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Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease. [Cancer Res 2007;67(1):398–407]

Supplementary Material and Methods from Establishment of Human Colon Cancer Cell Lines from Fresh Tumors versus Xenografts: Comparison of Success Rate and Cell Line Features

International audience; Cancer and malaria exemplify two maladies historically assigned to separated research spaces. Cancer, on the one hand, ranks among the top priorities in the research agenda of developed countries. Its rise is mostly explained by the ageing of these populations and linked to environment and lifestyle. Malaria, on the other hand, represents a major health burden for developing countries in the Southern Hemisphere. These two diseases also belong to separate fields of medicine: non-communicable diseases for cancer and communicable diseases for malaria.

International audience; Background - We aimed to assess the prognostic value of CYFRA 21-1 in a series of patients with anal canal squamous cell carcinoma treated by radiation-based therapy. Methods - All patients with anal cancer referred between September 2005 and July 2013 were considered. Patients with diagnosis of anal squamous cell carcinoma and in whom pre- and post-treatment serum CYFRA 21-1 levels were available were included. Serum CYFRA 21-1 levels at initial workup and after therapy were collected. Survival rates were estimated using the Kaplan-Meier method. Cox regression analysis was used to evaluate prognostic variables for prediction of outcomes. Results - Eighty-two patients were included. Median follow-up was 60 months (range: 8-128). Pre-treatment serum CYFRA 21-1 levels were significantly correlated with tumour stage (p

BackgroundThe placenta relies on phenotypes that are characteristic of cancer to successfully implant the embryo in the uterus during early pregnancy. Notably, it has to invade its host tissues, promote angiogenesis, while surviving hypoxia, and escape the immune system. Similarities in DNA methylation patterns between the placenta and cancers suggest that common epigenetic mechanisms may be involved in regulating these behaviors.ResultsWe show here that megabase-scale patterns of hypomethylation distinguish first from third trimester chorionic villi in the placenta, and that these patterns mirror those that distinguish many tumors from corresponding normal tissues. We confirmed these findings in villous cytotrophoblasts isolated from the placenta and identified a time window at the end of the first trimester, when these cells come into contact with maternal blood as the likely time period for the methylome alterations. Furthermore, the large genomic regions affected by these patterns of hypomethylation encompass genes involved in pathways related to epithelial-mesenchymal transition (EMT), immune response and inflammation. Analyses of expression profiles corresponding to genes in these hypomethylated regions in colon adenocarcinoma tumors point to networks of differentially expressed genes previously implicated in carcinogenesis and placentogenesis, where nuclear factor kappa B (NF-kB) is a key hub.ConclusionTaken together, our results suggest the existence of epigenetic switches involving large-scale changes of methylation in the placenta during pregnancy and in tumors during neoplastic transformation. The characterization of such epigenetic switches might lead to the identification of biomarkers and drug targets in oncology as well as in obstetrics and gynecology.

International audience; The placenta relies on phenotypes that are characteristic of cancer to successfully implant the embryo in the uterus during early pregnancy. Notably, it has to invade its host tissues, promote angiogenesis—while surviving hypoxia—, and escape the immune system. Similarities in DNA methylation patterns between the placenta and cancers suggest that common epigenetic mechanisms may be involved in regulating these behaviors. We show here that megabase-scale patterns of hypomethylation distinguish first from third trimester chorionic villi in the placenta, and that these patterns mirror those that distinguish many tumors from corresponding normal tissues. We confirmed these findings in villous cytotrophoblasts isolated from the placenta and identified a time window at the end of the first trimester, when these cells come into contact with maternal blood, as the likely time period for the methylome alterations. Furthermore, the large genomic regions affected by these patterns of hypomethylation encompass genes involved in pathways related to epithelial-mesenchymal transition, immune response, and inflammation. Analyses of expression profiles corresponding to genes in these hypomethylated regions in colon adenocarcinoma tumors point to networks of differentially expressed genes previously implicated in carcinogenesis and placentogenesis, where nuclear factor kappa B is a key hub. Taken together, our results suggest the existence of epigenetic switches involving large-scale changes of methylation in the placenta during pregnancy and in tumors during neoplastic transformation. The characterization of such epigenetic switches might lead to the identification of biomarkers and drug targets in oncology as well as in obstetrics and gynecology.

Inflammatory breast cancer (IBC) is the most aggressive type of breast cancer with a poor prognosis. Locoregional staging is based on dynamic contrast-enhanced (DCE) CT or MRI. The aim of this study was to compare the performances of FDG PET/CT and DCE CT in locoregional staging of IBC and to assess their respective prognostic values. The study group comprised 50 women (median age: 51 ± 11 years) followed in our institution for IBC who underwent FDG PET/CT and DCE CT scans (median interval 5 ± 9 days). CT enhancement parameters were net maximal enhancement, net early enhancement and perfusion. The PET/CT scans showed intense FDG uptake in all primary tumours. Concordance rate between PET/CT and DCE CT for breast tumour localization was 92 %. No significant correlation was found between SUVmax and CT enhancement parameters in primary tumours (p > 0.6). PET/CT and DCE CT results were poorly correlated for skin infiltration (kappa = 0.19). Ipsilateral foci of increased axillary FDG uptake were found in 47 patients (median SUV: 7.9 ± 5.4), whereas enlarged axillary lymph nodes were observed on DCE CT in 43 patients. Results for axillary node involvement were fairly well correlated (kappa = 0.55). Nineteen patients (38 %) were found to be metastatic on PET/CT scan with a significant shorter progression-free survival than patients without distant lesions (p = 0.01). In the primary tumour, no statistically significant difference was observed between high and moderate tumour FDG uptake on survival, using an SUVmax cut-off of 5 (p = 0.7 and 0.9), or between high and low tumour enhancement on DCE CT (p > 0.8). FDG PET/CT imaging provided additional information concerning locoregional involvement to that provided by DCE CT on and allowed detection of distant metastases in the same whole-body procedure. Tumour FDG uptake or CT enhancement parameters were not correlated and were not found to have any prognostic value.

