Marie-Christine Copin

Expression of human mucin genes in respiratory, digestive, and reproductive tracts ascertained by in situ hybridization.

In recent years considerable advances have been made in our knowledge of the peptide moiety of human mucins through cDNA cloning. In many diseases disorders in mucin biosynthesis are observed, which result either from changes in the synthesis of the carbohydrate side chains or from differences in the relative expression of the different apomucins, each of which may affect physical properties of the viscous gel. We describe in situ hybridization studies on healthy human mucosae with five different oligonucleotide probes corresponding to each of the human genes known to date that encode secreted mucins, i.e., MUC 2, 3, 4 (HGM nomenclature) and 5B, 5C (proposed name). These genes present a nucleic tandem repeat organization. The choice of oligonucleotide probes was made to amplify the signal by hybridization of many small probes on the same mRNA molecules. A characteristic pattern of mucin gene expression was observed for each mucosa.

Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Sp1.

The 11p15 mucin genes (MUC2, MUC5AC, MUC5B and MUC6) possess a cell-specific pattern of expression in normal lung that is altered during carcinogenesis. Growth factors of the epidermal growth factor family are known to target key genes that in turn may affect the homeostasis of lung mucosae. Our aim was to study the regulation of the 11p15 mucin genes both at the promoter and protein levels to assess whether their altered expression may represent a key event during lung carcinogenesis. Studies were performed in the mucoepidermoid NCI-H292 lung cancer cell line. Cell treatment with epidermal growth factor (EGF), transforming growth factor α (TGF-α), or tumor necrosis factor α (TNF-α) resulted in a dramatic increase of MUC2 and MUC5ACmRNAs levels, promoter activity, and apomucin expression, whereas those of MUC5B and MUC6 were unchanged. pGL3 deletion mutants of MUC2, MUC5AC, andMUC5B promoters were constructed and used in transient transfection assays to characterize EGF- and TGF-α-responsive regulatory regions within the promoters. They were located in the −2627/−2097 and −202/−1 regions of MUC2 andMUC5AC promoters, respectively. Finally, we demonstrate that transcription factor Sp1 not only binds and activatesMUC2 and MUC5AC promoters but also participates to their EGF- and TGF-α-mediated up-regulation. We also show that Sp3 is a strong inhibitor of 11p15 mucin gene transcription. In conclusion, MUC2 and MUC5AC are two target genes of EGFR ligands in lung cancer cells, and up-regulation of these two genes goes through concomitant activation of the EGFR/Ras/Raf/Extracellular Signal-regulated Kinase-signaling pathway and Sp1 binding to their promoters.

Utility of osteopontin and serum mesothelin in malignant pleural mesothelioma diagnosis and prognosis assessment.

Purpose: Malignant mesothelioma is a highly aggressive tumor and is often diagnosed too late for a curative treatment. We compared diagnostic and prognostic values of mesothelin and osteopontin in 172 patients suspected of malignant pleural mesothelioma (MPM) and in a control group of 112 asymptomatic asbestos-exposed subjects. Experimental Design: Osteopontin and mesothelin were assayed with commercial ELISA kits in a series of 43 patients with pleural metastases of various carcinomas, 33 patients with benign pleural lesions associated with asbestos exposure, 96 patients with MPMs, and 112 asbestos-exposed healthy subjects. Results were correlated with patients diagnosis and survival. Results: Serum osteopontin level was higher in MPM patients compared with healthy asbestos-exposed subjects and had a good capability to distinguish between these two populations. However, osteopontin was unable to distinguish between MPM and pleural metastatic carcinoma or benign pleural lesions associated with asbestos exposure. Neither plasma nor pleural fluid osteopontin were more powerful in this respect. Serum mesothelin had a good ability for diagnosing MPM but was unable to identify patients with nonepithelioid mesothelioma subtypes. Survival analysis identified tumor histologic subtype along with serum osteopontin and serum mesothelin as independent prognostic factors in mesothelioma patients. Conclusions: Osteopontin has a lower diagnostic accuracy than mesothelin in patients suspected of MPM. Insufficient specificity limits osteopontin utility as diagnostic marker. Both molecules have a potential value as prognostic markers.

miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.

As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFβ exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFβ signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.

