Publications by authors named "Hélène Blons"

97 Publications

Role of circulating tumor DNA in the management of patients with colorectal cancer.

Clin Res Hepatol Gastroenterol 2018 10 5;42(5):396-402. Epub 2018 Apr 5.

INSERM UMR-S1147, CNRS SNC5014, Paris Descartes University, Equipe labellisée Ligue Nationale contre le cancer, Paris, France; Department of Gastroenterology and Digestive Oncology, European Georges Pompidou Hospital, AP-HP, Paris Descartes University, Paris, France. Electronic address:

Colorectal cancer is a major health burden with a prognosis that has been improved with the progresses in diagnosis and the advance of chemotherapy and personalized medicine. However, because of intra-tumor heterogeneity, clonal evolution and selection, tumors often develop resistance to treatments. "Liquid biopsy" is a minimally invasive method, based on analysis of tumor-specific material in peripheral blood samples of patients. Analysis of tumor specific genetic or epigenetic alterations in cell-free circulating nucleic acids may reflect the molecular heterogeneity of the underlying disease process and serial testing could allow to monitor its temporal genomic changing without using re-biopsy. In this review, we focused on the role of circulating tumor DNA (ctDNA) as a biomarker in the management of patients with colorectal cancer at early and advanced stages. Through recent studies, we described its promising clinical applications for diagnosis, detection of recurrence after surgery and monitoring for tumor response or therapeutic resistance in metastatic setting. Such recent developments offer new perspectives for personalized medicine in colorectal cancer but still needs some standardized detection methods and further studies to validate its use in clinical routine.
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http://dx.doi.org/10.1016/j.clinre.2018.03.002DOI Listing
October 2018

Cost-effectiveness of , and testing for decision making in advanced nonsmall cell lung carcinoma: the French IFCT-PREDICT.amm study.

Eur Respir J 2018 03 15;51(3). Epub 2018 Mar 15.

Service de Pneumologie, Assistance Publique Hôpitaux de Paris, Hôpital Tenon, Sorbonne Universités, UPMC Université Paris 06, GRC 04, Theranoscan, Paris, France.

rearrangement and / mutations constitute the primary biomarkers tested to provide targeted or nontargeted therapies in advanced nonsmall cell lung cancer (NSCLC) patients. Our objective was to assess the cost-effectiveness of biomarker testing for NSCLC.Between 2013 and 2014, 843 treatment-naive patients were prospectively recruited at 19 French hospitals into a longitudinal observational cohort study. Two testing strategies were compared, with "at least one biomarker status known" and "at least status known", in addition to "no biomarker testing" as the reference strategy. The Kaplan-Meier approach was employed to assess restricted mean survival time. Direct medical costs incurred by hospitals were estimated with regard to treatment, inpatient care and biomarker testing.Compared with "no biomarker testing", the "at least one biomarker status known" strategy yielded an incremental cost-effectiveness ratio of EUR13 230 per life-year saved, which decreased to EUR7444 per life-year saved with the "at least status known" testing strategy. In sensitivity analyses, biomarker testing strategies were less costly and more effective in 41% of iterations.In summary, molecular testing prior to treatment initiation proves to be cost-effective in advanced NSCLC management and may assist decision makers in defining conditions for further implementation of these innovations in general practice.
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http://dx.doi.org/10.1183/13993003.01467-2017DOI Listing
March 2018

Transplantation of Human Embryonic Stem Cell-Derived Cardiovascular Progenitors for Severe Ischemic Left Ventricular Dysfunction.

J Am Coll Cardiol 2018 01;71(4):429-438

Cell Therapy Unit, Assistance Publique-Hôpitaux de Paris, Hôpital Saint-Louis, Paris, France; INSERM, Clinical Investigation Center in Biotherapies (CBT-501) and U1160, Institut Universitaire d'Hématologie, Hôpital Saint-Louis, Paris, France; University Paris Diderot, Sorbonne Paris Cité, Paris, France.

Background: In addition to scalability, human embryonic stem cells (hESCs) have the unique advantage of allowing their directed differentiation toward lineage-specific cells.

Objectives: This study tested the feasibility of leveraging the properties of hESCs to generate clinical-grade cardiovascular progenitor cells and assessed their safety in patients with severe ischemic left ventricular dysfunction.

Methods: Six patients (median age 66.5 years [interquartile range (IQR): 60.5 to 74.7 years]; median left ventricular ejection fraction 26% [IQR: 22% to 32%]) received a median dose of 8.2 million (IQR: 5 to 10 million) hESC-derived cardiovascular progenitors embedded in a fibrin patch that was epicardially delivered during a coronary artery bypass procedure. The primary endpoint was safety at 1 year and focused on: 1) cardiac or off-target tumor, assessed by imaging (computed tomography and fluorine-18 fluorodeoxyglucose positron emission tomography scans); 2) arrhythmias, detected by serial interrogations of the cardioverter-defibrillators implanted in all patients; and 3) alloimmunization, assessed by the presence of donor-specific antibodies. Patients were followed up for a median of 18 months.

Results: The protocol generated a highly purified (median 97.5% [IQR: 95.5% to 98.7%]) population of cardiovascular progenitors. One patient died early post-operatively from treatment-unrelated comorbidities. All others had uneventful recoveries. No tumor was detected during follow-up, and none of the patients presented with arrhythmias. Three patients developed clinically silent alloimmunization. All patients were symptomatically improved with an increased systolic motion of the cell-treated segments. One patient died of heart failure after 22 months.

Conclusions: This trial demonstrates the technical feasibility of producing clinical-grade hESC-derived cardiovascular progenitors and supports their short- and medium-term safety, thereby setting the grounds for adequately powered efficacy studies. (Transplantation of Human Embryonic Stem Cell-derived Progenitors in Severe Heart Failure [ESCORT]; NCT02057900).
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http://dx.doi.org/10.1016/j.jacc.2017.11.047DOI Listing
January 2018

Mutational Diversity of Lung Cancer and Associated Lymph Nodes. An Exploratory Prospective Study of 4 Resected cIIIA-N2.

Pathol Oncol Res 2019 Jan 6;25(1):319-325. Epub 2017 Nov 6.

Thoracic Surgery and Lung Transplantation Department, Georges Pompidou European Hospital, Assistance Publique Hôpitaux de Paris, 20 rue Leblanc, 75908, Paris Cedex 15, France.

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.
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http://dx.doi.org/10.1007/s12253-017-0352-xDOI Listing
January 2019

[Lynch syndrome and endometrial cancer].

