Papers by Isabelle Renard
The immune system appears to play a role in the development of squamous intraepithelial lesions (... more The immune system appears to play a role in the development of squamous intraepithelial lesions (SIL) and their progression to cancer. We have developed a technique to isolate and cultivate lymphocytes either from the epithelium or from the underlying stroma of small biopsies from the normal exocervix, the transformation zone (TZ) and SIL. The majority of cells derived from the epithelium of all biopsies were CD8 + T cells. No major difference was observed for the lymphocyte phenotype. Among all cytokines tested by ELISA (IL10, IL4 and IFNγ), only IL10 was significantly higher in the TZ in comparison with the exocervix. In cultures derived from the stroma, a decreased percentage in T cells was observed in the TZ and SIL in comparison with the exocervix. This decrease in T cells concerned CD8 + (SIL) and TCRγδ + T cells (TZ and SIL). We did not observe any difference in IL10 or IL4 production, although patients with SIL produced more IFNγ. The higher levels of IL10 production by lymphocytes derived from the epithelium of the TZ might contribute to the predisposition of this region to cervical carcinogenesis.
Vaccine, Mar 1, 2001
The purpose of this study is to develop a reliable in vitro human model to test new immunotherape... more The purpose of this study is to develop a reliable in vitro human model to test new immunotherapeutic approaches for squamous cell carcinoma that develop on mucosal surfaces. The organotypic (raft) culture permits cells to proliferate and differentiate at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while human papillomavirus-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to (pre)neoplastic lesions seen in vivo. We have demonstrated the ability of these organotypic cultures to be manipulated by altering the epithelial stratification with cytokines (interferon-g and tumor necrosis factor-a) and by integrating activated lymphocytes or dendritic cells into the in vitro formed epithelial sheet. This model may provide a useful tool to investigate the factors contributing to the presence and function of immunocompetent cells within a neoplastic epithelium that develops on a mucosal surface.
Vaccine, 2001
The purpose of this study is to develop a reliable in vitro human model to test new immunotherape... more The purpose of this study is to develop a reliable in vitro human model to test new immunotherapeutic approaches for squamous cell carcinoma that develop on mucosal surfaces. The organotypic (raft) culture permits cells to proliferate and differentiate at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while human papillomavirus-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to (pre)neoplastic lesions seen in vivo. We have demonstrated the ability of these organotypic cultures to be manipulated by altering the epithelial stratification with cytokines (interferon-g and tumor necrosis factor-a) and by integrating activated lymphocytes or dendritic cells into the in vitro formed epithelial sheet. This model may provide a useful tool to investigate the factors contributing to the presence and function of immunocompetent cells within a neoplastic epithelium that develops on a mucosal surface.
Background: Accumulating evidence suggests that DNA methylation markers could serve as sensitive ... more Background: Accumulating evidence suggests that DNA methylation markers could serve as sensitive and specific cancer biomarkers. Objective: To determine whether a panel of methylated genes would have the potential to identify primary bladder cancer (BCa) in voided urine samples. Design, setting, and participants: A pharmacologic unmasking reexpression analysis in BCa cell lines was initially undertaken to unveil candidate methylated genes, which were then evaluated in methylation-specific polymerase chain reaction (MSP) assays performed on DNA extracted from noncancerous and cancerous bladder tissues. The most frequently methylated genes in cancerous tissues, with 100% specificity, were retained for subsequent MSP analysis in DNA extracted from urine samples to build and validate a panel of potential methylated gene markers. Urine samples were prospectively collected at three urologic centres from patients with histologically proven BCa and processed for use in real-time MSP and cytologic analysis. Patients with nonmalignant urologic disorders were included as controls. Measurements: A urine sample was classified as valid when !10 copies of the gene encoding ß-actin were measured in the urine sediment genomic DNA. Sensitivity, specificity, and predictive values of the MSP and cytology tests were assessed and compared. Results and limitations: MSP assays performed on 466 of the 496 (94%) valid urine samples identified two genes, TWIST1 and NID2, that were frequently methylated in urine samples collected from BCa patients, including those with early-stage and low-grade disease. The sensitivity of this two-gene panel (90%) was significantly better than that of cytology (48%), with comparable specificity (93% and 96%, respectively). The positive predictive value and negative predictive value of the two-gene panel was 86% and 95%, respectively. Conclusions: Detection of the methylated TWIST1 and NID2 genes in urine sediments using MSP provides a highly (!90%) sensitive and specific, noninvasive approach for detecting primary BCa.
