Establishing a Dedicated Lung Cancer Biobank at the University Center Hospital of Nice (France). Why and How?

Lung cancer is the major cause of death from cancer in the world and its incidence is increasing in women. Despite the progress made in developing immunotherapies and therapies targeting genomic alterations, improvement in the survival rate of advanced stages or metastatic patients remains low. Thus, urgent development of effective therapeutic molecules is needed. The discovery of novel therapeutic targets and their validation requires high quality biological material and associated clinical data. With this aim, we established a biobank dedicated to lung cancers. We describe here our strategy and the indicators used and, through an overall assessment, present the strengths, weaknesses, opportunities and associated risks of this biobank.

Expression of MET in circulating tumor cells correlates with expression in tumor tissue from advanced-stage lung cancer patients.

Given the difficulty in obtaining adequate tissue in NSCLC, we investigated the utility of circulating tumor cells (CTCs) for MET status assessment in NSCLC patients. We used two platforms for CTC capture, and assessed MET expression in CTCs and matched-bronchial biopsies in patients with advanced-stage III/IV lung adenocarcinoma. Baseline peripheral blood was collected from 256 advanced-stage III/IV NSCLC patients from Genentech clinical trials, and from 106 patients with advanced-stage III/IV lung adenocarcinoma treated at the Department of Pneumology, Pasteur Hospital, Nice. CTCs were enriched using CellSearch (Genentech), or ISET technologies (Pasteur Hospital). MET expression was evaluated by immunofluorescence on CellSearch, and by immunocytochemistry on ISET-enriched CTCs and on matched FFPE tissue sections (Pasteur Hospital). CTCs were detected in 83 of 256 (32%) patients evaluated on CellSearch, with 30 samples (12%) exhibiting ≥ 5 CTCs/7.5 ml blood. On ISET, CTC were observed in 80 of 106 patients (75%), and 79 patients (75%) exhibited ≥ 5 CTCs/4 ml blood. MET expression on ISET CTCs was positive in 72% of cases, and the MET expression on matched-patient tissue was positive in 65% patients using the Onartuzumab IHC scoring algorithm (93% concordance). Quantification of MET expression using H-scores showed strong correlation between MET expression in tissue and CTCs (Spearman correlation, 0.93). MET status in CTCs isolated on ISET filters from blood samples of advanced-stage NSCLC patients correlated strongly with MET status in tumor tissue, illustrating the potential for using CTCs as a non-invasive, real-time biopsy to determine MET status of patients entering clinical trials.

High expression of TRF2, SOX10, and CD10 in circulating tumor microemboli detected in metastatic melanoma patients. A potential impact for the assessment of disease aggressiveness.

Circulating tumors cells (CTCs) can be detected in the blood of metastatic melanoma patients (MMPs) both as isolated circulating tumor cells (iCTCs) and circulating tumor microemboli (CTMs), but their clinical significance remains unknown. The aim of this work was to evaluate the prognostic impact in metastatic cutaneous melanoma of CTMs and iCTCs identified by a cytomorphological approach using the isolation by size of tumor cell (ISET) method. We characterized the phenotype of CTCs using anti-PS100, anti-SOX10, anti-CD10, and anti-TRF2 antibodies. 128 MMPs and 37 control healthy individuals with benign nevi were included in this study. Results were compared to the follow-up of patients. 109/128 (85%) MMPs showed CTCs, 44/128 (34%) with 2 to 6 CTMs and 65/128 (51%) with 4 to 9 iCTCs. PS100 expression was homogeneous in iCTCs and heterogeneous in CTMs. SOX10, CD10, and TRF2 were mainly expressed in CTMs. None of the control subjects demonstrated circulating malignant tumor cells. Overall survival was significantly decreased in patients with CTMs, independently of the therapeutic strategies. In conclusion, the presence of CTMs is an independent predictor of shorter survival from the time of diagnosis of MMPs.

Setting up a wide panel of patient-derived tumor xenografts of non-small cell lung cancer by improving the preanalytical steps.