Bull Cancer 2017 Dec 21;104(12):1013-1021. Epub 2017 Oct 21.

Université Paris Descartes, Sorbonne Paris cité, faculté de médecine, 15, rue de l'École-de-médecine, 75006 Paris, France; Assistance publique-Hôpitaux de Paris, hôpital européen Georges-Pompidou, chirurgie cancérologique gynécologique et du sein, 20, rue Leblanc, 75015 Paris, France; Université Paris Descartes, Inserm UMR-S 1124, 45, rue des Saints-Pères, 75006 Paris, France.

Lynch syndrome is a hereditary predisposition to many tumors, in the forefront of which endometrial cancer in women. It is related to the mutation of a mismatch repair gene, involved in DNA mismatch repair. This mutation leads to a loss of expression of the corresponding protein, and to genome instability in tumor cells. Cumulative risk at the age of 70 years is over 40 %. Endometrial cancers related to Lynch syndrome are most of the time sentinel (They reveal the predisposition in half of families.) and are characterized by young age at onset (before 60 years) and low body mass index compared with patients presenting sporadic tumors. Pathological tumor characteristics are debated but it seems to be two types of tumors according to age, older patients having standard tumors and younger ones more aggressive pattern. Endometrial cancers related to Lynch syndrome can be synchronous of ovarian cancer. Therapeutic management does not present any particularity. Conservative treatment can be considered more frequently due to young age of patients but has to respect usual guidelines. Prognosis of these tumors is controversial. Gynaecological screening, although its benefit has not been proved, appears crucial in this population, as well as prophylactic surgery, which remains the best prevention.
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http://dx.doi.org/10.1016/j.bulcan.2017.06.018DOI Listing
December 2017

Epithelial-to-Mesenchymal Transition and MicroRNAs in Lung Cancer.

Cancers (Basel) 2017 Aug 3;9(8). Epub 2017 Aug 3.

INSERM UMR-S1147, CNRS SNC 5014, Saints-Pères Research Center, 45 rue des Saints-Pères Paris-Descartes University, Sorbonne Paris Cité University, 75006 Paris, France.

Despite major advances, non-small cell lung cancer (NSCLC) remains the major cause of cancer-related death in developed countries. Metastasis and drug resistance are the main factors contributing to relapse and death. Epithelial-to-mesenchymal transition (EMT) is a complex molecular and cellular process involved in tissue remodelling that was extensively studied as an actor of tumour progression, metastasis and drug resistance in many cancer types and in lung cancers. Here we described with an emphasis on NSCLC how the changes in signalling pathways, transcription factors expression or microRNAs that occur in cancer promote EMT. Understanding the biology of EMT will help to define reversing process and treatment strategies. We will see that this complex mechanism is related to inflammation, cell mobility and stem cell features and that it is a dynamic process. The existence of intermediate phenotypes and tumour heterogeneity may be debated in the literature concerning EMT markers, EMT signatures and clinical consequences in NSCLC. However, given the role of EMT in metastasis and in drug resistance the development of EMT inhibitors is an interesting approach to counteract tumour progression and drug resistance. This review describes EMT involvement in cancer with an emphasis on NSCLC and microRNA regulation.
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http://dx.doi.org/10.3390/cancers9080101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575604PMC
August 2017

Morphologic and molecular study of lung cancers associated with idiopathic pulmonary fibrosis and other pulmonary fibroses.

Respir Res 2017 06 15;18(1):120. Epub 2017 Jun 15.

Département de Pathologie, Hôpital Bichat-Claude Bernard, Assistance Publique-Hôpitaux de Paris, 46 rue Henri Huchard, 75018, Paris, France.

Background: Primitive lung cancers developed on lung fibroses are both diagnostic and therapeutic challenges. Their incidence may increase with new more efficient lung fibrosis treatments. Our aim was to describe a cohort of lung cancers associated with idiopathic pulmonary fibrosis (IPF) and other lung fibrotic disorders (non-IPF), and to characterize their molecular alterations using immunohistochemistry and next-generation sequencing (NGS).

Methods: Thirty-one cancer samples were collected from 2001 to 2016 in two French reference centers for pulmonary fibrosis - 18 for IPF group and 13 for non-IPF group. NGS was performed using an ampliseq panel to analyze hotspots and targeted regions in 22 cancer-associated genes. ALK, ROS1 and PD-L1 expressions were assessed by immunohistochemistry.

Results: Squamous cell carcinoma was the most frequent histologic subtype in the IPF group (44%), adenocarcinoma was the most frequent subtype in the non-IPF group (62%). Forty-one mutations in 13 genes and one EGFR amplification were identified in 25 samples. Two samples had no mutation in the selected panel. Mutations were identified in TP53 (n = 20), MET (n = 4), BRAF (n = 3), FGFR3, PIK3CA, PTEN, STK11 (n = 2), SMAD4, CTNNB1, DDR2, ERBB4, FBXW7 and KRAS (n = 1) genes. No ALK and ROS1 expressions were identified. PD-L1 was expressed in 10 cases (62%) with only one (6%) case >50%.

Conclusions: This extensive characterization of lung fibrosis-associated cancers evidenced molecular alterations which could represent either potential therapeutic targets either clues to the pathophysiology of these particular tumors. These findings support the relevance of large molecular characterization of every lung fibrosis-associated cancer.
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http://dx.doi.org/10.1186/s12931-017-0605-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472872PMC
June 2017

Early Evaluation of Circulating Tumor DNA as Marker of Therapeutic Efficacy in Metastatic Colorectal Cancer Patients (PLACOL Study).

Clin Cancer Res 2017 Sep 2;23(18):5416-5425. Epub 2017 Jun 2.

INSERM UMR-S1147, CNRS SNC5014; Paris Descartes University, Equipe Labellisée Ligue Nationale Contre le Cancer, Paris, France.

