could be a tumour suppressor protein. The C22orf2 gene coding Chibby is located on chromosome 22, a region recurrently lost in colorectal cancer. Activation of the Wnt pathway is a major feature of colorectal cancer and occurs through inactivation of APC or activation of beta-catenin. All of this led us to analyse the possible implication of Chibby in colorectal carcinogenesis., Methods: First, 36 tumour and matched normal colonic mucosa DNA were genotyped with five microsatellite markers located on chromosome 22 to search for loss of heterozygosity. Then, mutation screening of the C22orf2 coding sequence and splice sites was performed in the 36 tumour DNA. Finally, expression of Chibby was analysed by quantitative RT-PCR on 10 patients, 4 with loss of heterozygosity (LOH) on chromosome 22., Results: Loss of heterozygosity involving the C22orf2 region was detected in 11 out of 36 patients (30%). Sequencing analysis revealed a known variant, rs3747174, in exon 5: T321C leading to a silent amino acid polymorphism A107A. Allelic frequencies were 0.69 and 0.31 for T and C variants respectively. No other mutation was detected. Among the 10 patients studied, expression analysis revealed that Chibby is overexpressed in 2 tumours and underexpressed in 1. No correlations were found with 22q LOH status., Conclusion: As no somatic mutation was detected in C22orf2 in 36 colorectal tumour DNA, our results do not support the implication of Chibby as a tumour suppressor in colorectal carcinogenesis. This was supported by the absence of underexpression of Chibby among the tumour samples with 22q LOH. The implication of other Wnt pathway members remains to be identified to explain the part of colorectal tumours without mutation in APC and beta-catenin.

Background: The HNPCC syndrome (hereditary non polyposis colon cancer) is an inherited condition defined by clinical and genealogical information, known as Amsterdam criteria. In about 70% of cases, HNPCC syndrome is caused by germline mutations in MMR genes, leading to microsatellite instability of tumor DNA (MSI phenotype). Patients affected by the disease are at high risk for colorectal and endometrial carcinomas, but also for small intestine, urothelial, ovary, stomach and biliary tract carcinomas. HNPCC syndrome is responsible for 5% of colorectal cancers. Identification and management of this disease are part of a multidisciplinary procedure., Methods: 12 experts have been mandated by the French Health Ministry to analyze and synthesize their consensus position, and the resulting document has been reviewed by an additional group of 4 independent experts., Main Recommendations: The lack of sensitivity of Amsterdam criteria in recognizing patients carrying a MMR germline mutation led to an enlargement of these criteria for the recruitment of possible HNPCC patients, and to a 2-steps strategy, asking first for a tumor characterization according to MSI phenotype, especially in case of early-onset sporadic cases. The identification of germline MMR mutations has no major consequence on the cancer treatments, but influences markedly the long-term follow-up and the management of at-risk relatives. Gene carriers will enter a follow-up program regarding their colorectal and endometrial cancer risks, but other organs being at low lifetime risk, no specific surveillance will be proposed.

The influence of p53 mutations on the response to ionizing radiation and survival was retrospectively evaluated in patients treated with preoperative radiotherapy for rectal carcinoma. From 1989 to 1991, 86 rectal cancer patients treated by preoperative radiotherapy were included in this series. For all patients, endorectal sonography (to define ultrasonography TNM [uTNM]) was performed before treatment; 19 patients were classified as stage 1, 27 as stage 2 and 40 as stage 3. Response to radiotherapy (39 Gy in 13 fractions delivered in 17 days) was assessed by comparing the uT and the T obtained by histologic examination of the resected specimen (TNM classification). A rectal cancer biopsy was performed before treatment and enabled the search for p53 mutations by denaturing gradient gel electrophoresis (DGGE) and sequencing. The status of the p53 gene was correlated with the response to radiotherapy and survival. Forty-nine percent of the tumors presented abnormal DGGE profiles. The prevalence of p53 mutations was significantly higher in patients who did not respond to radiotherapy (63%) than in those who did respond (34%) (p < 0.01). Presence of a p53 mutation was associated with significantly shorter 5-year survival compared to patients without mutations (p < 0.02). In a multivariate analysis, p53 mutation status remained a prognostic factor independent of tumor posttreatment staging (p < 0.05). p53 status is an independent prognostic factor of response to radiotherapy and survival in rectal carcinoma., (Copyright 2002 Wiley-Liss, Inc.)