Background: Ex vivo colospheres have been previously characterised as a colorectal cancer (CRC) well-rounded multicellular model, exclusively formed by carcinoma cells, and derived from fresh CRC tissue after mechanical dissociation. The ability to form colospheres was correlated with tumour aggressiveness. Their three-dimensional conformation prompted us to further investigate their potential interest as a preclinical cancer tool. Methods: Patient-derived CRC xenografts were used to produce numerous colospheres. Mechanism of formation was elucidated by confocal microscopy. Expression analysis of a panel of 64 selected cancer-related genes by real-time qRT–PCR and hierarchical clustering allowed comparison of colospheres with parent xenografts. In vitro and in vivo assays were performed for migration and chemosensitivity studies. Results: Colospheres, formed by tissue remodelling and compaction, remained viable several weeks in floating conditions, escaping anoikis through their strong cell–cell interactions. Colospheres matched the gene expression profile of the parent xenograft tissue. Colosphere-forming cells migrated in collagen I matrix and metastasised when subrenally implanted in nude mice. Besides, the colosphere responses to 5-fluorouracil and irinotecan, two standard drugs in CRC, reproduced those of the in vivo original xenografts. Conclusion: Colospheres closely mimic biological characteristics of in vivo CRC tumours. Consequently, they would be relevant ex vivo CRC models.

International audience; Background: The aim of this study is to evaluate a new tumour marker, HE4, and to compare it with CA125 in predicting optimal cytoreduction and response to chemotherapy. Thirty patients with advanced epithelial ovarian cancer and multiple sera harvested during neoadjuvant chemotherapy (NAC) were included. Results: Based on ROC curves analysis, CA125 ≤ 75 UI/ml and HE4 ≤ 252 pmol/L after the 3rd cycles of NAC, with a sensitivity of 93.7 % and a specificity of 92.3 % (PPV = 93.7 % and NPV = 92.3 %), offered the best combination for predicting optimal cytoreduction. In addition, the HE4 value of 115 pmol/L is the best cut-off level for identifying platinum-sensitive patients. Conclusions: The introduction of HE4 as a new tool for predicting platinum-sensitivity and interval optimal cytoreduction is promising.

International audience; Specific human papillomavirus genotypes are associated with most ano-genital carcinomas and a large subset of oro-pharyngeal carcinomas. Human papillomavirus DNA is thus a tumour marker that can be detected in the blood of patients for clinical monitoring. However, data concerning circulating human papillomavirus DNA in cervical cancer patients has provided little clinical value, due to insufficient sensitivity of the assays used for the detection of small sized tumours. Here we took advantage of the sensitive droplet digital PCR method to identify circulating human papillomavirus DNA in patients with human papillomavirus-associated carcinomas. A series of 70 serum specimens, taken at the time of diagnosis, between 2002 and 2013, were retrospectively analyzed in patients with human papillomavirus-16 or human papillomavirus-18-associated carcinomas, composed of 47 cases from the uterine cervix, 15 from the anal canal and 8 from the oro-pharynx. As negative controls, 18 serum samples from women with human papillomavirus-16-associated high-grade cervical intraepithelial neoplasia were also analyzed. Serum samples were stored at -80 degrees C (27 cases) or at -20 degrees C (43 cases). DNA was isolated from 200 mu l of serum or plasma and droplet digital PCR was performed using human papillomavirus-16 E7 and human papillomavirus-18 E7 specific primers. Circulating human papillomavirus DNA was detected in 61/70 (87%) serum samples from patients with carcinoma and in no serum from patients with cervical intraepithelial neoplasia. The positivity rate increased to 93% when using only serum stored at -80 degrees C. Importantly, the two patients with microinvasive carcinomas in this series were positive. Quantitative evaluation showed that circulating viral DNA levels in cervical cancer patients were related to the clinical stage and tumour size, ranging from 55 +/- 85 copies/ml (stage I) to 1774 +/- 3676 copies/ml (stage IV). Circulating human papillomavirus DNA is present in patients with human papillomavirus-associated invasive cancers even at sub-clinical stages and its level is related to tumour dynamics. Droplet digital PCR is a promising method for circulating human papillomavirus DNA detection and quantification. No positivity was found in patients with human papillomavirus-associated high grade cervical intraepithelial neoplasia.

Malignant tumours have the remarkable property to express cell surface antigens. Pressman was first reporting that radiolabeled antibodies were capable of organ localization. It was a promising challenge but the expected success and the development of this imaging method was limited by a poor imaging resolution despite a rather good specificity of the antibodies used. Identification of key cell surface markers is opening a new era as potential molecular imaging biomarkers in oncologic applications. Antibodies production has been promoted by the development of engineered fragments with preserved immunological properties and pharmacokinetics optimized for molecular imaging. A good compromise has to be obtained between the biological properties of the antibody and the physical half-life of the radionuclide. Several positron emission tomography (PET) radionuclides such as iodine-124, copper-64, yttrium-86 or zirconium-89 have been the focus of recent immuno-PET studies with inter- esting informative images in preclinical and clinical stud- ies. Thanks to the development of more sensitive new detectors and specific software, molecular imaging meth- ods, particularly PET imaging, allow nowadays the detec- tion of lesions smaller than 5 mm in human. Immuno-PET can potentially be used for tumour detection and identifi- cation at diagnosis, staging and restaging, for treatment selection and monitoring, and during follow-up. Moreover the availability of matched imaging or therapeutic radio- nuclide pairs, such as 124 I/ 131 I, 64 Cu/ 67 Cu and 86 Y/ 90 Y, make easier the quantification of tissue uptake and dosimetry calculation for radioimmunotherapy.