Markers of chemotherapy efficacy in metastatic colorectal cancer (mCRC) are essential for optimization of treatment strategies. We evaluated the applicability of early changes in circulating tumor DNA (ctDNA) as a marker of therapeutic efficacy. This prospective study enrolled consecutive patients with mCRC receiving a first- or second-line chemotherapy. CtDNA was assessed in plasma collected before the first (C), second (C) and/or third (C) chemotherapy cycle, using picodroplet-digital PCR assays based either on detection of gene mutation () or hypermethylation (). CT scans were centrally assessed using RECIST v1.1 criteria. Multivariate analyses were adjusted on age, gender, ECOG performance status (PS), metastatic synchronicity, and treatment line. Eighty-two patients with mCRC treated in first- (82.9%) or second- (17.1%) line chemotherapy were included. Patients with a high (>10 ng/mL) versus low (≤0.1 ng/mL) ctDNA concentration at C had a shorter overall survival (OS; 6.8 vs. 33.4 months: adjusted HR, 5.64; 95% CI, 2.5-12.6; < 0.0001). By analyzing the evolution of the ctDNA concentration between C and C or C (C), we classified the patients in two groups (named "good" or "bad ctDNA responders"). In multivariate analysis, patients belonging to the group called "good ctDNA responder" ( = 58) versus "bad ctDNA responder" () had a better objective response rate ( < 0.001), and a longer median progression-free survival (8.5 vs. 2.4 months: HR, 0.19; 95% CI, 0.09-0.40; < 0.0001) and OS (27.1 vs. 11.2 months: HR, 0.25; 95% CI, 0.11-0.57; < 0.001). This study suggests that early change in ctDNA concentration is a marker of therapeutic efficacy in patients with mCRC. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-3155DOI Listing
September 2017

Composite biomarkers defined by multiparametric immunofluorescence analysis identify ALK-positive adenocarcinoma as a potential target for immunotherapy.

Oncoimmunology 2017;6(4):e1286437. Epub 2017 Feb 21.

INSERM U970, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

Anaplastic lymphoma kinase (ALK) inhibitors have been successfully developed for non-small cell lung carcinoma (NSCLC) displaying chromosomal rearrangements of the gene, but unfortunately resistance invariably occurs. Blockade of the PD-1-PD-L1/2 inhibitory pathway constitutes a breakthrough for the treatment of NSCLC. Some predictive biomarkers of clinical response to this therapy are starting to emerge, such as PD-L1 expression by tumor/stromal cells and infiltration by CD8 T cells expressing PD-1. To more effectively integrate all of these potential biomarkers of clinical response to immunotherapy, we have developed a multiparametric immunofluorescence technique with automated immune cell counting to comprehensively analyze the tumor microenvironment of -positive adenocarcinoma (ADC). When analyzed as either a continuous or a dichotomous variable, the mean number of tumor cells expressing PD-L1 ( = 0.012) and the percentage of tumor cells expressing PD-L1 were higher in -positive ADC than in ADC or WT (non--mutated and non--mutated) NSCLC. A very strong correlation between PD-L1 expression on tumor cells and intratumoral infiltration by CD8 T cells was observed, suggesting that an adaptive mechanism may partly regulate this expression. A higher frequency of tumors combining positive PD-L1 expression and infiltration by intratumoral CD8 T cells or PD-1CD8 T cells was also observed in -positive lung cancer patients compared with -mutated ( = 0.03) or WT patients ( = 0.012). These results strongly suggest that a subgroup of -positive lung cancer patients may constitute good candidates for anti-PD-1/-PD-L1 therapies.
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http://dx.doi.org/10.1080/2162402X.2017.1286437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414864PMC
February 2017

Women Epidemiology Lung Cancer (WELCA) study: reproductive, hormonal, occupational risk factors and biobank.

BMC Public Health 2017 Apr 17;17(1):324. Epub 2017 Apr 17.

Université Paris Descartes, Unité de cancérologie thoracique, Groupe Hospitalier Paris Saint-Joseph, Paris, France.

Background: Lung cancer aetiology and clinical aspects have been mainly studied in men, although specific risk factors probably exist in women. Here we present the rationale, design and organization of the WELCA study (Women Epidemiology Lung CAncer) that has been launched to investigate lung cancer in women, focusing particularly on hormonal and occupational factors.

Methods/design: WELCA is a population based case-control study and planned to recruit 1000 cases and 1000 controls in three years, based on study power calculation. Eligible cases are female patients newly diagnosed with lung cancer, living in Paris and the Ile de France area and aged up to 75 years. Almost all Parisian pneumology and oncology clinical departments are involved. The control group is a random sample of the population living in the same area, frequency-matched on age and additionally stratified on the distribution of socio-professional categories of women residing there. After acquisition of written consent, research nurses administer standardized computer assisted questionnaires to all the subjects in face-to-face interviews and acquire anthropometric measures. Besides usual socio-demographic characteristics, information is gathered about menstrual and reproductive factors, hormonal treatments, lifestyle and leisure characteristics, occupational history, personal and familial medical history. Biological samples are also collected, in order to establish a biobank for molecular epidemiology studies. Molecular characteristics of the tumours will be obtained and patients will be followed up for five years.

Discussion: The WELCA study aims to answer key questions in lung cancer aetiology and clinical characteristics specifically in women. The role of hormonal impregnation is investigated, and the interactions with cigarette smoking or body mass index (BMI) will be analyzed in detail. The occupational history of the subjects is carefully reconstructed, focusing in particular on the service sector. The creation of a biobank for collection of serum, plasma, DNA and tumour tissue will allow the genetic and biochemical characterization of both the subjets and the tumours. The follow-up of the patients will help in disentangling the role of hormonal factors and tumour molecular characteristics in survival.
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http://dx.doi.org/10.1186/s12889-017-4191-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5392991PMC
April 2017

Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study.

PLoS Med 2016 Dec 27;13(12):e1002199. Epub 2016 Dec 27.

INSERM UMR-S1147, CNRS SNC 5014, Equipe labélisée Ligue Contre le Cancer, Université Sorbonne Paris Cité, Paris, France.

Background: Circulating tumor DNA (ctDNA) is an approved noninvasive biomarker to test for the presence of EGFR mutations at diagnosis or recurrence of lung cancer. However, studies evaluating ctDNA as a noninvasive "real-time" biomarker to provide prognostic and predictive information in treatment monitoring have given inconsistent results, mainly due to methodological differences. We have recently validated a next-generation sequencing (NGS) approach to detect ctDNA. Using this new approach, we evaluated the clinical usefulness of ctDNA monitoring in a prospective observational series of patients with non-small cell lung cancer (NSCLC).

