Search

Supplementary Tables S1-S5.

Purpose: Medullary thyroid carcinoma (MTC), an aggressive rare tumor due to activating mutations in the proto-oncogene RET, requires new therapeutic strategies. Sunitinib, a potent inhibitor of RET, VEGF receptor (VEGFR)-1, VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor (PDGFR)α/β, has been reported as clinically effective in some patients with advanced MTC. In this study, we examine molecular mechanisms of action of sunitinib and identify candidate soluble biomarkers of response.Experimental Design: Both in vitro and in vivo assays, using the human TT RETC634W MTC cell line, were done to assess the activity of sunitinib. Kinetic microarray studies were used to analyze molecular pathways modified by sunitinib and to identify candidate biomarkers that were subsequently investigated in the serum of patients.Results: Sunitinib displayed antiproliferative and antiangiogenic activities and inhibited RET autophosphorylation and activation of downstream signaling pathways. We showed that sunitinib treatment induced major changes in the expression of genes involved in tissue invasion and metastasis including vimentin (VIM), urokinase plasminogen (PLAU), tenascin-C (TN-C), SPARC, and CD44. Analyzing downregulated genes, we identified those encoding secreted proteins and, among them, interleukin (IL)-8 was found to be modulated in the serum of xenografted mice under sunitinib treatment. Furthermore, we demonstrated that metastatic MTC patients presented increased serum levels of IL-8 and TGF-β2.Conclusions: Experimental models confirm the clinical efficacy of sunitinib observed in a few studies. Molecular pathways revealed by genomic signatures underline the impact of sunitinib on tissue invasion. Selected soluble candidate biomarkers could be of value for monitoring sunitinib response in metastatic MTC patients. Clin Cancer Res; 17(7); 2044–54. ©2011 AACR.

Background: We aimed to understand the dependence of MEK and m-TOR inhibition in EGFRWT/ALKnon-rearranged NSCLC cell lines. Methods: In a panel of KRASM and KRASWT NSCLC cell lines, we determined growth inhibition (GI) following maximal reduction in p-ERK and p-S6RP caused by trametinib (MEK inhibitor) and AZD2014 (m-TOR inhibitor), respectively. Results: GI caused by maximal m-TOR inhibition was significantly greater than GI caused by maximal MEK inhibition in the cell line panel (52% vs 18%, P

Simple Summary Cytopathology cannot distinguish benign from malignant follicular lesions in 20–30% of cases. These indeterminate cases includes the so-called follicular neoplasms (FNs) according to The Bethesda System for Reporting Thyroid Cytopathology. Frozen samples from 66 classic follicular adenomas (cFAs) and carcinomas (cFTCs) studied by array-comparative genomic hybridization identified three specific alterations of cFTCs (losses of 1p36.33-35.1 and 22q13.2-13.31, and gain of whole chromosome X) confirmed by fluorescent in situ hybridization (FISH) in a second independent series of 60 touch preparations from frozen samples of cFAs and cFTCs. In a third independent set of 27 cases of already stained pre-operative fine-needle aspiration cytology samples diagnosed as FNs and histologically verified, FISH analysis using these three markers identified half of cFTCs. Specificity of our assay for identifying cFTCs is higher than 98% which might be comparable with BRAF600E testing in cases of suspicion of classic papillary thyroid carcinomas. Abstract Although fine-needle aspiration cytology (FNAC) is helpful in determining whether thyroid nodules are benign or malignant, this distinction remains a cytological challenge in follicular neoplasms. Identification of genomic alterations in cytological specimens with direct and routine techniques would therefore have great clinical value. A series of 153 cases consisting of 72 and 81 histopathologically confirmed classic follicular adenomas (cFAs) and classic follicular thyroid carcinomas (cFTCs), respectively, was studied by means of different molecular techniques in three different cohorts of patients (pts). In the first cohort (training set) of 66 pts, three specific alterations characterized by array comparative genomic hybridization (aCGH) were exclusively found in half of cFTCs. These structural abnormalities corresponded to losses of 1p36.33-35.1 and 22q13.2-13.31, and gain of whole chromosome X. The second independent cohort (validation set) of 60 pts confirmed these data on touch preparations of frozen follicular neoplasms by triple DNA fluorescent in situ hybridization using selected commercially available probes. The third cohort, consisting of 27 archived cytological samples from an equal number of pts that had been obtained for preoperative FNAC and morphologically classified as and histologically verified to be follicular neoplasms, confirmed our previous findings and showed the feasibility of the DNA FISH (DNA fluorescent in situ hybridization) assay. All together, these data suggest that our triple DNA FISH diagnostic assay may detect 50% of cFTCs with a specificity higher than 98% and be useful as a low-cost adjunct to cytomorphology to help further classify follicular neoplasms on already routinely stained cytological specimens.