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Revue médicale de Liège, 2009
Bladder cancer mainly affects patients aged 50 years or more and requires close and repeated surv... more Bladder cancer mainly affects patients aged 50 years or more and requires close and repeated surveillance. Flexible cystoscopy associated with urinary cytology are the currently recommended diagnostic and follow-up methods. Because medical imaging techniques remain rather unsatisfying for bladder carcinoma detection, research efforts have focused on urinary markers of the disease. Various approaches were tested with results generally too unconsistant to replace cystoscopy. Recently, the department of Urology at the University of Liège together with the Biotechnology Company OncoMethylome Sciences have been interested in testing whether the detection of hypermethylated genes in voided urine samples would be of value for the detection of bladder cancer. The method is based on the Methylation-Specific PCR technology (MSP). This approach has the theoretical advantage of being non invasive, reproducible and based on DNA, whose stability, in urine, is higher than that of proteins. The res...
The immune system appears to play a role in the development of squamous intraepithelial lesions (... more The immune system appears to play a role in the development of squamous intraepithelial lesions (SIL) and their progression to cancer. We have developed a technique to isolate and cultivate lymphocytes either from the epithelium or from the underlying stroma of small biopsies from the normal exocervix, the transformation zone (TZ) and SIL. The majority of cells derived from the epithelium of all biopsies were CD8 T cells. No major difference was observed for the lymphocyte phenotype. Among all cytokines tested by ELISA (IL10, IL4 and IFNγ), only IL10 was significantly higher in the TZ in comparison with the exocervix. In cultures derived from the stroma, a decreased percentage in T cells was observed in the TZ and SIL in comparison with the exocervix. This decrease in T cells concerned CD8+ (SIL) and TCRγδ+ T cells (TZ and SIL). We did not observe any difference in IL10 or IL4 production, although patients with SIL produced more IFNγ. The higher levels of IL10 production by lymphocy...
Revue Medicale De Liege, 2009
Histopathology, 2008
High frequency of RASSF1A and RARb2 gene promoter methylation in morphologically normal endometri... more High frequency of RASSF1A and RARb2 gene promoter methylation in morphologically normal endometrium adjacent to endometrioid adenocarcinoma Aims: To identify a DNA methylation signature of endometrioid carcinoma of the endometrium (EEC) in the early stages of endometrial carcinogenesis. Methods and results: Archival biopsy specimens of 39 EECs, 14 cases of atypical hyperplasia (AH), 11 histologically normal endometrial tissues adjacent to EECs and 24 normal control endometrial samples were retrieved. The cases were tested by quantitative methylation-specific polymerase chain reaction with primers hybridizing in the promoter regions of five genes frequently methylated in human cancer (RASSF1A, RARb2, P16, MGMT and GSTPi). Twenty-nine of 39 (74%) EECs and 7 ⁄ 14 (50%) AHs were methylated for the RASSF1A gene, whereas 17 ⁄ 39 (44%) EECs and 6 ⁄ 14 (43%) AHs were positive for the methylation of the RARb2 gene. No significant results were obtained for the other genes (P16, MGMT and GSTPi). Interestingly, 4 ⁄ 11 (36%) and 6 ⁄ 11 (55%) histologically normal endometrial tissues adjacent to EEC showed, respectively, RASSF1A and RARb2 gene methylation. Furthermore, these 11 specimens were microsatellite stable and showed similar proliferative, cell cycle and apoptotic mean labelling indices as the normal endometrial control tissues. Conclusions: Promoter region methylation of RASSF1A and RARb2 genes is an early event in endometrial carcinogenesis.
European Urology, 2010
Background: Accumulating evidence suggests that DNA methylation markers could serve as sensitive ... more Background: Accumulating evidence suggests that DNA methylation markers could serve as sensitive and specific cancer biomarkers. Objective: To determine whether a panel of methylated genes would have the potential to identify primary bladder cancer (BCa) in voided urine samples. Design, setting, and participants: A pharmacologic unmasking reexpression analysis in BCa cell lines was initially undertaken to unveil candidate methylated genes, which were then evaluated in methylation-specific polymerase chain reaction (MSP) assays performed on DNA extracted from noncancerous and cancerous bladder tissues. The most frequently methylated genes in cancerous tissues, with 100% specificity, were retained for subsequent MSP analysis in DNA extracted from urine samples to build and validate a panel of potential methylated gene markers. Urine samples were prospectively collected at three urologic centres from patients with histologically proven BCa and processed for use in real-time MSP and cytologic analysis. Patients with nonmalignant urologic disorders were included as controls. Measurements: A urine sample was classified as valid when !10 copies of the gene encoding ß-actin were measured in the urine sediment genomic DNA. Sensitivity, specificity, and predictive values of the MSP and cytology tests were assessed and compared. Results and limitations: MSP assays performed on 466 of the 496 (94%) valid urine samples identified two genes, TWIST1 and NID2, that were frequently methylated in urine samples collected from BCa patients, including those with early-stage and low-grade disease. The sensitivity of this two-gene panel (90%) was significantly better than that of cytology (48%), with comparable specificity (93% and 96%, respectively). The positive predictive value and negative predictive value of the two-gene panel was 86% and 95%, respectively. Conclusions: Detection of the methylated TWIST1 and NID2 genes in urine sediments using MSP provides a highly (!90%) sensitive and specific, noninvasive approach for detecting primary BCa. Trial registration: BlCa-001 study-EudraCt 2006-003303-40.