Methods And Findings: We recruited 124 patients with newly diagnosed advanced NSCLC for ctDNA monitoring. The primary objective was to analyze the prognostic value of baseline ctDNA on overall survival. ctDNA was assessed by ultra-deep targeted NGS using our dedicated variant caller algorithm. Common mutations were validated by digital PCR. Out of the 109 patients with at least one follow-up marker mutation, plasma samples were contributive at baseline (n = 105), at first evaluation (n = 85), and at tumor progression (n = 66). We found that the presence of ctDNA at baseline was an independent marker of poor prognosis, with a median overall survival of 13.6 versus 21.5 mo (adjusted hazard ratio [HR] 1.82, 95% CI 1.01-3.55, p = 0.045) and a median progression-free survival of 4.9 versus 10.4 mo (adjusted HR 2.14, 95% CI 1.30-3.67, p = 0.002). It was also related to the presence of bone and liver metastasis. At first evaluation (E1) after treatment initiation, residual ctDNA was an early predictor of treatment benefit as judged by best radiological response and progression-free survival. Finally, negative ctDNA at E1 was associated with overall survival independently of Response Evaluation Criteria in Solid Tumors (RECIST) (HR 3.27, 95% CI 1.66-6.40, p < 0.001). Study population heterogeneity, over-representation of EGFR-mutated patients, and heterogeneous treatment types might limit the conclusions of this study, which require future validation in independent populations.

Conclusions: In this study of patients with newly diagnosed NSCLC, we found that ctDNA detection using targeted NGS was associated with poor prognosis. The heterogeneity of lung cancer molecular alterations, particularly at time of progression, impairs the ability of individual gene testing to accurately detect ctDNA in unselected patients. Further investigations are needed to evaluate the clinical impact of earlier evaluation times at 1 or 2 wk. Supporting clinical decisions, such as early treatment switching based on ctDNA positivity at first evaluation, will require dedicated interventional studies.
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http://dx.doi.org/10.1371/journal.pmed.1002199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5189949PMC
December 2016

Analysis of Base-Position Error Rate of Next-Generation Sequencing to Detect Tumor Mutations in Circulating DNA.

Clin Chem 2016 11 13;62(11):1492-1503. Epub 2016 Sep 13.

INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer;

Background: Detecting single-nucleotide variations and insertions/deletions in circulating tumor DNA is challenging because of their low allele frequency. The clinical use of circulating tumor DNA to characterize tumor genetic alterations requires new methods based on next-generation sequencing.

Methods: We developed a method based on quantification of error rate of each base position [position error rate (PER)]. To identify mutations, a binomial test was used to compare the minor-allele frequency to the measured PER at each base position. This process was validated in control samples and in 373 plasma samples from patients with lung or pancreatic cancer.

Results: Minimal mutated allele frequencies were 0.003 for single-nucleotide variations and 0.001 for insertions/deletions. Independent testing performed by droplet digital PCR (n = 231 plasma samples) showed strong agreement with the base-PER method (κ = 0.90).

Conclusions: Targeted next-generation sequencing analyzed with the base-PER method represents a robust and low cost method to detect circulating tumor DNA in patients with cancer.
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http://dx.doi.org/10.1373/clinchem.2016.258236DOI Listing
November 2016

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

PLoS One 2016 14;11(7):e0159094. Epub 2016 Jul 14.

Université Sorbonne Paris Cité (USPC), INSERM UMR-S1147, CNRS SNC 5014, Centre Universitaire des Saints-Pères, Paris, France.

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0159094PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945036PMC
July 2017

Intratumoral Immune Cell Densities Are Associated with Lung Adenocarcinoma Gene Alterations.

Am J Respir Crit Care Med 2016 12;194(11):1403-1412

1 INSERM UMRS 1138, Centre de Recherche des Cordeliers, Paris, France.

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.
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http://dx.doi.org/10.1164/rccm.201510-2031OCDOI Listing
December 2016

[Lung cancer molecular testing, what role for Next Generation Sequencing and circulating tumor DNA].

Ann Pathol 2016 Jan 20;36(1):80-93. Epub 2016 Jan 20.

Inserm UMR-S1147, université Paris Sorbonne Cité, 75006 Paris, France; Unité de pharmacogénétique et oncologie moléculaire, service de biochimie, hôpital européen Georges-Pompidou (HEGP), Assistance publique-Hôpitaux de Paris, 75015 Paris, France. Electronic address:

Molecular screening has become a standard of care for patients with advanced cancers and impacts on how to treat a patient. Advances in genomic technologies with the development of high throughput sequencing methods will certainly improve the possibilities to access a more accurate molecular diagnosis and to go beyond the identification of validated targets as a large number of genes can be screened for actionable changes. Moreover, accurate high throughput testing may help tumor classification in terms of prognosis and drug sensitivity. Finally, it will be possible to assess tumor heterogeneity and changes in molecular profiles during follow-up using ultra-deep sequencing technologies and circulating tumor DNA characterization. The accumulation of somatic ADN alterations is considered as the main contributing factor in carcinogenesis. The alterations can occur at different levels: mutation, copy number variations or gene translocations resulting in altered expression of the corresponding genes or impaired protein functions. Genes involved are mainly tumor suppressors, oncogenes or ADN repair genes whose modifications in tumors will impinge cell fate and proliferation from tumor initiation to metastasis. The entire genome of various tumor types, have now been sequenced. In lung cancer, the average number of mutations is very high with more than 8.9 mutations/Mb (Network TCGAR, 2014) that is to say more than 10,000 mutations/genome. These alterations need to be classified, indeed, some are true drivers that directly impact proliferation and some are passenger mutations linked to genetic instability. The development of targeted therapies relies on the identification of oncogenic drivers. The identification of genotype-phenotype associations as in the case of EGFR-TKI (Epidermal growth factor receptor-tyrosine kinase inhibitor) and EGFR mutations in lung cancer led to the restriction of drugs to patients for which tumor genotype predicts efficacy. Tumor-molecular directed therapy based on validated targets (EGFR, ALK) is in the clinics, rapidly, with the developments of multi-targets or multi-drug assays there will be a need for tumor-molecular-profile directed therapy. Today, there are practical challenges to a successful implementation of NGS technologies for clinical applications. Broadly, some are linked to the tumor (heterogeneity), to the tissue (availability, storage, fixative), to the design of specific assays or set of genes, to the interpretation of non-driver mutations and to a possible access to drugs once a target is identified. Technical challenges are solved, NGS (at least targeted-NGS) plateforms have been validated by INCa labeled laboratories, in this context, we will address different questions: How, for whom, what kind of profiling and what can we expect?
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http://dx.doi.org/10.1016/j.annpat.2015.11.012DOI Listing
January 2016

[What future for circulating tumor DNA? Current data and prospects in colorectal, non-small cell lung and pancreatic cancers].