Hybrid systems associating the sharpness of anatomic images coming from computed tomography (CT) and radionuclide functional imaging (SPET or PET) are opening a new era in oncology. This multimodal imaging method is now routinely used for the diagnosis, extent, follow up, treatment response and detection of occult disease in different types of malignancies with a significant impact on the treatment strategy leading for a change for more than 68% of all investigated patients.

BACKGROUND: Breast cancer recurrence is often suspected on tumor marker rising in asymptomatic patients. The value of fluorine-18 fluorodeoxyglucose (18FDG)–positron emission tomography/computed tomography (PET/CT) imaging to detect recurrence and its subsequent impact on patient management were retrospectively assessed. METHODS: PET/CT scans were performed on 228 asymptomatic patients (mean, 60.8 years; range, 30-91 years) presenting with rising CA 15-3 and/or CEA serum levels. RESULTS: PET/CT scans were positive in 181 patients (79.5%) and normal in 47 patients, whereas 187 true recurrences were diagnosed. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of PET/CT imaging for detection of breast cancer recurrence were 93.6%, 85.4%, 96.7%, 74.5%, and 92.1%, respectively. When compared with the standard workup available in 67 patients, PET/CT imaging had a higher sensitivity and accuracy (94.5% vs 33% and 94% vs 48%, respectively). Recurrences were confirmed by pathology, conventional imaging techniques, or radiological and clinical follow-up beyond 1 year (mean, 34 months; range, 12-67 years) in 32, 130, and 25 patients, respectively. The diagnosis of recurrence led to a treatment modification in 123 patients (54%). CONCLUSIONS: 18FDG-PET/CT imaging is an efficient technique to detect breast cancer recurrence suspected on tumor marker rising in asymptomatic patients. It may thus contribute to improve patient management, providing an earlier diagnosis with complete whole-body staging as a “one-stop shop” procedure. Cancer 2011. © 2010 American Cancer Society.

Inflammatory disorders account for a significant percentage of gynecologic disease, particularly in reproductive age women. Inflammation is a basic method by which we respond to infection, irritation, or injury. Inflammation is now recognized as a type of nonspecific immune response, either acute or chronic. In gynecology, inflammation leads to anatomic disorders primarily as a result of infectious disease; however inflammation can affect ovulation and hormone production as well as be associated with endometriosis. Similarly, immune cell trafficking is an important component of cyclic endometrial development in each menstrual cycle. These immune cells are required for endometrial function, producing a vast array of inflammatory cytokines. Inflammation alters endometrial receptivity, however it may also play a role in tissue repair and remodeling. Finally, inflammation affects the trophoblast and trophoblast-endometrial interaction. Some components of the immune response are required for optimal fertility and normal tissue remodeling. A better understanding of the necessary role of inflammation in reproduction will allow more rational and targeted treatment of inflammatory disorders in reproductive medicine.

International audience; Background: The sequence of the beta-subunit of human chorionic gonadotropin (hCG beta varies depending on whether hCG beta is encoded by type I or type II genes. Type II genes are upregulated in trophoblast and cancer but hCG beta can be detected in the serum of nonpregnant women and healthy individuals. We aimed to determine whether monoclonal antibody (mAb) FBT11-II specifically detects hCG beta encoded by type II genes (type II hCG beta). Methods: Competitive inhibition assays with synthetic peptides, immunocytochemical and immunohistochemical studies, type II hCG beta dosing immunoassays and sequencing of CGB genes were performed. Results: Competitive inhibition assays determined that mAb FBT11-II recognizes the type II hCG beta derived peptide. CGB mRNA sequencing of JEG-3 (trophoblastic) and 124 (bladder) cell lines confirmed that JEG-3 expresses type II genes while T24 expresses exclusively type I. FBT11-II only recognizes JEG-expressed hCG beta. Placenta immunohistochemical studies confirmed that type II hCG beta expression is restricted to the syncytiotrophoblast. Immunoassays detected type II hCG beta in serum of patients with either nontrophoblastic cancers or fetal Down syndrome. Conclusion: Type II gene expression can be detected using FBT11-II. This specific recognition could improve the clinical usefulness of assays aimed at either managing aggressive tumors or screening for Down syndrome.

Au cours des deux dernieres decennies, le nombre de nouveaux cas de cancers en France a augmente de 63% : en 2000, 278 000 nouveaux cas de cancers ont ete diagnostiques contre 170 000 en 1980 (chiffres de l’Institut national de veille sanitaire, bulletin epidemiologique hebdomadaire du 21 octobre 2003). Si des progres notables ont ete faits dans la prise en charge de la maladie cancereuse tant au niveau du diagnostic que du traitement, le nombre de deces par cancer ne cesse d’augmenter dans notre pays et, en 2000, le cancer a ete responsable de 150 000 deces. Dans ce contexte, il faut rappeler qu’en France comme partout en Europe ou aux Etats-Unis, la survenue de metastases reste la principale cause de deces chez les malades atteints de cancer (1). De ce fait, l’etude de la biologie des metastases devrait etre un des objectifs majeurs de la recherche en cancerologie, et le developpement de nouveaux moyens diagnostiques et therapeutiques des metastases apparait essentiel. Cet article a pour objectif de faire le point sur les avancees recentes de nos connaissances sur la biologie des metastases et de voir comment ces avancees peuvent conduire au developpement de nouveaux examens biologiques pour predire la survenue de metastases.