With the ongoing need to improve therapy for non-small cell lung cancer (NSCLC) there has been increasing interest in developing reliable preclinical models to test novel therapeutics. Patient-derived tumor xenografts (PDX) are considered to be interesting candidates. However, the establishment of such model systems requires highly specialized research facilities and introduces logistic challenges. We aimed to establish an extensive well-characterized panel of NSCLC xenograft models in the context of a long-distance research network after careful control of the preanalytical steps. One hundred fresh surgically resected NSCLC specimens were shipped in survival medium at room temperature from a hospital-integrated biobank to animal facilities. Within 24 h post-surgery, tumor fragments were subcutaneously xenografted into immunodeficient mice. PDX characterization was performed by histopathological, immunohistochemical, aCGH and next-generation sequencing approaches. For this model system, the tumor take rate was 35%, with higher rates for squamous carcinoma (60%) than for adenocarcinoma (13%). Patients for whom PDX tumors were obtained had a significantly shorter disease-free survival (DFS) compared to patients for whom no PDX tumors (P = 0.039) were obtained. We established a large panel of PDX NSCLC models with a high frequency of mutations (29%) in EGFR, KRAS, NRAS, MEK1, BRAF, PTEN, and PI3KCA genes and with gene amplification (20%) of c-MET and FGFR1. This new patient-derived NSCLC xenograft collection, established regardless of the considerable time required and the distance between the clinic and the animal facilities, recapitulated the histopathology and molecular diversity of NSCLC and provides stable and reliable preclinical models for human lung cancer research.

Current challenges for detection of circulating tumor cells and cell-free circulating nucleic acids, and their characterization in non-small cell lung carcinoma patients. What is the best blood substrate for personalized medicine?

The practice of "liquid biopsy" as a diagnostic, prognostic and theranostic tool in non-small cell lung cancer (NSCLC) patients is an appealing approach, at least in theory, since it is noninvasive and easily repeated. In particular, this approach allows patient monitoring during treatment, as well as the detection of different genomic alterations that are potentially accessible to targeted therapy or are associated with treatment resistance. However, clinical routine practice is slow to adopt the liquid biopsy. Several reasons may explain this: (I) the vast number of methods described for potential detection of circulating biomarkers, without a consensus on the ideal technical approach; (II) the multiplicity of potential biomarkers for evaluation, in particular, circulating tumor cells (CTCs) vs. circulating tumor DNA (ctDNA); (III) the difficulty in controlling the pre-analytical phase to obtain robust and reproducible results; (IV) the present cost of the currently available techniques, which limits accessibility to patients; (V) the turnaround time required to obtain results that are incompatible with the urgent need for delivery of treatment. The purpose of this review is to describe the main advances in the field of CTC and ctDNA detection in NSCLC patients and to compare the main advantages and disadvantages of these two approaches.

"Sentinel" circulating tumor cells allow early diagnosis of lung cancer in patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer. Migration of circulating tumor cells (CTCs) into the blood stream is an early event that occurs during carcinogenesis. We aimed to examine the presence of CTCs in complement to CT-scan in COPD patients without clinically detectable lung cancer as a first step to identify a new marker for early lung cancer diagnosis. The presence of CTCs was examined by an ISET filtration-enrichment technique, for 245 subjects without cancer, including 168 (68.6%) COPD patients, and 77 subjects without COPD (31.4%), including 42 control smokers and 35 non-smoking healthy individuals. CTCs were identified by cytomorphological analysis and characterized by studying their expression of epithelial and mesenchymal markers. COPD patients were monitored annually by low-dose spiral CT. CTCs were detected in 3% of COPD patients (5 out of 168 patients). The annual surveillance of the CTC-positive COPD patients by CT-scan screening detected lung nodules 1 to 4 years after CTC detection, leading to prompt surgical resection and histopathological diagnosis of early-stage lung cancer. Follow-up of the 5 patients by CT-scan and ISET 12 month after surgery showed no tumor recurrence. CTCs detected in COPD patients had a heterogeneous expression of epithelial and mesenchymal markers, which was similar to the corresponding lung tumor phenotype. No CTCs were detected in control smoking and non-smoking healthy individuals. CTCs can be detected in patients with COPD without clinically detectable lung cancer. Monitoring "sentinel" CTC-positive COPD patients may allow early diagnosis of lung cancer.

[Setting up a department of molecular pathology in oncology in a pathology laboratory (LPCE, CHU de Nice)]. / Mise en place d'un secteur de pathologie moléculaire en oncologie au sein d'un laboratoire d'anatomie pathologique (LPCE, CHU de Nice).