Participants of the Second International Workshop (WS) on human chorionic gonadotropin (hCG) of the International Society of Oncology and Biomarkers Tissue Differentiation 7 (ISOBM TD-7) have characterized in detail a panel of 69 antibodies (Abs) directed against hCG and hCG-related variants that were submitted by eight companies and research groups. Specificities of the Abs were determined using the First WHO International Reference Reagents for six hCG variants, i.e., hCG, hCGn, hCGβ, hCGβn, hCGβcf, and hCGα, which are calibrated in SI units, and hLH. Molecular epitope localizations were assigned to the ISOBM-mAbs by comparing ISOBM-Ab specificity, sandwich compatibility, and mutual inhibition profiles, to those of 17 reference monoclonal (m)Abs of known molecular epitope specificities. It appeared that 48 Abs recognized hCGβ-, 8 hCGα-, and 13 αβ-heterodimer-specific epitopes. Twenty-seven mAbs were of pan hCG specificity, two thereof with no (>1 %; epitopes β3/5) hLH cross-reactivity. The majority of hCGβ epitopes recognized were located in two major antigenic domains, one on the peptide chain of the tips of β-sheet loops 1 and 3 (epitopes β2–6; 27 mAbs) and the second around the cystine knot (e.g., epitopes β1, β7, and β10; 9 mAbs). Four mAbs recognized epitopes on hCGβcf-only (e.g., epitopes β11 and β13) and six mAbs epitopes on the remote hCGβ-carboxyl-terminal peptide (epitopes β8 and β9 corresponding to amino acids 135–144 and 111–116, respectively). For routine diagnostic measurements, methods are used that either detect hCG-only, hCGβ-only, or hCG together with hCGβ or hCG together with hCGβ and hCGβcf. Sandwich assays that measure hCG plus hCGβ and eventually hCGβcf should recognize the protein backbone of the analytes preferably on an equimolar basis, should not cross-react with hLH and not be susceptible to blunting of signal by nonmeasured variants like hCGβcf. Such assays can be constructed using pairs of mAbs directed against the cystine knot-associated epitope β1 (Asp10, Asp60, and Gln89) in combination with epitopes β2 or β4 located at the top of β-sheet loops 1 + 3 of hCGβ involving aa hCGβ20-25 + 68-77. In summary, the results of the First and Second ISOBM TD-7 WSs on hCG provide the basis for harmonization of specificities and epitopes of mAbs to be used in multifunctional and selective diagnostic hCG methods for different clinical purposes. Electronic supplementary material The online version of this article (doi:10.1007/s13277-013-0994-6) contains supplementary material, which is available to authorized users.

Medullary thyroid carcinoma (MTC) is characterized by proto-oncogene RET mutations in almost all hereditary cases as well as in more than 40% of sporadic cases. Recently, a high prevalence of RAS mutations was reported in sporadic MTC, suggesting an alternative genetic event in sporadic MTC tumorigenesis.This study aimed to extend this observation by screening somatic mutational status of RET, BRAF, and the three RAS proto-oncogenes in a large series of patients with MTC.Direct sequencing of RET (exons 8, 10, 11, 13, 14, 15, 16), BRAF (exons 11 and 15), and KRAS, HRAS, and NRAS genes (exons 2, 3, and 4) was performed on DNA prepared from 50 MTC samples, including 30 sporadic cases.Activating RET mutations were detected in the 20 hereditary cases (germline mutations) and in 14 sporadic cases (somatic mutations). Among the 16 sporadic MTC without any RET mutation, eight H-RAS mutations and five K-RAS mutations were found. Interestingly, nine RAS mutations correspond to mutation hot spots in exons 2 and 3, but the other four mutations were detected in exon 4. The RET and RAS mutations were mutually exclusive. No RAS gene mutation was found in hereditary MTC, and no BRAF or NRAS mutation was observed in any of the 50 samples.Our study confirms that RAS mutations are frequent events in sporadic MTC. Moreover, we showed that RAS mutation analysis should not be limited to the classical mutational hot spots of RAS genes and should include analysis of exon 4.

Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22(phox) that produces H(2)O(2). Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H(2)O(2), we detected an increase in H(2)O(2) in the nucleus correlated with NOX4-p22(phox) perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1β foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence.

Context: Thyroperoxidase (TPO) and dual oxidase (DUOX) are present at the apical membrane of thyrocytes, where TPO catalyzes thyroid hormone biosynthesis in the presence of H2O2 produced by DUOX. Both enzymes are colocalized and associated, but the consequences of this interaction remain obscure.Objective: The objective of this study was to evaluate the functional consequences of TPO-DUOX interaction at the plasma membrane.Design: The functional consequences of DUOX-TPO interaction were studied by measuring extracellular H2O2 concentration and TPO activity in a heterologous system. For this purpose, HEK293 cells were transiently transfected with a combination of human TPO with human DUOX1 or DUOX2 in the presence of their respective maturation factors, DUOXA1 or DUOXA2. The effect of human DUOX2 mutants in which cysteine residues in the N-terminal domain were replaced by glycines was also analyzed.Results: We observed that production of H2O2 decreases both TPO and DUOX activities. We show that TPO presents a catalase-like effect that protects DUOX from inhibition by H2O2. This catalase-like effect depends on the association between both enzymes, which probably occurs through the DUOX peroxidase-like domain because this effect was not observed with human DUOX2 mutants.Conclusion: The DUOX-TPO association at the plasma membrane is relevant for normal enzyme properties. Normally, TPO consumes H2O2 produced by DUOX, decreasing the availability of this substance at the apical membrane of thyrocytes and, in turn, probably decreasing the oxidative damage of macromolecules.

Background: There is a need to develop blood-based bioassays for breast cancer (BC) screening. In this study, differential gene expression between BC samples and benign tumours was used to identify candidate biomarkers for blood-based screening. Methods: We identified two proteins (Fibronectin 1 and CXCL9) from a gene expression data set that included 120 BC samples and 45 benign lesions. These proteins fulfil the following criteria: differential gene expression between cancer and benign lesion, protein released in the extracellular medium and stable in the serum, commercially available ELISA kit, ELISA accuracy in a feasibility study. Protein concentrations were determined by ELISA. Blood samples were from normal volunteers (n=119) and early BC patients (n=133). Results: Seventy-three per cent of patients had cT1-T2 tumour. Patients had higher CXCL9 and Fibronectin 1 concentrations than volunteers. CXCL9 mean concentration was 851 and 635 pg ml−1 for patients and volunteers respectively (P=0.013). CXCL9 concentration was significantly higher in patients with estrogen receptor (ER)-negative compared with volunteers (P=0.003), data consistent with gene expression profile. Fibronectin 1 mean concentration was 190 μg ml−1 for patients and 125 μg ml−1 for volunteers (P

Ionizing radiation (IR) causes not only acute tissue damage, but also late effects in several cell generations after the initial exposure. The thyroid gland is one of the most sensitive organs to the carcinogenic effects of IR, and we have recently highlighted that an oxidative stress is responsible for the chromosomal rearrangements found in radio-induced papillary thyroid carcinoma. Using both a human thyroid cell line and primary thyrocytes, we investigated the mechanism by which IR induces the generation of reactive oxygen species (ROS) several days after irradiation. We focused on NADPH oxidases, which are specialized ROS-generating enzymes known as NOX/DUOX. Our results show that IR induces delayed NADPH oxidase DUOX1-dependent H2O2 production in a dose-dependent manner, which is sustained for several days. We report that p38 MAPK, activated after IR, increased DUOX1 via IL-13 expression, leading to persistent DNA damage and growth arrest. Pretreatment of cells with catalase, a scavenger of H2O2, or DUOX1 down-regulation by siRNA abrogated IR-induced DNA damage. Analysis of human thyroid tissues showed that DUOX1 is elevated not only in human radio-induced thyroid tumors, but also in sporadic thyroid tumors. Taken together, our data reveal a key role of DUOX1-dependent H2O2 production in long-term persistent radio-induced DNA damage. Our data also show that DUOX1-dependent H2O2 production, which induces DNA double-strand breaks, can cause genomic instability and promote the generation of neoplastic cells through its mutagenic effect.