Molecular markers enabling the prediction of sensitivity/resistance to rapamycin may facilitate further clinical development of rapamycin and its derivatives as anticancer agents. In this study, several human ovarian cancer cell lines (IGROV1, OVCAR-3, A2780, SK-OV-3) were evaluated for susceptibility to rapamycin-mediated growth inhibition. The differential expression profiles of genes coding for proteins known to be involved in the mTOR signaling pathway, cell cycle control and apoptosis were studied before and after drug exposure by RT-PCR. In cells exposed to rapamycin, we observed a dose-dependent downregulation of CCND1 (cyclin D1) and CDK4 gene expression and late G1 cell cycle arrest. Among these cell lines, SK-OV-3 cells resistant to both rapamycin and RAD001 were the sole to show the expression of the anti-apoptotic gene Bcl-2. Bcl-2/bclxL-specific antisense oligonucleotides restored the sensitivity of SK-OV-3 cells to apoptosis induction by rapamycin and RAD001. These results indicate that baseline Bcl-2 expression and therapy-induced downexpression of CCND1 and CDK4 may be regarded as molecular markers enabling the prediction and follow-up of the cellular effects on cell cycle and apoptosis induction of rapamycin in ovarian cancer. Furthermore, strategies to down regulate Bcl-2 in ovarian cancer may prove useful in combination with rapamycin or RAD001 for ovarian cancer.

Once initiated, invasion of trophoblast cells must be tightly regulated, particularly in early pregnancy. The mechanisms necessary for the invasion and migration of trophoblast cells are thought to be related to those involved in the invasive and metastatic properties of cancer cells. Quantitative PCR was used to measure, in trophoblast cells, the transcriptional expression profiles of four genes, INSL4, BRMS1, KiSS-1 and KiSS-1R, reported to be implicated in tumor invasion and metastasis. Laser capture microdissection and purification of trophoblast cells demonstrate that, as already known for INSL4, BRMS1, KiSS-1 and KiSS-1R are expressed by the trophoblast subset of placental tissues. Expression profiles of these genes studied in early placentas (7-9 weeks, n=55) and term placentas (n=11) showed that expression levels of BRMS1 are higher in term than in early placentas, while expression levels of KiSS-1R are higher in early than in term placentas. Low levels of expression of BRMS1 were observed in normal pregnancies, in molar pregnancies and in choriocarcinoma cell lines BeWo, JAR and JEG3 while, in striking contrast, the expression levels of INSL4, KiSS-1 and Kiss-1R were increased in both early placentas and molar pregnancies and were reduced in choriocarcinoma cells. These transcriptional expression profiles are in favor of a predominant role of INSL4, KiSS-1 and KiSS-1R in the control of the invasive and migratory properties of trophoblast cells.

Immunotherapy of cancer has always been a very attractive fourth-modality therapeutic approach. Over the past few years, advances in the identification of tumor antigens have offered new perspectives and provided new opportunities for more accurate immunotherapy for cancer. However, when applied to patients with established tumors, it rarely leads to an objective response. This is partly due to the fact that tumors evade host immunity at both the induction and effector phases. Thus, understanding tumor escape mechanisms may be the key to successful immunotherapy for cancer. In the present review, we will focus on how the expression of killer Ig receptors (KIR) on tumor infiltrating lymphocytes can compromise their function and how tumors evade apoptotic death - two additional mechanisms of tumor escape.

In order to assess the effect of in vitro models on the expression of key genes known to be implicated in the development or progression of cancer, we quantified by real-time quantitative PCR the expression of 28 key genes in three bladder cancer tissue specimens and in their derived cell lines, studied either as one-dimensional single cell suspensions, two-dimensional monolayers or three-dimensional spheroids. Global analysis of gene expression profiles showed that in vitro models had a dramatic impact upon gene expression. Remarkably, quantitative differences in gene expression of 2–63-fold were observed in 24 out of 28 genes among the cell models. In addition, we observed that the in vitro model which most closely mimicked in vivo mRNA phenotype varied with both the gene and the patient. These results provide evidence that mRNA expression databases based on cancer cell lines, which are studied to provide a rationale for selection of therapy on the basis of molecular characteristics of a patient's tumour, must be carefully interpreted. British Journal of Cancer (2002) 86, 1283–1289. DOI: 10.1038/sj/bjc/6600239 www.bjcancer.com © 2002 Cancer Research UK

To investigate the influence of tumor cell architecture on T-cell activation, we used an autologous human model based on 2 bladder tumor cell lines as targets for cytotoxic tumor-infiltrating lymphocytes (TILs). These tumor cell lines were grown in vitro as either standard 2-dimensional (2D) monolayers or 3-dimensional (3D) spheroids. T-cell activation was determined by measuring the production of three major cytokines (tumor necrosis factor, granulocyte/macrophage colony-stimulating factor and interferon-gamma), known to be secreted by most activated TILs. Changes in the architecture of target cells from 2D to 3D induced a dramatic decrease in their capacity for stimulating TILs. Interestingly, neither TIL infiltration nor MHC class I, B7.1 costimulatory or lymphocyte function-associated factor-3 adhesion molecule downregulation played a major role in this decrease. These findings demonstrate that tumor architecture has a major impact on T-cell activation and might be implicated in the escape of tumor cells from the immune system.