The advent of targeted therapies and personalized medicine in oncology has led in France to the settlement and organisation of a network of hospital molecular genetic platforms under the impetus of the National Cancer Institute (INCa). These platforms are, according to the concerned sites, integrated or not in pathology laboratories. The development of molecular biology methods, the choice of the procedures, the establishment of sample workflow, the quality control and the selection of the genomic alterations to be detected on each platform, have been left to the discretion of the different laboratories. Based on calls for project made by the INCa, hospital molecular genetic platforms were able to adapt their activity according to the assigned budgets. While the presence of some genomic alterations (i.e. KRAS gene mutations in metastatic colon adenocarcinoma or EGFR gene mutations in lung adenocarcinomas), may lead to administration of targeted therapies under the Marketing Authorization Application (MAA), others are associated with therapeutic clinical trials. However, increasing number of MAA for new molecules targeting genomic alterations is likely in the near future. In this context, it is necessary to quickly adapt the organisation of work of the hospital pathology laboratories performing molecular biology tests in order to meet the growing demand of oncologists in the field of targeted therapies. The purpose of this article is to describe the different steps of the settlement of a molecular genetic platform in an academic pathology laboratory (LPCE, CHU de Nice) and to show the experience of this laboratory specifically oriented on the support of the morphological and molecular diagnosis of lung cancer, thyroid cancer and malignant melanoma.

Significance of circulating tumor cell detection using the CellSearch system in patients with locally advanced head and neck squamous cell carcinoma.

The objective of this study was to evaluate the potential detection of circulating tumor cells (CTCs) using the CellSearch (CS) Assay™ in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) and then to identify the clinical factors predictive of the presence of CTCs. The presence and number of CTCs were determined using the CS system before treatment, and in 10 healthy individuals (control group). The CS system was able to successfully identify the presence of CTCs in 8 of 49 patients (16 %) before therapy. No CTC was found in the control group. CTCs were detected before therapy in 1 of 19 patients (5 %) with N0 tumor and in 7 of 30 patients (23 %) with N1-2c tumor (p = 0.12; Fisher's exact test). CTCs were identified in a relatively low proportion of patients with locally advanced HNSCC.

[Setting up indicators in biobanking: why and how?]. / Mise en place d'indicateurs de suivi au sein d'une tumorothèque et/ou d'un centre de ressources biologiques : pourquoi et comment ?

The biobanking area is highly complex, and its complexity is increasing along with its growth and demand. Due to the advancements in genetic research, stem cell research and regenerative medicine, biobanking has become ever more important and plays a key role in biomedical research. The robustness and the reproducibility of research results depend greatly on the quality and on the number of the samples used, and thus on the expertise of biobanks having supplied these samples. Undoubtedly, the recognition of a research biobank depends on the impact of the research projects conducted with samples obtained from tumour bank(s), but also on many other criteria. It thus seems important to determine a number of indicators within a biobank to estimate objective criteria for the performance of these structures. These indicators can allow to make some strategic decisions knowing that biobanks are expensive structures to maintain in the present hospital context. The use of these indicators could also contribute to the elaboration of an "biobank impact factor of" or so called "bioresource research impact factor" (BRIF). We describe here four major categories of indicators (quality, activity, scientific production, visibility), which seem to be useful for the evaluation of a biobank by making a proposition of allocation of coefficients for the various considered items.

miR-483-3p controls proliferation in wounded epithelial cells.

The mechanisms that regulate keratinocyte migration and proliferation in wound healing remain largely unraveled, notably regarding possible involvements of microRNAs (miRNAs). Here we disclose up-regulation of miR-483-3p in 2 distinct models of wound healing: scratch-injured cultures of human keratinocytes and wounded skin in mice. miR-483-3p accumulation peaks at the final stage of the wound closure process, consistent with a role in the arrest of "healing" progression. Using an in vitro wound-healing model, videomicroscopy, and 5-bromo-2'-uridine incorporation, we observed that overexpression of miR-483-3p inhibits keratinocyte migration and proliferation, whereas delivery of anti-miR-483-3p oligonucleotides sustains keratinocyte proliferation beyond the closure of the wound, compared with irrelevant anti-miR treatment. Expression profiling of keratinocytes transfected with miR-483-3p identified 39 transcripts that were both predicted targets of miR-483-3p and down-regulated after miR-483-3p overexpression. Luciferase reporter assays, Western blot analyses, and silencing by specific siRNAs finally established that kinase MK2, cell proliferation marker MKI67, and transcription factor YAP1 are direct targets of miR-483-3p that control keratinocyte proliferation. miR-483-3p-mediated down-regulation of MK2, MKI67, and YAP1 thus represents a novel mechanism controlling keratinocyte growth arrest at the final steps of reepithelialization.