Bull Cancer 2016 Jan 11;103(1):55-65. Epub 2016 Jan 11.

Université Paris Sorbonne Cité, centre universitaire des Saints-Pères, CNRS SNC5014, Inserm UMR-S1147 MEPPOT, 75006 Paris, France; Assistance publique-Hôpitaux de Paris, groupe hospitalier Pitié-Salpêtrière, pôle 3I, service d'hépatogastroentérologie, 75013 Paris, France; Université Sorbonne, université Pierre-et-Marie-Curie, Paris 06, 75005 Paris, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bulcan.2015.10.017DOI Listing
January 2016

Routine molecular profiling of patients with advanced non-small-cell lung cancer: results of a 1-year nationwide programme of the French Cooperative Thoracic Intergroup (IFCT).

Lancet 2016 Apr 15;387(10026):1415-1426. Epub 2016 Jan 15.

Assistance Publique Hôpitaux de Paris, Hôpital Tenon, Service de Pneumologie, Sorbonne Universités, UPMC Université Paris 06, Paris, France.

Background: The molecular profiling of patients with advanced non-small-cell lung cancer (NSCLC) for known oncogenic drivers is recommended during routine care. Nationally, however, the feasibility and effects on outcomes of this policy are unknown. We aimed to assess the characteristics, molecular profiles, and clinical outcomes of patients who were screened during a 1-year period by a nationwide programme funded by the French National Cancer Institute.

Methods: This study included patients with advanced NSCLC, who were routinely screened for EGFR mutations, ALK rearrangements, as well as HER2 (ERBB2), KRAS, BRAF, and PIK3CA mutations by 28 certified regional genetics centres in France. Patients were assessed consecutively during a 1-year period from April, 2012, to April, 2013. We measured the frequency of molecular alterations in the six routinely screened genes, the turnaround time in obtaining molecular results, and patients' clinical outcomes. This study is registered with ClinicalTrials.gov, number NCT01700582.

Findings: 18,679 molecular analyses of 17,664 patients with NSCLC were done (of patients with known data, median age was 64·5 years [range 18-98], 65% were men, 81% were smokers or former smokers, and 76% had adenocarcinoma). The median interval between the initiation of analysis and provision of the written report was 11 days (IQR 7-16). A genetic alteration was recorded in about 50% of the analyses; EGFR mutations were reported in 1947 (11%) of 17,706 analyses for which data were available, HER2 mutations in 98 (1%) of 11,723, KRAS mutations in 4894 (29%) of 17,001, BRAF mutations in 262 (2%) of 13,906, and PIK3CA mutations in 252 (2%) of 10,678; ALK rearrangements were reported in 388 (5%) of 8134 analyses. The median duration of follow-up at the time of analysis was 24·9 months (95% CI 24·8-25·0). The presence of a genetic alteration affected first-line treatment for 4176 (51%) of 8147 patients and was associated with a significant improvement in the proportion of patients achieving an overall response in first-line treatment (37% [95% CI 34·7-38·2] for presence of a genetic alteration vs 33% [29·5-35·6] for absence of a genetic alteration; p=0·03) and in second-line treatment (17% [15·0-18·8] vs 9% [6·7-11·9]; p<0·0001). Presence of a genetic alteration was also associated with improved first-line progression-free survival (10·0 months [95% CI 9·2-10·7] vs 7·1 months [6·1-7·9]; p<0·0001) and overall survival (16·5 months [15·0-18·3] vs 11·8 months [10·1-13·5]; p<0·0001) compared with absence of a genetic alteration.

Interpretation: Routine nationwide molecular profiling of patients with advanced NSCLC is feasible. The frequency of genetic alterations, acceptable turnaround times in obtaining analysis results, and the clinical advantage provided by detection of a genetic alteration suggest that this policy provides a clinical benefit.

Funding: French National Cancer Institute (INCa).
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http://dx.doi.org/10.1016/S0140-6736(16)00004-0DOI Listing
April 2016

Prognostic Effect of BRAF and KRAS Mutations in Patients With Stage III Colon Cancer Treated With Leucovorin, Fluorouracil, and Oxaliplatin With or Without Cetuximab: A Post Hoc Analysis of the PETACC-8 Trial.

JAMA Oncol 2016 May;2(5):643-653

Centre de Recherché UMR-S 1147, Médecine Personnalisée, Pharmacogénomique, Optimisation Thérapeutique, Institut National de la Santé et de la Recherche Médicale, Paris, France6Paris Descartes University, Department of Biology, European Georges Pompidou Ho.

Importance: The prognostic value of BRAF and KRAS mutations in patients who have undergone resection for colon cancer and have been treated with combination leucovorin, fluorouracil, and oxaliplatin (FOLFOX)-based adjuvant chemotherapy is controversial, possibly owing to a lack of stratification on mismatch repair status.

Objective: To examine the prognostic effect of BRAF and KRAS mutations in patients with stage III colon cancer treated with adjuvant FOLFOX with or without cetuximab.

Design, Setting, And Participants: This study included patients with available tumor blocks of resected stage III colon adenocarcinoma who participated between December 2005 and November 2009 in the PETACC-8 phase III randomized trial. Mismatch repair, BRAF V600E, and KRAS exon 2 mutational status were determined on prospectively collected tumor blocks from 2559 patients enrolled in the PETACC-8 trial. The data were analyzed in April 2015.

Intervention: Patients were randomly assigned to receive 6 months of FOLFOX4 or FOLFOX4 plus cetuximab after surgical resection for stage III colon cancer.

Main Outcomes And Measures: Associations between these biomarkers and disease-free survival (DFS) and overall survival (OS) were analyzed with Cox proportional hazards models. Multivariate models were adjusted for covariates (age, sex, tumor grade, T/N stage, tumor location, Eastern Cooperative Oncology Group performance status).