4th International …, 2000
The immune system appears to play a role in the development of squamous intraepithelial lesions (... more The immune system appears to play a role in the development of squamous intraepithelial lesions (SIL) and their progression to cancer. We have developed a technique to isolate and cultivate lymphocytes either from the epithelium or from the underlying stroma of small biopsies from the normal exocervix, the transformation zone (TZ) and SIL. The majority of cells derived from the epithelium of all biopsies were CD8 + T cells. No major difference was observed for the lymphocyte phenotype. Among all cytokines tested by ELISA (IL10, IL4 and IFNγ), only IL10 was significantly higher in the TZ in comparison with the exocervix. In cultures derived from the stroma, a decreased percentage in T cells was observed in the TZ and SIL in comparison with the exocervix. This decrease in T cells concerned CD8 + (SIL) and TCRγδ + T cells (TZ and SIL). We did not observe any difference in IL10 or IL4 production, although patients with SIL produced more IFNγ. The higher levels of IL10 production by lymphocytes derived from the epithelium of the TZ might contribute to the predisposition of this region to cervical carcinogenesis.
Virchows Archiv, 1998
The aim of our study was to establish the relevance of an in vitro model for analysing the abili... more The aim of our study was to establish the relevance of an in vitro model for analysing the ability of human lymphocytes to infiltrate human papillomavirus (HPV)-associated (pre)neoplastic lesions of the uterine cervix. To mimic these lesions, we have used the organotypic raft culture of HPV-transformed keratinocytes (SiHa). The SiHa organotypic raft culture was co-cultured with resting or prestimulated (IL-2 or IL-2+anti-CD3 mAb) allogeneic peripheral blood mononuclear cells (PBMC) for 24 and 72 h. The majority of infiltrating cells were T lymphocytes. Occasional NK cells were also identified. The stimulation with IL-2+anti-CD3 mAb induced the highest number of infiltrating cells, with the maximum lymphocyte infiltration observed after 24 h of co-culture. The lymphocyte infiltration was associated with an increased number of apoptotic cells in the organotypic cultures. The ability of PBMC and purified T cell and NK cell populations to lyse HPV-transformed keratinocytes was also inv...
The American Journal of Pathology, 2002
Local cellular immune defects have been described in several tumors including human papillomaviru... more Local cellular immune defects have been described in several tumors including human papillomavirus (HPV)-associated cervical cancer. This observation suggests the potential therapeutic benefit of immune manipulations that restore cellular immunity. Here, we evaluated the ability of bispecific monoclonal antibodies (bimAbs) to redirect T cells against keratinocytes transformed in vitro by HPV in an autologous three-dimensional culture model (organotypic cultures). The epidermal growth factor receptor (EGFR) was chosen as target for an anti-CD3/anti-EGFR bimAb because it is overexpressed in many malignant epithelial lesions and only weakly expressed in the basal layers of normal squamous epithelium. Interestingly, in organotypic cultures, the pattern of expression of EGFR was similar to that observed in vivo. The ability of T cells retargeted by CD3/EGFR bimAb to lyse HPV-transformed cell lines was confirmed in monolayer cultures. In autologous organotypic cultures, an increase in apoptotic HPV+ keratinocytes and a significant decrease in the thickness of HPV+ organotypic cultures were observed when activated lymphocytes and bimAbs were added to the cultures, whereas organotypic cultures of normal keratinocytes were not significantly affected. These data were similar to those obtained in the allogeneic model. These results suggest the potential usefulness of CD3-EGFR bimAb-retargeted lymphocytes in immunotherapeutic protocols for malignant epithelial lesions.
The Journal of Urology, 2011
RESULTS: When injected into the damaged kidney, luciferaselabeled hAFSC can be tracked using biol... more RESULTS: When injected into the damaged kidney, luciferaselabeled hAFSC can be tracked using bioluminescence. Moreover, we show that hAFSC provide a protective effect, ameliorating ATN in the acute injury phase as reflected by decreased creatinine and BUN blood levels and by a decrease in the number of damaged tubules and apoptosis therein, as well as by promoting proliferation of tubular epithelial cells. We show significant immunomodulatory effects of hAFSC, over the course of ATN.