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA in peripheral blood leukocytes (PBLs) of bone marrow transplantation patients. Unlike other teams, we quantified CMV and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using a plasmid containing both sequences as an external standard. Tenfold serial dilutions of this plasmid yielded overlapping standard curves that allowed the quantification of CMV and GAPDH gene copies in an efficient and accurate manner. Sequential blood samples (164 specimens) were collected from 16 patients. PBLs were tested by the pp65 antigenemia assay and quantitative CMV and GAPDH gene PCRs. CMV DNA was detected by PCR in 13 patients a mean of 15 days prior to the appearance of antigenemia. The administration of anti-CMV drugs led to a rapid decrease in the numbers of viral copies and positive nuclei. Real-time PCR assay results correlated with those of the CMV pp65 antigenemia assay ( P < 0.00001). The TaqMan assay may be a useful tool for rapid quantification of CMV infection and for monitoring of CMV reactivation in bone marrow transplantation recipients.

Unite´ de Pharmacologie Chimique et Ge´ne´tique et Imagerie, Universite´ Paris Descartes; Unite´ 1022, Insti-tut National de la Sante´ et de la Recherche Me´dicale; Unite´ Mixte de Recherche 8151, Centre National de la RechercheScientifique; and Ecole Nationale Supe´rieure de Chimie de Paris, Paris, France

Placental iodide transport is critical for the fetal thyroid function, but the molecular mechanisms of this transport are not understood. The expression of two recently identified iodide transporters, namely the sodium/iodide symporter (NIS) and pendrin, the product of the gene responsible for the Pendred syndrome (PDS), was studied using real-time kinetic quantitative PCR and immunohistochemistry 1) in placental tissues collected at different gestational ages and 2) in primary cultures of villous cytotrophoblast cells (VCT) that differentiate and fuse over 2-3 days in vitro to form villous syncytiotrophoblast (VSCT) cells. Both NIS and PDS genes are expressed in placenta, albeit at low levels compared with those in thyroid tissue. NIS gene expression in placental samples from first trimester and term pregnancies was similar. In contrast, the expression of PDS gene was higher in term than in first trimester pregnancy samples. In vitro, NIS gene was expressed at a high level in VCT obtained from first trimester pregnancy, and its expression decreased by 3- to 4-fold during the differentiation of VCT in VSCT. Expression of NIS was lower (up to 30-fold) in VCT obtained in placental samples from third trimester than from first trimester pregnancy. In contrast, the expression of PDS gene was low in VCT and increased by 5- to 10-fold during VSCT formation; this was observed in cells isolated from placental samples of both first trimester and term pregnancies. Immunohistochemical analysis showed that NIS protein was present on the entire membrane of VCT, whereas pendrin was mainly located at the brush border membrane of VSCT, facing the mother. In conclusion, 1) NIS and PDS genes are differently expressed in the placenta during gestation; and 2) whereas pendrin is expressed at the brush border membrane of syncytiotrophoblast cells, NIS protein is mainly located in the cytotrophoblast layer.

The syncytiotrophoblast (ST) is one of the major components of the human placenta, as it is involved in feto-maternal exchanges and the secretion of pregnancy-specific hormones. The aim of this study was to elucidate the formation and function of the ST in trisomy 21 (Down's syndrome). We first used the in vitro model of cytotrophoblast differentiation into ST. Cytotrophoblasts were isolated from 15 trisomy 21-affected placentas (12-35 weeks gestation) and 10 gestational age-matched control placentas. In vitro cytotrophoblasts isolated from normal placenta fused to form the ST. This was associated with an increase in transcript levels and in the secretion of hCG, human placental lactogen, placental GH, and leptin. In trisomy 21-affected placentas, we observed a defect (or a delay) in ST formation and a dramatic decrease in the synthesis and secretion of these hormones compared to those in cultured cells isolated from control age-matched placentas. These results were confirmed by a significant (P < 0.001) decrease in gene expression in total homogenates of trisomy 21-affected placentas compared to controls. These results will be of help in understanding the maternal hormonal markers of fetal trisomy 21 and the consequences of placental defects for fetal development.

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Polypeptide growth factors play an important role in the regulation of human embryonic development. Insulin-like 4 gene (INSL4) is a member of the insulin family, which includes insulin, IGF-I, IGF-II, relaxin, and INSL3. Using RT-PCR, we previously found abundant INSL4 mRNA in the human placenta. In this study, we examined the chronology and spatial expression of this gene in sections of human placenta and conceptus by means of in situ hybridization. Expression of the IGF-II gene was studied as a positive control. INSL4 distribution was tissue- and cell-specific. Indeed, INSL4 mRNA was most abundant in syncytiotrophoblast cells. In fetal tissues, INSL4 mRNA was identified in the perichondrium of all four limbs, vertebrae, and ribs. Moreover, INSL4 mRNA was abundant in interbone ligaments. These findings indicate that the INSL4 gene may play an important role in trophoblast development and regulation of bone formation. IGF-II mRNA, in agreement with the literature, are mainly located in the mesodermal core in the villous trophoblast and in most embryonic tissues. Mol. Reprod. Dev. 51:123-129, 1998. © 1998 Wiley-Liss, Inc.