Results: Among the 2559 patients enrolled in the PETACC-8 trial (42.9% female; median [range] age, 60.0 [19.0-75.0] years), microsatellite instability (MSI) phenotype, KRAS, and BRAF V600E mutations were detected in, respectively, 9.9% (177 of 1791), 33.1% (588 of 1776), and 9.0% (148 of 1643) of cases. In multivariate analysis, MSI (hazard ratio [HR] for DFS: 1.10 [95% CI, 0.73-1.64], P = .67; HR for OS: 1.02 [95% CI, 0.61-1.69], P = .94) and BRAF V600E mutation (HR for DFS: 1.22 [95% CI, 0.81-1.85], P = .34; HR for OS: 1.13 [95% CI, 0.64-2.00], P = .66) were not prognostic, whereas KRAS mutation was significantly associated with shorter DFS (HR, 1.55 [95% CI, 1.23-1.95]; P < .001) and OS (HR, 1.56 [95% CI, 1.12-2.15]; P = .008). The subgroup analysis showed in patients with microsatellite-stable tumors that both KRAS (HR for DFS: 1.64 [95% CI, 1.29-2.08], P < .001; HR for OS: 1.71 [95% CI, 1.21-2.41], P = .002) and BRAF V600E mutation (HR for DFS: 1.74 [95% CI, 1.14-2.69], P = .01; HR for OS: 1.84 [95% CI, 1.01-3.36], P = .046) were independently associated with worse clinical outcomes. In patients with MSI tumors, KRAS status was not prognostic, whereas BRAF V600E mutation was associated with significantly longer DFS (HR, 0.23 [95% CI, 0.06-0.92]; P = .04) but not OS (HR, 0.19 [95% CI, 0.03-1.24]; P = .08).

Conclusions And Relevance: BRAF V600E and KRAS mutations were significantly associated with shorter DFS and OS in patients with microsatellite-stable tumors but not in patients with MSI tumors. Future trials in the adjuvant setting will have to take into account mismatch repair, BRAF, and KRAS status for stratification.

Trial Registration: EudraCT 2005-003463-23.
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http://dx.doi.org/10.1001/jamaoncol.2015.5225DOI Listing
May 2016

Three Rounds of External Quality Assessment in France to Evaluate the Performance of 28 Platforms for Multiparametric Molecular Testing in Metastatic Colorectal and Non-Small Cell Lung Cancer.

J Mol Diagn 2016 Mar 2;18(2):205-14. Epub 2016 Jan 2.

Department of Pathology, Radboud University Medical Center, Nijmegen, the Netherlands.

Personalized medicine has gained increasing importance in clinical oncology, and several clinically important biomarkers are implemented in routine practice. In an effort to guarantee high quality of molecular testing in France, three subsequent external quality assessment rounds were organized at the initiative of the National Cancer Institute between 2012 and 2014. The schemes included clinically relevant biomarkers for metastatic colorectal (KRAS, NRAS, BRAF, PIK3CA, microsatellite instability) and non-small cell lung cancer (EGFR, KRAS, BRAF, PIK3CA, ERBB2), and they represent the first multigene/multicancer studies throughout Europe. In total, 56 laboratories coordinated by 28 regional molecular centers participated in the schemes. Laboratories received formalin-fixed, paraffin-embedded samples and were asked to use routine methods for molecular testing to predict patient response to targeted therapies. They were encouraged to return results within 14 calendar days after sample receipt. Both genotyping and reporting were evaluated separately. During the three external quality assessment rounds, mean genotype scores were all above the preset standard of 90% for all biomarkers. Participants were mainly challenged in case of rare insertions or deletions. Assessment of the written reports showed substantial progress between the external quality assessment schemes on multiple criteria. Several essential elements such as the clinical interpretation of test results and the reason for testing still require improvement by continued external quality assessment education.
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http://dx.doi.org/10.1016/j.jmoldx.2015.09.004DOI Listing
March 2016

Different prognostic impact of STK11 mutations in non-squamous non-small-cell lung cancer.

Oncotarget 2017 Apr;8(14):23831-23840

INSERM UMR-S1147, Paris Sorbonne Cité Université, Paris, France.

STK11 is commonly mutated in lung cancer. In light of recent experimental data showing that specific STK11 mutants could acquire oncogenic activities due to the synthesis of a short STK11 isoform, we investigated whether this new classification of STK11 mutants could help refine its role as a prognostic marker. We conducted a retrospective high-throughput genotyping study in 567 resected non-squamous non-small-cell lung cancer (NSCLC) patients. STK11 exons 1 or 2 mutations (STK11ex1-2) with potential oncogenic activity were analyzed separately from exons 3 to 9 (STK11ex3-9). STK11ex1-2 and STK11ex3-9 mutations occurred in 5% and 14% of NSCLC. STK11 mutated patients were younger (P = .01) and smokers (P< .0001). STK11 mutations were significantly associated with KRAS and inversely with EGFR mutations. After a median follow-up of 7.2 years (95%CI 6.8-.4), patients with STK11ex1-2 mutation had a median OS of 24 months (95%CI 15-57) as compared to 69 months (95%CI 56-93) for wild-type (log-rank, P = .005) and to 91 months (95%CI 57-unreached) for STK11ex3-9 mutations (P = .003). In multivariate analysis, STK11ex1-2 mutations remained associated with a poor prognosis (P = .002). Results were validated in two public datasets. Western blots showed that STK11ex1-2 mutatedtumors expressed short STK11 isoforms. Finally using mRNAseq data from the TCGA cohort, we showed that a stroma-derived poor prognosis signature was enriched in STK11ex1-2 mutated tumors. All together our results show that STK11ex1-2 mutations delineate an aggressive subtype of lung cancer for which a targeted treatment through STK11 inhibition might offer new opportunities.
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http://dx.doi.org/10.18632/oncotarget.6379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410347PMC
April 2017

Sustained response to vemurafenib in a low grade serous ovarian cancer with a BRAF V600E mutation.

Invest New Drugs 2015 Dec 21;33(6):1267-70. Epub 2015 Oct 21.

Centre des cancers de la femme et recherche clinique, GH Paris Centre, site Hôtel-Dieu, 1 place du Parvis Notre-Dame, 75181, Paris cedex 04, France.

Low-grade serous ovarian adenocarcinomas (LGSOC) make up approximately 10 % of serous ovarian carcinomas. While rarely aggressive, this slow-growing tumor is well known to respond poorly to chemotherapy. Specific treatments for this ovarian subtype are lacking, with the same global approaches used for high grade cases being applied for LGSOC patients. LGSOCs have been reported to have a specific genetic profile, with notable implication of the MAPK pathway. This has opened up opportunities for novel therapeutic strategies, with in particular the use of targeted therapies. We report here the case of a heavily pretreated unresectable BRAF p.V600E-mutated LGSOC, which we treated vemurafenib, a BRAF inhibitor specific for V600E mutations. We saw impressive efficacy, with a long-term partial response along with CA125 reductions and symptom relief. Although this mutation is present in LGSOC at very a low incidence, we recommend routine testing for BRAF and other targetable mutations in this patient population, along with further evaluation in the increasingly popular basket trial approach.
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http://dx.doi.org/10.1007/s10637-015-0297-4DOI Listing
December 2015

French multicentric validation of ALK rearrangement diagnostic in 547 lung adenocarcinomas.

