Virginie Gavric-Tanga

Usefulness of tissue microarrays for assessment of protein expression, gene copy number and mutational status of EGFR in lung adenocarcinoma

Specific inhibitors targeting the epidermal growth factor receptor (EGFR) can increase survival rates in certain lung adenocarcinoma patients with mutations in the EGFR gene. Although such EGFR-targeted therapies have been approved for use, there is no general consensus among surgical pathologists on how the EGFR status should be tested in lung adenocarcinoma tissues and whether the results of immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and mutational analysis by molecular methods correlate. We evaluated the EGFR status in 61 lung adenocarcinomas by IHC (using total and mutant-specific antibodies against EGFR), by FISH analysis on tissue microarrays (TMAs), and by direct sequencing. The results of each method were compared using χ2 and κappa statistics. The sensitivity and negative predictive value estimating the presence of abnormal EGFR for each test was calculated. The results show that, with respect to expression patterns and clinicopathological parameters, the total and mutant-specific EGFR detected by immunohistochemistry and FISH analysis on TMAs are valid and are equivalent to conventional methods performed on whole-tissue sections. Abnormal EGFR was detected in 52.4% of patients by IHC, FISH, and sequencing. The best sensitivity (100%) and negative predictive value (100%) was determined by evaluating the EGFR status with all methods. Testing for molecular changes in EGFR using a single test is likely to underestimate the presence of EGFR abnormalities. Taken together, these results demonstrate the high potential of TMAs to test for the major mechanisms of EGFR activation in patients with lung adenocarcinoma.

Assessment of Morphology, Antigenicity, and Nucleic Acid Integrity for Diagnostic Thyroid Pathology Using Formalin Substitute Fixatives

BACKGROUNDnWith the advent of the formaldehyde standard law in France, and because of the impact of new methods for diagnosis and prognosis in pathology, formalin replacement in surgical pathology laboratories is currently being discussed in France. However, a set of criteria must be assessed before introducing a formalin substitute fixative. The objective of this study was to compare formalin substitute fixation with formalin fixation and cryoconservation of tissues from several benign and malignant thyroid pathologies with respect to morphology, antigenicity, and nucleic acid (RNA, DNA, microRNA) integrity.nnnMETHODSnCalibrated specimens (200 mg, 1 cm(2) each) from four conventional papillary thyroid carcinomas, four follicular variant of papillary thyroid carcinomas, three minimally invasive follicular carcinomas, four thyroid adenomas, five thyroid nodular hyperplasias, and five normal thyroid tissues were fixed for 6, 12, or 24 hours, in different fixatives (formalin, Glyo-Fixx, FineFIX, ExcellPlus, RCL2) at room temperature or at 4 degrees C. Tissues were stained (hematoxylin-eosin, periodic acid Schiff, trichromic Masson, and Sweet-Gordon staining) and their antigenicity determined by immunohistochemistry (performed with HBME-1, galectin-3, CK19, vimentin, CD31, and KL1 antibodies). Evaluation by four pathologists was made blinded. The quantity and quality of DNA, RNA, and two representative microRNA extracted from deparaffinized sections of paraffin embedded specimen were compared with that of cryosections.nnnRESULTSnThe staining and morphology were not altered by the use of different fixatives. However, formalin, FineFIX, and RCL2 gave the best results for immunohistochemistry. Moreover, FineFIX and RCL2 gave the highest amount of nucleic acids and of the best quality.nnnCONCLUSIONSnAll the formalin substitute fixatives used in this study provided good histomorphologic quality for the different stained thyroid tissues, but individually, some fixatives performed better for immunohistochemical and molecular biological procedures for different thyroid pathologies.

Rôle du laboratoire d’anatomie pathologique dans l’approche pré-analytique des examens de biologie moléculaire réalisés en pathologie tumorale

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

Accréditation de l’activité de pathologie moléculaire selon la norme ISO 15189. Principales étapes à respecter et principaux écueils possibles

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

Article originalAccréditation de l’activité de pathologie moléculaire selon la norme ISO 15189. Principales étapes à respecter et principaux écueils possiblesAccreditation of the activity of molecular pathology according to ISO 15189: Key steps to follow and the main potential pitfalls

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

Setting up a Prospective Thyroid Biobank for Translational Research: Practical Approach of a Single Institution (2004–2009, Pasteur Hospital, Nice, France)

In the last few years, conditions for setting up a human biobank in France have been upgraded by taking into account (1) the new laws and regulations that integrate the ethical and societal dimension of biobanking and delineate the risks for patients associated with the procurement of human cells and tissues, (2) the increasing request by scientists for human samples with proven biological quality and sophisticated sets of annotations, including information produced through the evergrowing use of molecular biology in pathology, and (3) establishment of procedures concerning the safety of the personnel working with biological products. For this purpose, health authorities and national research institutes in France have provided significant support for the set up of biobanks. The present work was conducted to describe how we set up a biobank targeting diseases of a specific organ (thyroid gland), with the aim of rapidly developing translational research projects. The prospective experience of a single institution (Pasteur Hospital, Nice, France) over a 6-year period (2004-2009) is presented from the practical point of view of a surgical pathology laboratory. We describe different procedures required to obtain high-quality thyroid biological resources and clinical annotations. The procedures were established for the management of biological products obtained from 1454 patients who underwent thyroid surgery. The preanalytical steps leading to the storage of frozen specimens were carried out in parallel with diagnostic procedures. As the number of international networks for research programs using biological products is steadily increasing, it is crucial to harmonize the procedures used by biobanks. In this regard, the described thyroid biobank has been set up using criteria established by the French National Cancer Institute (Institut National du Cancer) to guarantee the quality of different collections stored in biobanks.

Les méthodes de séquençage de « nouvelle génération » (NGS) et le cancer broncho-pulmonaire: principales technologies, applications et limites actuelles en pathologie

Resume Durant la derniere decennie, des progres spectaculaires ont ete menes dans la comprehension des mecanismes moleculaires et cellulaires gouvernant l’initiation et la progression tumorale des cancers bronchopulmonaires non a petites cellules (CBNPC). En parallele, des progres therapeutiques majeurs ont ete realises avec la decouverte de plusieurs cibles moleculaires et le developpement des therapies ciblees. Actuellement, ces approches sont parfaitement illustrees par le traitement des patients atteints de CBNPC avec des inhibiteurs de tyrosine kinase lorsque la tumeur presente une mutation activatrice sur le gene de l’ EGFR ou un rearrangement du gene ALK . La liste des biomarqueurs analyses en genetique somatique est amenee a s’accroitre a court terme en raison du nombre croissant de therapies ciblees en cours de developpement. Des avancees technologiques recentes se traduisant par le developpement du sequencage de nouvelle generation (NGS) offrent la possibilite d’interroger rapidement et de maniere exhaustive le genome des CBNPC, meme sur des biopsies de petite taille. C’est dans ce contexte que les pathologistes moleculaires doivent faire face au defi de determiner comment selectionner, interpreter et appliquer progressivement dans leur laboratoire ces nouvelles technologies de genomique. Cette revue rapporte l’etat actuel des principales technologies de NGS, leurs applications et leurs limites actuelles, en particulier dans le cadre des CBNPC.