Expression of E-Selectin, ICAM-1 and VCAM-1 on Bronchial Biopsies from Allergic and Non-Allergic Asthmatic Patients

The bronchial mucosa of asthmatic patients is characterized by a large influx of eosinophils, monocytes and lymphocytes. Leucocyte migration and accumulation is thought to involve adhesion molecules, as shown in an animal of allergic asthma and in humans with other allergic diseases. The aim of this study was to evaluate the expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1) on endothelium and epithelium in bronchial biopsies obtained from patients with allergic (n = 17) and non-allergic asthma (n = 18). Bronchial biopsies were taken in asthmatic patients and control subjects (n = 10) by fiberoptic bronchoscopy and embedded in paraffin. The cellular infiltrate was evaluated by May-Grünwald-Giemsa staining. Adhesion molecule expression was analyzed by immunohistochemistry using mouse monoclonal antibodies; the results were expressed as the percentage of positive cells. For each patient, we evaluated the severity of asthma as defined by the AAS score and the treatment. In controls, low expression of ICAM-1 was observed on the epithelium and endothelium (9.6 +/- 2.7 and 11.2 +/- 4.1%, respectively), while E-selectin and VCAM-1 were not expressed. In patients with allergic asthma, a significant increase of ICAM-1 expression was observed on epithelium and endothelium (28 +/- 5.3 and 35.6 +/- 5%, respectively), whereas E-selectin (17.4 +/- 4.8%) and VCAM-1 (12.8 +/- 3.6%) were overexpressed only on endothelium. In allergic asthmatic patients, adhesion molecule expression on endothelium was correlated with eosinophil and total leucocyte infiltrate (p < 0.05). In contrast, adhesion molecule expression in biopsies from patients with non-allergic asthma (14.1 +/- 5.2 and 15.3 +/- 3.6% for ICAM-1 expression on epithelium and endothelium, respectively) was not significantly different from the control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocan Expression and Relationship with Survival in Human Non–Small Cell Lung Cancer

Purpose: We evaluated the expression of endocan, a soluble lung- and kidney-selective endothelial cell-specific dermatan sulfate proteoglycan, in non–small cell lung tumors compared with normal lung and studied the significance of high levels of circulating endocan in patients with non–small cell lung cancer. Material and Methods: Endocan and vascular endothelial growth factor mRNA expression were evaluated by semiquantitative PCR in tumoral and nontumoral lung tissue samples from a first series of 24 patients submitted to curative surgery. Relationships between survival, time to tumor progression, and serum levels of endocan were evaluated in a second series of 30 previously untreated patients addressed for staging. Results: In non–small cell lung cancers, endocan mRNA was overexpressed compared with control lung. Immunohistochemistry shows that endocan was expressed only by tumor endothelium in all cases, especially in the periphery of the tumors, with no differences between adenocarcinoma and squamous cell carcinoma. Endocan and vascular endothelial growth factor mRNA expression was positively correlated in lung tumors. Serum endocan levels, as well as tumor, node, and metastasis status, were correlated with both survival and time to tumor progression. However, endocan serum level was not an independent prognostic factor due to its correlation with the presence of metastasis. Conclusion: Endocan is overexpressed in non–small cell lung tumors compared with healthy lung and probably represents a response of tumoral endothelium to proangiogenic growth factor stimulation. Circulating levels of endocan might reflect tumor angiogenic stimulation and present prognostic significance.

Bronchogenic cysts of the lung

BACKGROUND The clinical presentation of lung bronchogenic cysts (BC) is variable, from respiratory distress at birth to late appearance of symptoms. METHODS This study of BC was based on a retrospective review of 41 cases: 21 infants and children and 20 adults, aged 1 day to 68 years. The diagnosis was antenatal in 4 cases. Three infants required mechanical ventilation, and 2 had their cyst drained before resection. Twenty infants and children and 17 adults underwent operations. RESULTS Compression was the most important complication in infants and children. Cough, infection, and hemoptysis occurred later in life; 80% of the total population was symptomatic. Seven cysts were infected. There were no deaths after resection, and there was no recurrence of symptoms during the follow-up period (13 months to 21 years). CONCLUSIONS Bronchogenic cysts originate from the foregut. Differentiation from other acquired or congenital lesions can be difficult. It is uncertain what proportion of BC remain asymptomatic. More than half of patients are diagnosed after the age of 15 years, and complications may appear late. Clinical findings and plain chest radiograms are often sufficient for diagnosis. Lobectomy is the standard treatment, whereas drainage is a temporary, palliative, and risky procedure in cases of life-threatening compression. We conclude that a symptomatic BC is an indication for resection and that the long-term prognosis of an asymptomatic BC is unpredictable. Thus, there is a role for preventive operations.