Eur Respir J 2015 Jul 30;46(1):207-18. Epub 2015 Apr 30.

Département d'Anatomie et de Cytologie Pathologiques, Pôle de Biologie et de Pathologie, CHU A. Michallon, and Université Joseph Fourier, INSERM U823, Institut Albert Bonniot, Grenoble, France.

Anaplastic lymphoma kinase (ALK) gene rearrangements in lung adenocarcinoma result in kinase activity targetable by crizotinib. Although fluorescence in situ hybridisation (FISH) is the reference diagnostic technique, immunohistochemistry (IHC) could be useful for pre-screening. Diagnostic yields of ALK IHC, FISH and quantitative reverse transcriptase PCR performed in 14 French pathology/molecular genetics platforms were compared. 547 lung adenocarcinoma specimens were analysed using 5A4 and D5F3 antibodies, two break-apart FISH probes and TaqMan kits. Clinicopathological data were recorded. 140 tumours were ALK rearranged (FISH with ≥15% of rearranged cells) and 400 were ALK FISH negative (<15%). FISH was not interpretable for seven cases. ALK patients were young (p=0.003), mostly females (p=0.007) and light/nonsmokers (p<0.0001). 13 cases were IHC negative but FISH ≥15%, including six cases with FISH between 15% and 20%; eight were IHC positive with FISH between 10% and 14%. Sensitivity and specificity for 5A4 and D5F3 were 87% and 92%, and 89% and 76%, respectively. False-negative IHC, observed in 2.4% of cases, dropped to 1.3% for FISH >20%. Variants were undetected in 36% of ALK tumours. Discordances predominated with FISH ranging from 10% to 20% of rearranged cells and were centre dependent. IHC remains a reliable pre-screening method for ALK rearrangement detection.
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http://dx.doi.org/10.1183/09031936.00119914DOI Listing
July 2015

Beyond KRAS status and response to anti-EGFR therapy in metastatic colorectal cancer.

Pharmacogenomics 2014 May;15(7):1043-52

Institut National de la Santé et de la Recherche Médicale (INSERM), Unité Mixte de Recherche (UMR)-S1147, Personalized Medicine, Pharmacogenomics, Therapeutic Optimization, University Paris Descartes, 45 rue des Saints Pères, Paris 75006, France.

In patients with metastatic colorectal cancer, overall survival has improved over the last decade mainly due to the use of effective targeted therapies such as anti-EGFR. However, survival improvement is linked to proper selection of patients expected to benefit from these treatments. KRAS codons 12 and 13 mutation status was the first validated molecular biomarker for anti-EGFR antibodies. Today, rare KRAS alterations and NRAS mutations were implemented, defining the 'RAS' status as the new validated marker of response to anti-EGFR antibodies. Moreover, other biomarkers are under investigation to screen for other targets and help with patients selection. Here, we reviewed these promising biomarkers: mutations in the RAS-MAPK and PI3K-AKT pathways genes, MET activation, HER/ErbB receptors activation (EGFR, HER2 and HER3), EGFR ligands, antibody-dependent cell-mediated cytotoxicity) and miRNAs. Further data are needed to define their impact for the treatment of patients with metastatic colorectal cancer.
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http://dx.doi.org/10.2217/pgs.14.66DOI Listing
May 2014

Oxaliplatin, fluorouracil, and leucovorin with or without cetuximab in patients with resected stage III colon cancer (PETACC-8): an open-label, randomised phase 3 trial.

Lancet Oncol 2014 Jul 11;15(8):862-73. Epub 2014 Jun 11.

Hepato-Gastroenterology Department Dijon University Hospital and INSERM U 866, France.

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.
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http://dx.doi.org/10.1016/S1470-2045(14)70227-XDOI Listing
July 2014

Microsatellite instability analysis in uterine cavity washings to detect endometrial cancer in Lynch syndrome.

Anticancer Res 2014 Jun;34(6):3211-5

Université Paris Descartes, Sorbonne Paris Cité, Faculté de Médecine, Paris, France Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges-Pompidou, Chirurgie Cancérologique Gynécologique et du Sein, Paris, France INSERM UMR-S 747, Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

Aim: To assess the feasibility of Microsatellite Instability (MSI) analysis in uterine cavity washings for detecting endometrial cancer in Lynch syndrome.

Materials And Methods: This was a proof-of-concept study in Lynch syndrome patients, scheduled for hysterectomy. At the beginning of surgical procedure, uterine cavity washings were performed, and sent for MSI analysis. Pathological examination of the uterus was associated with mismatch repair protein expression and MSI analysis.

Results: Nine patients were included in the study. Uterine cavity washings were feasible and interpretable in all cases. Final histological report identified 2 endometrial cancers and 7 benign specimens. There was no atypical hyperplasia. Sensitivity, specificity, positive predictive value, and negative predictive value of MSI analysis in uterine washings reached 100% in all cases. Concordance of MSI presence or absence was absolute between uterine washings and final histology.

Conclusion: MSI analysis in uterine cavity washings may be a promising screening tool for Lynch syndrome-associated endometrial cancer diagnosis.
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June 2014

Intratumoral distribution of EGFR mutations and copy number in metastatic lung cancer, what impact on the initial molecular diagnosis?

J Transl Med 2014 May 16;12:131. Epub 2014 May 16.

Université Paris Descartes, Sorbonne, Paris cité, France.

Background: Activating epidermal growth factor receptor (EGFR) mutations characterize a subgroup of non-small-cell lung cancer that benefit from first line EGFR tyrosine kinase inhibitors (EGFR-TKI). However, the existence of polyclonal cell populations may hinder personalized-medicine strategies as patients' screening often depends upon a single tumor-biopsy sample. The purpose of this study is to clarify and to validate in clinical testing conditions the accuracy of EGFR genotyping using different tumor sites and various types of samples (transthoracic, surgical or endoscopic biopsies and cytology specimens).