The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway.

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca²âº/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.

Cytopathologic detection of circulating tumor cells using the isolation by size of epithelial tumor cell method: promises and pitfalls.

Detection of circulating tumor cells (CTCs) morphologically may be a promising new approach in clinical oncology. We tested the reliability of a cytomorphologic approach to identify CTCs: 808 blood samples from patients with benign and malignant diseases and healthy volunteers were examined using the isolation by size of epithelial tumor cell (ISET) method. Cells having nonhematologic features (so-called circulating nonhematologic cells [CNHCs]) were classified into 3 categories: CNHCs with malignant features, CNHCs with uncertain malignant features, and CNHCs with benign features. CNHCs were found in 11.1% and 48.9% of patients with nonmalignant and malignant pathologies, respectively (P < .001). CNHCs with malignant features were observed in 5.3% and in 43.1% of patients with nonmalignant and malignant pathologies, respectively. Cytopathologic identification of CTCs using the ISET method represents a promising field for cytopathologists. The possibility of false-positive diagnosis stresses the need for using ancillary methods to improve this approach.

Preoperative circulating tumor cell detection using the isolation by size of epithelial tumor cell method for patients with lung cancer is a new prognostic biomarker.

PURPOSE: Pathologic TNM staging is currently the best prognostic factor for non-small cell lung carcinoma (NSCLC). However, even in early-stage NSCLC, the recurrence rates after surgery range from 25% to 50%. The preoperative detection of circulating tumor cells (CTC) could be useful to tailor new therapeutic strategies in NSCLC. We assessed the presence of CTC in NSCLC patients undergoing surgery, using cytologic analyses, after their isolation by size of epithelial tumor cells (ISET method). The presence and the number of CTCs were considered and correlated with clinicopathologic parameters including patient follow-up. EXPERIMENTAL DESIGN: Of the 247 blood samples tested, 208 samples were from patients with resectable NSCLC and 39 from healthy subjects. The mean follow-up was 24 months. An image of detected cells with presumably nonhematologic features [initially defined as "circulating nonhematologic cells" (CNHC)] was recorded. The presence of CNHC was assessed blindly and independently by 10 cytopathologists, using cytologic criteria of malignancy on stained filters. The count of detected CNHCs was made for each filter. RESULTS: One hundred two of 208 (49%) patients showed CNHCs corresponding to CNHC with malignant cytopathologic features in 76 of 208 (36%) cases. CNHCs were not detected in the control group. A level of 50 or more CNHCs corresponding to the third quartile was associated with shorter overall and disease-free-survival, independently of disease staging, and with a high risk of recurrence and death in early-stage I + II-resectable NSCLC. CONCLUSION: A high percentage of NSCLC patients show preoperative detection of CNHC by the ISET method. The presence and level of 50 or more CNHCs are associated with worse survival of patients with resectable NSCLC.

Detection of circulating tumor cells as a prognostic factor in patients undergoing radical surgery for non-small-cell lung carcinoma: comparison of the efficacy of the CellSearch Assay™ and the isolation by size of epithelial tumor cell method.

Comparison of the efficacy of different enrichment methods for detection of circulating tumor cells (CTCs) before radical surgery is lacking in non-small-cell lung carcinoma (NSCLC) patients. Detection and enumeration of CTCs in 210 consecutive patients undergoing radical surgery for NSCLC were evaluated with the CellSearch Assay™ (CS), using the CellSearch Epithelial Cell Kit, and by the isolation by size of epithelial tumor (ISET) method, using double immunolabeling with anti-cytokeratin and anti-vimentin antibodies. CTCs were detected in 144 of 210 (69%) patients using CS and/or ISET and in 104 of 210 (50%) and 82 of 210 (39%) patients using ISET and CS, respectively. Using ISET, 23 of 210 (11%) patients had vimentin-positive cells with cytological criteria of malignancy. Disease-free survival (DFS) was worse for patients with CTCs compared to patients without CTCs detected by CS alone (p < 0.0001; log rank = 30.59) or by ISET alone (p < 0.0001; log rank = 33.07). The presence of CTCs detected by both CS and ISET correlated even better with shorter DFS at a univariate (p < 0.0001; log rank = 42.15) and multivariate level (HR, 1.235; 95% CI, 1.056-1.482; p < 0.001). CS and ISET are complementary methods for detection of CTCs in preoperative radical surgery for NSCLC. CTC detection in resectable NSCLC patients using CS and/or ISET could be a prognostic biomarker of great interest and may open up new avenues into improved therapeutic strategies for lung carcinoma patients.