Human lutropin (hLH) exhibits both carbohydrate and peptidic heterogeneities that affect its biological potency and the duration of its activity in vivo. Peptidic changes within the hLH beta-subunit are characterized as intrachain proteolytic nicking and carboxyl terminus heterogeneity. To date, the carboxyl terminus of hLHbeta appears to end at either position Gln114 or Gly117, as determined by sequencing of purified subunit. Furthermore, the complementary DNA for hLHbeta predicts a protein containing an additional peptidic stretch, which would make the beta-subunit 121 residues long. This extension may be responsible for the particular intracellular behavior of hLHbeta. To investigate the carboxyl terminus polymorphism of natural hLHbeta, monoclonal antipeptide antibodies were raised against a synthetic peptide mimicking the 104-119 portion of hLHbeta. One antibody, designated LHP09, was found to specifically react with the recombinant hLHbeta ending at position hLHbeta[Leu119] but not with other recombinant forms ending at [Ser116], [Phe120] or [Leu121]. Immunochemical analysis of hLH, either pituitary or urinary in origin, indicated that only pituitary hLH contains a Leu119-ending form of hLHbeta. Finally, immunohistochemical detection was performed using LHP09 and showed specific staining of a normal adult pituitary gland. These observations support the in vivo existence of intrapituitary molecular forms of hLHbeta ending at various positions between Gln114 and Leu121.

Recently, a new member of the insulin gene superfamily termed insulin-like 4 (INSL4) was identified in the human placenta and uterus. The present study investigated whether placenta translates INSL4 mRNA into a putative peptide named early placenta insulin-like (EPIL). Among antibodies elicited against the C chain of pro-EPIL, one antibody (AB7381) was specifically directed against the C chain 59-88 portion, and among those elicited against the A and B chains of EPIL, one antibody (Ab1661) was directed against the A chain 115-139 and the B chain 23-52 portions. Immunohistochemistry based on antibody 7381 to pro-EPIL and antibody 1661 to EPIL demonstrated that the cytotrophoblast from early placenta preferentially expresses the pro-EPIL peptide, whereas the EPIL peptide is expressed by both the cytotrophoblast and the syncytiotrophoblast. At term, the pro-EPIL peptide was detected in villous cytotrophoblast cells, whereas the EPIL peptide was not detected. Moreover, in vitro experiments performed on term placenta showed that the steady state levels of INLS-4 mRNA in the cytotrophoblast are 10 times (one log unit) lower than in the differentiated villous syncytiotrophoblast cells. Taken together, these findings reveal that expression of EPIL peptides in the villous cytotrophoblast is different from that displayed by the syncytiotrophoblast. Finally, these data are the first demonstration that INSL4 mRNA are translated into pro-EPIL and EPIL peptides.

Tumor-infiltrating lymphocytes (TIL) were grown from 23 urothelial carcinomas. Phenotyping analysis showed that the TIL cultures were mainly CD3+. Although CD4+ and CD8+ T-cell sub-sets were grown in culture, CD4+ T-cell sub-sets predominated over CD8+ T cells. Immunohistochemical studies performed on 5 tumor specimens confirmed this observation, and indicated that CD4+ T cells surrounded the tumor islets, whereas CD8+ T lymphocytes were localized among the tumor cells. Five short-term carcinoma cell lines established from these urothelial tumors were used as target cells in cytolysis assays in order to investigate the functional anti-tumor activity of autologous TIL. TIL from 4/5 tumors were lytic and 3 TIL lines displayed MHC-class-I-dependent cytotoxicity directed against autologous tumor cells. CD4+ T-cell-depletion experiments performed on TIL line 07 confirmed that CD8+ MHC-class-I-dependent CTL were the predominant effectors. Finally, experiments performed on 6 allogeneic urothelial-cancer cell lines matched for HLA-class-I molecules showed that TIL07 exhibited selective lytic activity toward tumor 07. These data indicate that CD8+ MHC-class-I-dependent CTL present in urothelial carcinomas are functional and may participate in the anti-tumor immune response.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alterations found in human tumors. There are mainly point mutations that lead to single amino acid substitutions. The mutated proteins have a longer half-life than wild-type p53 and accumulate in the nucleus of tumor cells. Anti-p53 antibodies have been found in sera of patients with several types of cancers including breast cancer. This report describes a T cell immune response in three patients with breast tumors who had mutated p53 gene and accumulated p53 protein. All showed a humoral response to p53 protein and the T cells of these patients recognized the wild-type p53 protein and proliferated in response to it. The data reported here are relevant to the immune processes leading to autoimmunity and have a bearing on anti-p53 vaccine development in tumor immunology.