The Ah receptor (AhR) is a ligand-dependent transcription factor that positively regulates the expression of the CYP1A1 gene. We investigated the genetic polymorphisms of the AhR gene including the promoter, and examined the link between these polymorphisms, CYP1A1 inducibility and the lung cancer incidence. The AhR promoter region and the 11 exons of 30 subjects were screened. Among the three polymorphisms found, two [(2417)(A/G) ((157)G/A)] have never been described previously. The (1721)(G/A) and (2417)(A/G) are localized in exon 10 and lead to Arg(554)Lys and Met(786)Val substitutions, respectively. The other polymorphism was found in the 5'-untranslated region, resulting in the substitution of a G by an A at position 157 (157)(G/A). To evaluate the frequency of this allelic variant found, a DNA library of a case-control study of lung cancer (162 controls and 177 patients) was studied. There is no significant association between (1721)(G/A), (157)(G/A) and lung cancer: (1721)(G/A) and (157)(G/A) were detected at the same allele frequency of 0.086 and 0.25, respectively in both controls and patients. (2417)(A/G) was found in only one control of 100 (allele frequency 0.005). Statistical analysis did not show any relationship between both (1721)(G/A) and (157)(G/A) polymorphisms found and CYP1A1 inducibility. Considering the rareness of the (2417)(A/G) allelic variant we were not able to evaluate its association with inducibility. In conclusion, none of the polymorphisms were found to play a key role in the CYP1A1 inducibility or in the susceptibility to develop lung cancer.

Intensive screening for genetic alteration in colorectal cancer led to the identification of two types of colorectal tumours that are distinct by their carcinogenesis processes. The first group, named LOH (for loss of heterozygosity)-positive, is characterized by hyperploidy and allelic losses involving preferentially chromosome 18q and chromosome 17p. More than two-thirds of colorectal cancers belong to this group. The second group, called multiple microsatellite loci (MSI)-positive cancers, is characterized by genetic instability at microsatellite loci. Although colorectal cancer cells are characterized by specific microsatellite alterations, the same four different signalling pathways, WNT/Wingless pathway, K-ras pathway, transforming growth factor (TGF)beta pathway and p53 pathway, could be implicated in tumour progression. The WNT/Wingless pathway could be altered in two different ways according to whether the cancer cells belong to the group of LOH-positive or MSI-positive tumours. LOH-positive tumours activate the WNT/Wingless signalling pathway through an adenomatous polyposis coli (APC) mutation, whereas the MSI-positive tumours activate this pathway through a beta-catenin stabilizing mutation. Beta-catenin and APC mutations were observed as early as the adenomatous stage of colorectal neoplasia. In TGFbeta pathways LOH-positive tumours inactivated SMAD2 (similar to mother against decapentaplegic drosophilia) or SMAD4, whereas in MSI-positive tumours the TGFbeta type II receptor is frequently deleted. Alteration of these genes correlated closely with the progression of the adenoma to cancer. In the p53 pathway LOH-positive tumours showed frequent p53 mutation, whereas MSI-positive tumours demonstrated BAX (BCL-2-associated X protein)-inactivating mutation. These alterations contribute to the adenoma-carcinoma transition.

Recurrent allelic losses on chromosome 22q have been reported in colorectal cancer, distal to the NF2 gene, suggesting that another tumor suppressor gene might be involved. We report here the typing of 256 sporadic colorectal tumors and 18 colonic cancer cell lines using a set of chromosome 22 polymorphisms, ranging from 20 to 45. A panel of somatic cell hybrids, that allows to distinguish 11 bins in the 22q13 region, was used to localize 19 of the 45 selected markers and the putative tumor suppressor gene BZRP. Allelic-loss was observed in 43% of tumors. The minimal region of deletion that could be determined, telomeric to locus D22S270, refines significantly the position of the gene. The localization of the BZRP gene in this region led to a systematic screening for somatic point mutation. Direct sequencing of its coding sequence in 36 tumors hemizygous for chromosome 22 allowed the identification of three polymorphisms but failed to detect somatic mutation.