Follicular thyroid carcinomas (FTC) arise through oncogenic pathways distinct from those involved in the papillary histotype. Recently, a t(2;3)(q13;p25) rearrangement, which juxtaposes the thyroid transcription factor PAX8 to the peroxisome proliferator-activated receptor (PPAR) gamma1, was described in FTCs. In this report, we describe gene expression in 11 normal tissues, 4 adenomas, and 8 FTCs, with or without the PAX8-PPARgamma1 translocation, using custom 60-mer oligonucleotide microarrays. Results were confirmed by quantitative real-time polymerase chain reaction of 65 thyroid tissues and by immunohistochemistry. Statistical analysis revealed a pattern of 93 genes discriminating FTCs, with or without the translocation, that were morphologically undistinguishable. Although the expression of thyroid-specific genes was detectable, none appeared to be differentially regulated between tumors with or without the translocation. Differentially expressed genes included genes related to lipid/glucose/amino acid metabolism, tumorigenesis, and angiogenesis. Surprisingly, several PPARgamma target genes were up-regulated in PAX8-PPARgamma-positive FTCs such as angiopoietin-like 4 and aquaporin 7. Moreover many genes involved in PAX8-PPARgamma expression profile presented a putative PPARgamma-promoter site, compatible with a direct activity of the fusion product. These data identify several differentially expressed genes, such as FGD3, that may serve as potential targets of PPARgamma and as members of novel molecular pathways involved in the development of thyroid carcinomas.

The dual oxidase (Duox)2 flavoprotein is strongly expressed in the thyroid gland, where it plays a critical role in the synthesis of thyroid hormones by providing thyroperoxidase with H2O2. DUOX2 mRNA was recently detected by RT-PCR and in-situ hybridization experiments in other tissues, such as rat colon and rat and human epithelial cells from the salivary excretory ducts and rectal glands. We examined Duox2 expression at the protein level throughout the porcine digestive tract and in human colon. Western blot analysis identified Duox2 as the same two molecular species ( Mr 165 and 175 kDa) as detected in the thyroid. It was expressed in all the tissues tested, but the highest levels were found in the cecum and sigmoidal colon. Immunohistochemical studies showed that Duox2 protein is mainly present in these parts of the gut and located at the apical membrane of the enterocytes in the brush border, indicating that it is expressed only in highly differentiated cells. A Ca2+/NADPH-dependent H2O2-generating system was associated with Duox2 protein expression, which had the same biochemical characteristics as the NADPH oxidase in the thyroid. Indeed, treatment of the thyroid and cecum particulate fractions with phenylarsine oxide resulted in complete calcium desensitization of both enzymes. A marked increase in DUOX2 expression was also found during spontaneous differentiation of postconfluent Caco-2 cells. The discovery of Duox2 as a novel source of H2O2 in the digestive tract, particularly in the cecum and colon, makes it a new candidate mediator of physiopathological processes.

Iodide transport by thyrocytes involves two transporters, namely the Na(+)/I (-) symporter located at the basolateral pole and possibly pendrin in the apical membranes of the cell. Recently, we identified a human gene and its protein product, designated hAIT, as a putative new transporter involved in iodide transfer across the apical membrane of thyrocytes. In the present report, we analyzed both hAIT gene and protein expressions in a large series of benign and malignant human thyroid tissues. Using immunohistochemistry, hAIT staining was detected in normal thyroid tissue in about 10% of follicles; in positive follicles, 10-40% of thyrocytes, mostly the tall cells, were stained. In thyroid tissues obtained from patients with Graves' disease and toxic adenomas, hAIT mRNA and protein levels were similar to those found in normal tissue. In hypofunctioning adenomas, hAIT mRNA levels were slightly decreased, and apical iodide transporter (AIT) immunostaining was similar to that observed in normal thyroid tissue. AIT staining was stronger in Hürthle cell adenomas and in microfollicular adenomas. In thyroid carcinomas, the mean and median hAIT mRNA levels were significantly decreased. Expression of AIT protein was undetectable in most papillary carcinomas and was weak but detectable in most follicular carcinomas; it was negative in anaplastic carcinomas.