Identifying common patterns of regulation in cancer and placental cells might shed new light on cellular programs allowing aggressive tumor development. Indeed, as it has been described for the first time more than a century ago, cancer and placental cells (trophoblasts) share astonishingly similar phenotypes : migration and invasion, immune escape, and angiogenesis induction. These common phenotypes, relying on common genomic sequences and transcriptomic profiles, may result from a shared epigenomic regulation program. This might be especially true for tumor cells leading eventually to metastasis and cytotrophoblasts (a trophoblasts subset) during placental implantation at the beginning of pregnancy. In order to investigate such common epigenomic patterns, we carried out comparisons of DNA methylation in cancer and placental cell genomes. This study involved: cancer samples (primary tumor vs. normal tissue) across various tissues (including breast, colon, liver, lung, prostate, thyroid, uterus), on one hand; and placenta samples (early vs. late term) either heterogeneous (chorionic villi) or homogenous (ex vivo cytotrophoblasts), on the other hand. Cancer data were data downloaded from The Cancer Genome Atlas web portal. Placenta data were downloaded from the Gene Expression Omnibus web portal. In addition, our group generated original data from ex vivo cytotrophoblasts samples. All data were generated on the Illumina Infinium 450K array. Data analysis was carried out thanks to computational methods for epigenomics recently described. This first direct comparison of cancer and placental cells epigenomes, leveraging both published data and original data, led to the identification of large hypomethylated blocks common to cancer and placental cells. Such common patterns have recently been described as a universal defining epigenetic alteration in human solid tumors. These megabase-scale DNA methylation marks differentiate primary tumors from normal tissues. Likewise, they differentiate early term placentas from late term placentas. Moreover, genes belonging to genomic regions displaying common large hypomethylated blocks overlapping in cancers and placentas are enriched for pathways involved in migration and invasion, immune escape, and angiogenesis induction. Common DNA methylation patterns in cancer and placental cells identified in this pilot study might contribute to the epigenomic regulation of cellular programs allowing aggressive tumor development. Further analyses of these common patterns, as well as analyses of differences in cancer and placental cell epigenomes, could eventually lead to the identification of critical epigenomic switches that prevent healthy placentas to degenerate into tumors, while they allow aggressive tumors to develop. Ultimately, such epigenomic switches could also represent innovative targets in oncology. Citation Format: Akpeli V. Nordor, Sophie Richon, Thierry Fournier, Dominique Bellet, Virginie Dangles-Marie, Martin J. Aryee. Common DNA methylation patterns in cancer and placental cells involved in migration and invasion, immune escape, and angiogenesis induction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2762.

Inflammatory breast cancer (IBC) is the most aggressive type of breast cancer with a poor prognosis. Locoregional staging is based on dynamic contrast-enhanced (DCE) CT or MRI. The aim of this study was to compare the performances of FDG PET/CT and DCE CT in locoregional staging of IBC and to assess their respective prognostic values.The study group comprised 50 women (median age: 51 ± 11 years) followed in our institution for IBC who underwent FDG PET/CT and DCE CT scans (median interval 5 ± 9 days). CT enhancement parameters were net maximal enhancement, net early enhancement and perfusion.The PET/CT scans showed intense FDG uptake in all primary tumours. Concordance rate between PET/CT and DCE CT for breast tumour localization was 92%. No significant correlation was found between SUVmax and CT enhancement parameters in primary tumours (p0.6). PET/CT and DCE CT results were poorly correlated for skin infiltration (kappa = 0.19). Ipsilateral foci of increased axillary FDG uptake were found in 47 patients (median SUV: 7.9 ± 5.4), whereas enlarged axillary lymph nodes were observed on DCE CT in 43 patients. Results for axillary node involvement were fairly well correlated (kappa = 0.55). Nineteen patients (38%) were found to be metastatic on PET/CT scan with a significant shorter progression-free survival than patients without distant lesions (p = 0.01). In the primary tumour, no statistically significant difference was observed between high and moderate tumour FDG uptake on survival, using an SUVmax cut-off of 5 (p = 0.7 and 0.9), or between high and low tumour enhancement on DCE CT (p0.8).FDG PET/CT imaging provided additional information concerning locoregional involvement to that provided by DCE CT on and allowed detection of distant metastases in the same whole-body procedure. Tumour FDG uptake or CT enhancement parameters were not correlated and were not found to have any prognostic value.

The purpose of this pilot study was to assess whether outcome in metastatic or recurrent breast cancer patients is related to metabolic response to endocrine therapy determined by (18)F-FDG PET/CT.The study group comprised 22 patients with breast cancer (age 58 ± 11 years, mean ± SD) who were scheduled to receive endocrine therapy. They were systematically assessed by PET/CT at baseline and after a mean of 10 ± 4 weeks for evaluation of response after induction. All patients demonstrated FDG-avid lesions on the baseline PET/CT scan. The metabolic response was assessed according to EORTC criteria and based on the mean difference in SUV(max) between the two PET/CT scans, and the patients were classified into four groups: complete or partial metabolic response, or stable or progressive metabolic disease (CMR, PMR, SMD and PMD, respectively). All patients were followed in our institution.Metastatic sites were localized in bone (n = 15), lymph nodes (n = 11), chest wall (n = 3), breast (n = 5), lung (n = 3), soft tissue (n = 1) and liver (n = 1). PMR was observed in 11 patients (50%), SMD in 5 (23%) and PMD in 6 (27%). The median progression-free survival (PFS) times were 20, 27 and 6 months in the PMR, SMD and PMD groups, respectively. PFS in the SMD group differed from that in the PMR and SMD groups (p0.0001).Metabolic response assessed by FDG PET/CT imaging in patients with metastatic breast cancer treated with endocrine therapy is predictive of the patients' PFS.

Hybrid systems associating the sharpness of anatomic images coming from computed tomography (CT) and radionuclide functional imaging (SPET or PET) are opening a new era in oncology. This multimodal imaging method is now routinely used for the diagnosis, extent, follow up, treatment response and detection of occult disease in different types of malignancies with a significant impact on the treatment strategy leading for a change for more than 68% of all investigated patients.

Resume Le systeme immunitaire assure les defenses de l'organisme. Il produit des anticorps qui neutralisent les bacteries, des cellules qui tuent les cellules infectees par les virus et d'autres types de molecules et de cellules actives. La mise en place de ces reponses defensives, efficaces et specifiques est une mecanique complexe. Elle exige la cooperation precise entre divers types cellulaires, des contacts de cellule a cellule par l'intermediaire de recepteurs presents a leur surface et l'echange de messages moleculaires. Ces cooperations cellulaires permettent de rendre compte du fonctionnement du systeme immunitaire dans son ensemble.