Allelic losses on the short arm of chromosome 17 occur frequently in colorectal cancers. Despite the existence of other common molecular events such as loss of the long arms of chromosomes 18 and 5, it has been demonstrated that the former has the greatest prognostic significance. Of the various genes mapping to the commonly deleted sequence, the best candidate as a 'target' seems to be the p53 antioncogene. We applied our methods of detection of the p53 protein in a series of 78 colorectal cancers stored in a tumour bank from 1985 to 1989, for which the median follow-up was 42 months. Nuclear-attached p53 was quantified by flow cytometry and soluble p53 was assayed by ELISA. Both assays used a monoclonal antibody considered to be specific for a conformational epitope present only on the mutated protein. Fifty of the 78 tumours (64%) were found to present significant levels of p53 attached to the nucleus. A further two tumours contained high levels of p53 only in their soluble fraction. Thus, 52 out of 78 cancers (67%) were considered to be positive for p53. The p53 content correlated with 17p loss (P < 0.002), hyperdiploid DNA content (P < 0.001) and tumour site (P < 0.03), but not Dukes' stage (P = 0.15). p53 negative cases had a better overall survival than p53 positive ones (P < 0.03). When the 14 stage D tumours were excluded from the analysis, p53 was no longer significantly predictive of survival (P < 0.07), but remained predictive of recurrence (P < 0.02) and metastasis (P < 0.03). Multivariate analysis was not performed because of the small number of cases. Overall, disease-free and metastasis-free survival were compared to the positivity obtained either with pAb 421 and/or 1801 or pAb 240 since all three were used in the flow cytometric analysis, defining subsets of 421-, 1801+ and 421-, 1801-, 240+. The presence of nuclear protein presenting the mutation-specific epitope, recognised by pAb 240, was found to be the most discriminant. It must be noted that univariate survival analysis demonstrated that more than 80% of patients with p53-negative tumours were alive at 3 years vs less than 50% in the p53-positive group. A large prospective study should be conducted to define the exact prognostic significance of the p53 content of colorectal carcinomas.

The p53 transformation-related gene is located on 17p, a chromosomal segment frequently under-represented in colorectal adenocarcinoma karyotypes. We have developed a flow cytometric method for detection of its gene product on isolated nuclei by indirect immunofluorescence. Criteria for defining the presence of p53 were established, using parameters related to the difference of fluorescence obtained by incubating nuclei with a specific monoclonal antibody (MAB) versus isotypic control. This method allowed simultaneous quantitation of p53 and DNA in tumor nuclei to be made. Twenty-two of the 41 tumors analyzed (54%) were found to overexpress p53 when compared to 13 normal mucosa specimens. Repeated preparations from different fragments of the same tumor gave reproducible results for the 21 cases tested. p53 was detected in a higher proportion of disseminated tumors (64%) compared to localized disease (39%), but the difference did not reach statistical significance (chi 2 = 2.4, p greater than 0.10). Tumors containing aneuploid cell subpopulations were also more frequently positive (65%) than diploid ones (20%), the difference being significant (Fisher's exact test, p less than 0.03). This dual parameter flow cytometric method, evaluating both DNA ploidy and p53 expression, may prove useful in identifying different biological subgroups of colorectal cancer.

Twenty-five colorectal tumors (rectum 6, left colon 13, right colon 6) were studied with respect to the overexpression of p53 and the activation by point mutation of the Ki-ras oncogene. Single point mutations on codon 12 and codon 13 were analyzed after PCR amplivication, dotblotting and sequential hybridization with 12 different oligonucleotides. The intranuclear concentration of p53 protein was measured by flow cytometry after immunofluorescence staining with monoclonal antibody Pab 421. Twelve tumors were found to significantly overexpress p53 and 6 of them had an activated Ki-ras (5 on codon 12, 1 on codon 13). Of 13 tumors which failed to demonstrate over expression of p53, 8 had an activated Ki-ras (5 on codon 12, 3 on codon 13). In our series, p53 overexpression and ki-ras activation appeared to be independent.