Postresection Irradiation for T2 N0 M0 Non–Small Cell Carcinoma: A Prospective, Randomized Study

BACKGROUND Stage I nonirradiated T2 N0 non-small cell lung carcinoma has a postoperative prognosis not very different from stage II irradiated T1 N1 carcinoma. The hypothesis was that more locoregional malignant sites are overlooked in T2 N0 M0 than in T1 N0 M0 tumors, considering the better prognosis of this last group, and that T2 N0 cancer might benefit from postresection irradiation. METHODS From 1985 to 1991, 163 non-small cell lung carcinomas were classified T2 N0 M0 and randomized for irradiation or nonirradiation after operation. After revision of all the cases, 132 were included in this study: 60 were irradiated and 72 were not irradiated. All were followed up. The study was closed in October 1995. Statistical analysis was then performed considering volume, location, cell type, survival, and recurrence in the two groups. RESULTS One hundred thirteen patients were followed up during a minimum of 5 years: the survival was 44.2%. There was no significant difference considering cell type or irradiation. There was no recurrence-free survivor beyond 5 years with a tumor invading the visceral pleura. At the close of the study (follow up, 4 years 3 months to 10 years 1 month), 49 of 132 patients were alive. The median survival was 3 years 11 months. Fifty-nine patients had died of local (21) or distant (40) recurrences (2 patients had both local and distant recurrence). There was again no significant difference considering cell type or irradiation, either in the survival or in the mode of recurrence. CONCLUSIONS Stage I T2 N0 M0 non-small cell lung carcinoma tends to manifest distant metastasis. Prospective studies of stratified systemic adjuvant therapy should improve the present moderate result of radical resection in this group of tumors.

Utility of cytokeratin 7 for distinguishing Chromophobe renal cell carcinoma from renal oncocytoma

Objective: Chromophobe renal cell carcinoma is a relatively uncommon variant of renal carcinoma described in 1985. The main differential diagnosis is renal oncocytoma. Hale’s colloidal iron staining is a powerful adjunct to morphological interpretation but it is not specific and is sometimes difficult to interpret. We studied the immunohistochemical expression of cytokeratin 7 to determine its value in distinguishing chromophobe renal cell carcinoma from renal oncocytoma.Methods: Immunostaining was performed on paraffin–embedded tumor tissue of 6 chromophobe renal cell carcinomas and 11 oncocytomas with an antibody to cytokeratin 7 (clone OV–TL 12/30, Dako, France) using a streptavidin–biotin method.Results: All chromophobe renal cell carcinomas showed strong cytoplasmic staining with peripheral cell accentuation. In contrast, 8 of 11 oncocytomas were entirely negative and 3 showed only weak and focal staining in less than 5% of the tumor cells.Conclusion: Immunohistochemical staining for cytokeratin 7 may be useful for the differential diagnosis of renal oncocytomas and chromophobe renal cell carcinomas when Hale’s colloidal iron staining is uncertain.

Morphologic Subtyping of Papillary Renal Cell Carcinoma: Correlation with Prognosis and Differential Expression of MUC1 between the Two Subtypes

Papillary renal cell carcinoma is now a well-established entity with distinct histological and cytogenetic features. A subdivision has been proposed in correlation with prognosis. Type 1 is the most frequent subtype and appears to have a better prognosis than Type 2. The subdivision is based on microscopic criteria. To investigate these 2 types of papillary renal cell carcinoma, we have compared the clinical features, ancillary factors (TNM stage, Fuhrman grade), survival and MUC1 expression in 25 Type 1 and 12 Type 2 papillary renal cell carcinomas. Type 2 tumors were significantly associated with a higher Fuhrman grade (Grade III frequent; P < .001). Type 2 tumors were also associated with a poorer prognosis than Type 1 (P < .005). Fuhrman grade was significantly associated with prognosis (P < .005). The type and the prognosis were not correlated with the TNM stage. We have shown a differential expression of MUC1 between Type 1 and Type 2 with a polarized expression in Type 1 and a rare expression in Type 2. In conclusion we confirm that the morphologic sub-typing and Fuhrman grade are valuable factors of outcome of papillary renal cell carcinomas and that MUC1 immunostaining is useful in differentiating Type 1 and Type 2 tumors.