The equine (e) placental glycoprotein hormone eCG plays a critical though not completely understood role during the first trimester of gestation in mares. In the present work, we have developed immunoradiometric assays (m-IRMAs) for detection of eCG, eCG alpha, and eCG beta using combinations of monoclonal antibodies (mAbs) specific for epitopes that reside on free and/or combined subunits. The free eCG alpha m-IRMA was based on AHT20 mAb, specific for the free alpha-subunit of all species, and 125I-labeled ECG01 mAb, which recognizes both free and combined alpha-subunit from equine and primate species. The free eCG beta was measured by the combination of FBT11 mAb, which binds to free beta-subunit from human and equine species, and radiolabeled 518B7 mAb, which detects CG/LH from diverse mammalian species. This assay provided a better sensitivity for eLH beta than for eCG beta. However, after treatment with neuraminidase, the latter molecule was recognized as well as eLH beta, indicating that the carbohydrate structure influenced the binding of mAbs. Detection of eCG was based on the combination of ECG01 mAb (anti-alpha) as capture antibody and 125I-labeled 518B7 mAb (anti-beta). Using such assays, we measured plasma and urinary concentrations of both eCG and free subunits in pregnant mares from mating to day 90 of gestation. eCG was constantly detectable in the serum between days 40-90, as previously reported, but small amounts of the dimeric hormone in the urine were also detectable. Further, m-IRMA specific for the free beta-subunit showed that low levels (5-100 ng/ml) of eCG beta may coexist with eCG in serum and urine during early pregnancy in mares. In contrast, free eCG alpha subunit was undetectable during the first 10 weeks of gestation. These results suggested that eCG and free subunit production in pregnant mares at the beginning of gestation is similar to that observed in pregnant women. These immunoassays, specific for either intact hormone or its free subunits, constitute useful diagnostic tools for investigating reproductive problems in mares.

Background Multicellular tumour sphere models have been shown to closely mimic phenotype characteristics of in vivo solid tumours, or to allow in vitro propagation of cancer stem cells (CSCs). CSCs are usually characterized by the expression of specific membrane markers using flow cytometry (FC) after enzymatic dissociation. Consequently, the spatial location of positive cells within spheres is not documented. Confocal microscopy is the best technique for the imaging of thick biological specimens after multi-labelling but suffers from poor antibody penetration. Thus, we describe here a new protocol for in situ confocal imaging of protein expression in intact spheroids. Methods Protein expression in whole spheroids (150 μm in diameter) from two human colon cancer cell lines, HT29 and CT320X6, has been investigated with confocal immunostaining, then compared with profiles obtained through paraffin immunohistochemistry (pIHC) and FC. Target antigens, relevant for colon cancer and with different expression patterns, have been studied. Results We first demonstrate that our procedure overcomes the well-known problem of antibody penetration in compact structures by performing immunostaining of EpCAM, a membrane protein expressed by all cells within our spheroids. EpCAM expression is detected in all cells, even the deepest ones. Likewise, antibody access is confirmed with CK20 and CD44 immunostaining. Confocal imaging shows that 100% of cells express β-catenin, mainly present in the plasma membrane with also cytoplasmic and nuclear staining, in agreement with FC and pIHC data. pIHC and confocal imaging show similar CA 19-9 cytoplasmic and membranar expression profile in a cell subpopulation. CA 19-9+ cell count confirms confocal imaging as a highly sensitive method (75%, 62% and 51%, for FC, confocal imaging and pIHC, respectively). Finally, confocal imaging reveals that the weak expression of CD133, a putative colon CSC marker, is restricted to the luminal cell surface of colorectal cancer acini, with CD133+ cellular debris into glandular lumina. Conclusion The present protocol enables in situ visualization of protein expression in compact three-dimensional models by whole mount confocal imaging, allowing the accurate localization and quantification of cells expressing specific markers. It should prove useful to study rare events like CSCs within tumour spheres.

Neuroendocrine (NE) cancers are usually suspected on clinical symptoms related to their metabolically active peptide secretion into the circulatory system. The most effective treatment is surgery and a preoperative accurate localization of these slow growing tumors is needed. Combined anatomical (CT) and molecular imaging modalities using single-photon emission computed tomography (SPET) with radiolabeled pentetreotide have been developed in routine, and we report here the potential of SPECT/CT image fusion for diagnosis, staging, and evaluation of treatment efficacy of NE cancers.

Antibodies were elicited against a synthetic peptide which encompassed two different regions of the human lutropin beta-subunit (hLH-beta). These antibodies were raised against either the peptide which was assembled using a conventional approach and conjugated to the tetanus toxoid, or with the peptide assembled using the multiple antigen peptide system approach. Automated simultaneous synthesis of the two forms of the immunizing peptide was successfully achieved. Animal injected with the peptide conjugated to tetanus toxoid produced high titers of antibodies to the synthetic peptide, but did not bind to the native hLH-beta subunit. In contrast, antisera induced by the peptide in its MAP form displayed reactivity with both the peptide and the native hLH-beta subunit; these latter antisera appeared to preferentially recognize the beta 47-55 portion of the molecule and were able to bind to the beta-subunit of human choriogonadotropin. Present results demonstrate that the beta 47-55 region is accessible to antibody binding and appears to be located at the surface of both hLH-beta and hLH. Moreover, this study confirms that the MAP approach provides a chemically unambiguous method for obtaining antibodies of predetermined specificity, capable of recognizing cognate sequences of various native proteins.

In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.

Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease. [Cancer Res 2007;67(1):398–407]