Methods: We conducted a retrospective review of 357 consecutive patients addressed for EGFR mutation screening in accordance with the directive of the European Medicines Agency (stage IV NSCLC). Fifty-seven samples were EGFR mutated and 40 had adequate tumor specimens for analysis on multiple spatially separated sites. Ten wild type samples were also analyzed. A total of 153 and 39 tumor fragments, from mutated and non-mutated cases respectively, were generated to analyze tumor heterogeneity or primary-metastatic discordances. After histological review of all fragments, EGFR genotyping was assessed using the routine diagnostic tools: fragment analysis for insertions and deletions and allele specific TaqMan probes for point mutations. EGFR copy number (CN) was evaluated by qPCR using TaqMan probes.

Results: The identification of EGFR mutations was independent of localization within primary tumor, of specimen type and consistent between primary and metastases. At the opposite, for half of the samples, tumor loci showed different EGFR copy number that may affect mutation detection cut-off.

Conclusions: This is the largest series reporting multiple EGFR testing in Caucasians. It validates the accuracy of EGFR mutation screening from single tumor-biopsy samples before first line EGFR-TKI. The unpredictable variability in EGFR CN and therefore in EGFR wild type/mutant allelic ratio justifies the implementation of sensitive methods to identify patients with EGFR mutated tumors.
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http://dx.doi.org/10.1186/1479-5876-12-131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041917PMC
May 2014

ERCC1, XRCC1 and GSTP1 Single Nucleotide Polymorphisms and Survival of Patients with Colon Cancer Receiving Oxaliplatin-Based Adjuvant Chemotherapy.

J Cancer 2014 2;5(6):425-32. Epub 2014 May 2.

1. INSERM, UMR_S 938, Saint-Antoine Research Centre, F-75012, Paris, France; ; 2. UPMC Univ Paris 06, UMR_S 938, Saint-Antoine Research Centre, F-75012, Paris, France;

Background: While single nucleotide polymorphisms (SNP) in genes involved in DNA repair or drug metabolism have been shown to influence survival of metastatic colon cancer patients treated with FOLFOX, data on adjuvant setting are scarce.

Methods: This study evaluated the correlation between disease-free survival (DFS) of 210 unselected stage III colon cancer patients receiving FOLFOX chemotherapy, and ERCC1-118 (rs11615, c.354T>C), XRCC1-399 (rs25487, c.1196G>A) and GSTP1-105 (rs1695, c.313A>G) polymorphisms. SNP were determined on tumor DNA using a PCR-based RFLP technique.

Results: In univariate analysis, a trend towards longer DFS was observed for ERCC1 (C/T + T/T) versus (C/C) (HR=2.29; p=0.06), and XRCC1 (A/A) versus (G/G + G/A) (HR=1.61; p=0.16), but not for GSTP1 genotypes; a statistically significant p value was obtained when combining ERCC1 and XRCC1 favorable genotypes (0 versus ≥ 1 favorable genotypes, HR=2.42; p=0.02). After adjustment on tumor stage, lymph node ratio and differentiation grade, multivariate analysis showed that combining ERCC1 and XRCC1 genotypes gave a p value slightly above the threshold for statistical significance (HR=2.03; p=0.06), which was lower than for tumor stage, lymph node ratio or differentiation grade.

Conclusion: The association of ERCC1 and XRCC1 polymorphisms may influence the prognosis of stage III colon cancer patients treated with FOLFOX adjuvant chemotherapy. Yet, these findings need to be confirmed in independent prospective studies.
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http://dx.doi.org/10.7150/jca.8594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026996PMC
May 2014

ERBB2 gene as a potential therapeutic target in small bowel adenocarcinoma.

Eur J Cancer 2014 Jul 2;50(10):1740-1746. Epub 2014 May 2.

Université Paris Descartes, Sorbone Paris Cité, Paris, France; Inserm UMR-S775 Bases Moléculaires de la réponse aux xénobiotiques, Paris, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ejca.2014.04.007DOI Listing
July 2014

The new histologic classification of lung primary adenocarcinoma subtypes is a reliable prognostic marker and identifies tumors with different mutation status: the experience of a French cohort.

Chest 2014 Sep;146(3):633-643

INSERM, U1138, Team Cancer, Immune Control, and Escape, Centre de Recherche des Cordeliers, Paris, France; Université Pierre et Marie Curie-Paris 6, Paris, France; Université Paris Descartes-Paris 5, Paris, France; Université Denis Diderot-Paris 7, Paris, France; Service de Pathologie, Paris, France. Electronic address:

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.
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http://dx.doi.org/10.1378/chest.13-2499DOI Listing
September 2014

A multicenter blinded study evaluating EGFR and KRAS mutation testing methods in the clinical non-small cell lung cancer setting--IFCT/ERMETIC2 Project Part 1: Comparison of testing methods in 20 French molecular genetic National Cancer Institute platforms.

J Mol Diagn 2014 Jan 30;16(1):45-55. Epub 2013 Oct 30.

the Francophone Intergroup of Thoracic Oncology, Paris, France; Pneumology Service, Assistance Publique Hôpitaux de Paris, Hôpital Tenon, GRC-04 Theranoscan, Université Paris VI, Paris Cedex 20, France.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors have limited use as first-line treatment for mutated EGFR metastatic non-small cell lung cancer. The French National Cancer Institute has installed molecular genetics platforms implementing EGFR and KRAS testing. However, there is considerable uncertainty as to which detection methods should be applied for routine diagnosis. This study aimed to compare the EGFR and KRAS genotyping methods developed by the IFCT/ERMETIC2 network platforms in two blind panels: 25 samples of serial dilutions of cell line DNA (20 centers) and 74 FFPE lung tumor samples (10 centers). The best threshold of mutation detection on cell lines was obtained using allele-specific amplification-based technologies. Nonamplifiable tissue samples were significantly less common when using alternative testing versus direct sequencing [15%; 95% confidence interval (CI), 14%-16% versus 40%; 95% CI, 39%-42%; P < 0.001]. Mutated cases increased from 42% (95% CI, 31%-54%) to 53% (95% CI, 41%-64%), with three supplementary EGFR mutations (p.G179A at exon 18 and p.L858R and p.L861Q at exon 21) and five supplementary KRAS mutations, when using alternative testing instead of direct sequencing. False-positive results were observed when using a PCR-based sizing assay, high-resolution melting, or pyrosequencing. Concordance analysis returned good kappa test scores for EGFR exon 19 and KRAS analysis when comparing sequencing with alternative methods and revealed no difference between alternative techniques themselves.
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http://dx.doi.org/10.1016/j.jmoldx.2013.07.009DOI Listing
January 2014