European Urology, 2010
E U R O P E A N U R O L O G Y 5 8 ( 2 0 1 0 ) 9 6 -1 0 4 a v a i l a b l e a t w w w . s c i e n ... more E U R O P E A N U R O L O G Y 5 8 ( 2 0 1 0 ) 9 6 -1 0 4 a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e u r o p e a n u r o l o g y . c o m Abstract Background: Accumulating evidence suggests that DNA methylation markers could serve as sensitive and specific cancer biomarkers. Objective: To determine whether a panel of methylated genes would have the potential to identify primary bladder cancer (BCa) in voided urine samples. Design, setting, and participants: A pharmacologic unmasking reexpression analysis in BCa cell lines was initially undertaken to unveil candidate methylated genes, which were then evaluated in methylation-specific polymerase chain reaction (MSP) assays performed on DNA extracted from noncancerous and cancerous bladder tissues. The most frequently methylated genes in cancerous tissues, with 100% specificity, were retained for subsequent MSP analysis in DNA extracted from urine samples to build and validate a panel of potential methylated gene markers. Urine samples were prospectively collected at three urologic centres from patients with histologically proven BCa and processed for use in real-time MSP and cytologic analysis. Patients with nonmalignant urologic disorders were included as controls. Measurements: A urine sample was classified as valid when !10 copies of the gene encoding ß-actin were measured in the urine sediment genomic DNA. Sensitivity, specificity, and predictive values of the MSP and cytology tests were assessed and compared. Results and limitations: MSP assays performed on 466 of the 496 (94%) valid urine samples identified two genes, TWIST1 and NID2, that were frequently methylated in urine samples collected from BCa patients, including those with early-stage and low-grade disease. The sensitivity of this two-gene panel (90%) was significantly better than that of cytology (48%), with comparable specificity (93% and 96%, respectively). The positive predictive value and negative predictive value of the two-gene panel was 86% and 95%, respectively. Conclusions: Detection of the methylated TWIST1 and NID2 genes in urine sediments using MSP provides a highly (!90%) sensitive and specific, noninvasive approach for detecting primary BCa. Trial registration: BlCa-001 study -EudraCt 2006-003303-40.
American Journal of Reproductive Immunology, 2003
The American Journal of Pathology, 2002
Local cellular immune defects have been described in several tumors including human papillomaviru... more Local cellular immune defects have been described in several tumors including human papillomavirus (HPV)associated cervical cancer. This observation suggests the potential therapeutic benefit of immune manipulations that restore cellular immunity. Here, we evaluated the ability of bispecific monoclonal antibodies (bimAbs) to redirect T cells against keratinocytes transformed in vitro by HPV in an autologous three-dimensional culture model (organotypic cultures). The epidermal growth factor receptor (EGFR) was chosen as target for an anti-CD3/anti-EGFR bimAb because it is overexpressed in many malignant epithelial lesions and only weakly expressed in the basal layers of normal squamous epithelium. Interestingly, in organotypic cultures, the pattern of expression of EGFR was similar to that observed in vivo. The ability of T cells retargeted by CD3/EGFR bimAb to lyse HPV-transformed cell lines was confirmed in monolayer cultures. In autologous organotypic cultures, an increase in apoptotic HPV ؉ keratinocytes and a significant decrease in the thickness of HPV ؉ organotypic cultures were observed when activated lymphocytes and bimAbs were added to the cultures, whereas organotypic cultures of normal keratinocytes were not significantly affected. These data were similar to those obtained in the allogeneic model. These results suggest the potential usefulness of CD3-EGFR bimAb-retargeted lymphocytes in immunotherapeutic protocols for malignant epithelial lesions. (Am J
The Journal of Molecular Diagnostics, 2008
Epigenetic silencing of the DNA repair protein O 6methylguanine-DNA methyltransferase (MGMT) by p... more Epigenetic silencing of the DNA repair protein O 6methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy , such as with temozolomide , in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test , suitable for high-throughput analysis of small amounts of formalin-fixed , paraffin-embedded tumor tissue. A direct , real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the -actin gene , provides a quantitative test result. We analyzed 134 clinical glioma samples , comparing the new test with the previously validated nested gel-based MSP assay , which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results , supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa ؍ 0.80; 95% confidence interval, 0.82؊0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gelbased MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy. (J Mol
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Papers by Isabelle Renard