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.

[The Nice CHU biobank experience to collect patients' informed consent for research context (2004-2009)]. / L'expérience de la tumorothèque du CHU de Nice pour le recueil des consentements éclairés dans le cadre de la recherche (2004-2009).

Over the last 10 years, significant financial support from the French National Institute of Cancer (INCa), the Ministry of Health (DGOS), and the Health and Research National Institute (Inserm) helped biobanks--of which tumour banks represent a prominent example of hospital-based infrastructures--to improve their operations, and in some instances to adopt the rules of Biological Ressource Centers as defined by OECD. Nowadays, the use of biological samples of human origin is strictly subordinated to regulations that integrate bioethical principles. However, in spite of the establishment of these regulations, requirement to obtain an authorisation and/or to register the biological collections with the Ministry of Research, many uncertainties persist. While French regulations mandate that samples can be used for research as long as patients did not oppose to such use, many biobank curators face practical and theoretical issues when establishing a Material Transfer Agreement with scientists, due to the lack of harmonization between national regulations--particularly due to a different perception of privacy and free will in anglo-american and other countries--and different demands on the side of private industry or editorial boards of scientific journals. The goal of this article is (1) to describe the procedure followed to collect patients' informed consent at the Biobank of CHU de Nice and (2) to assess the number of obtained consents in comparison to the number of collected samples between 01/09/2004 and 31/12/2009, the number of consents obtained before or after collecting the samples, and the number of patients' refusal to collect their biological resources. This balance-sheet is settled for the three major collections (thoracic, thyroid and head and neck tissues) from the Biobank of CHU de Nice. Results show that 88 % of consents were obtained during this period (82 % in a prospective manner and 6 % in a retrospective manner). Refusal was notified by writing in nine cases only. The percentage of consents varies slightly according to the collection involved and is stable from 2004 to 2009. Overall, our procedure is quite efficient at obtaining informed consents from a majority of patients for whom the tumour bank stores biological samples. This situation provides optimal conditions for the use of collected samples in the context of national and international research projects.

[Role of the surgical pathology laboratory in the pre-analytical approach of molecular biology techniques]. / 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.

Assessment of morphology, antigenicity, and nucleic acid integrity for diagnostic thyroid pathology using formalin substitute fixatives.

BACKGROUND: With 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. METHODS: Calibrated 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. RESULTS: The 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. CONCLUSIONS: All 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.

Differential expression and regulation of ADAM17 and TIMP3 in acute inflamed intestinal epithelia.

The acute phase of Crohn's disease (CD) is characterized by a large afflux of polymorphonuclear leukocytes (PMNL) into the mucosa and by the release of TNF-alpha. Conversion of inactive TNF-alpha into an active form requires the cleavage of a transmembrane TNF-alpha precursor by the TNF-alpha-converting enzyme (ADAM17), a protease mainly regulated by the tissue inhibitor of metalloproteinase 3 (TIMP3). The aim of the present study was to investigate in an in vitro model of PMNL transepithelial migration and in the intestinal mucosa of patients with CD the expression and regulation of ADAM17 and TIMP3 in intestinal epithelial cells (IEC). ADAM17 and TIMP3 expression was analyzed by Western blotting, RT-PCR, confocal microscopy, and immunohistochemistry by using the T84 model and digestive biopsies. ADAM17 expression in IEC was increased at a posttranscriptional level during the early phase (from 2 to 4 h) of PMNL transepithelial migration whereas TIMP3 was only increased 24 h later. TNF-alpha induced an early upregulation of ADAM17 in T84 cells, whereas PMNL adhesion, H(2)O(2), or epithelial tight junction opening alone did not affect the amount of ADAM17. Immunohistochemistry of intestinal biopsies revealed that strong expression of ADAM17 was associated with a high activity of CD. In contrast, TIMP3 was very poorly expressed in these biopsies. ADAM17 and TIMP3 profiling did not correlated with the NOD2/CARD15 status. The ADAM17 activity was higher both in the early phase of PMNL transepithelial migration and in active CD. These results showed early posttranscriptional upregulation of ADAM17 in IEC linked to PMNL transepithelial migration and a high activity of CD.