Summary objective The diagnostic impact of chromogranin A (CgA) measurement has been studied in various neuroendocrine tumours (NET) such as pheochromocytomas, gastrinomas and neuroblastomas. Clinically nonfunctioning pituitary adenomas (NFPA) are generally diagnosed on tumoural symptoms or hypopituitarism and, except for gonadotrophins and their free subunits which may be increased in the case of gonadotrophinomas, markers of endocrine secretory activity are lacking not only for diagnostic purpose but also in the postoperative follow-up of these patients. As the presence of CgA has been demonstrated by immunohistochemistry in pituitary adenomas, we performed this study to further assess the sensitivity of CgA measurement in sporadic pituitary adenomas using a new, specific, sandwich immunoassay. subjects We first completed a basal normative data set obtained using this assay by studying four healthy men (49 ± 13 years old), five healthy premenopausal women (35·8 ± 7·5 years old) and five healthy postmenopausal women (49·1 ± 4·6 years old) basally and after TRH administration. Twenty-seven patients [12 men (64·2 ± 11·8 years), even premenopausal women (38·4 ± 5·7 years) and eight postmenopausal women (67·7 ± 10·3 years)] with NFPA, 15 acromegalic patients [nine men (45 ± 13·3 years), six women (52 ± 14·9 years)] and 19 patients with a prolactin-secreting adenoma [four men (41·2 ± 18 years) and 15 women (31·2 ± 7·5 years), with a macroadenoma (n = 11) or a microadenoma (n = 8)] had basal and TRH-stimulated measurement of CgA. A gonadotrophin-releasing hormone (GnRH)-stimulation test was also performed in two, four and four patients, respectively. All patients had sporadic pituitary adenomas. measurements Serum CgA was measured using a solid-phase two-site immunoradiometric assay based on monoclonal antibodies that bind to two distinct contiguous epitopes within the 145–245 region of CgA. results Mean basal CgA concentration in 14 normal subjects was 80·2 ng/ml (SD: 31·7; range 19–124). A cut-off value for normal range was thus set at 125 ng/ml. TRH injection did not change significantly the CgA levels, peak values remaining less than 124 ng/ml. Three out of 27 subjects with NFPA (11%) had elevated basal CgA levels (576, 143, 241 ng/ml, respectively). Serum levels of CgA were not influenced by TRH in any of the NFPA subjects (including those three with increased basal levels). One out of 15 acromegalic patients (6·6%) and one out of 19 hyperprolactinemic patients (5·2%) had elevated serum basal CgA which did not significantly increase after TRH administration. In the remaining patients TRH-tests did not modify CgA levels. GnRH administration did not modify CgA levels. conclusions CgA serum levels measurement, assessed with a novel assay, does not provide a helpful marker for the clinical management of functioning and NFPA.

Summary Objective and Design Galectin-3 is a β-galactoside-binding protein that plays a role in cell adhesion and tumour progression. It was shown recently to diagnose malignant follicular thyroid lesions accurately. The reliability of this marker in the differential diagnosis between medullary thyroid carcinoma and C-cell hyperplasia was studied by immunohistochemistry. Patients Tissue specimens were obtained from 34 patients belonging to families with medullary thyroid carcinoma who underwent prophylactic thyroidectomy for RET gene mutation and/or abnormally increased plasma calcitonin levels. Results Galectin-3 was expressed in 23 of 25 cases of medullary thyroid carcinoma and in none of the nine cases of C-cell hyperplasia only, giving a sensitivity of 92% and a specificity of 100% for the diagnosis of carcinoma. A significant association was found between higher galectin-3 expression and occurrence of lymph node metastases (P

Iodide transport by thyrocytes is a two step process involving transporters located either in the basal or in the apical membranes of the cell. The sodium iodide symporter (NIS) is localized in the basolateral membrane facing the bloodstream and mediates iodide accumulation into thyrocytes. Pendrin has been proposed as an apical transporter. In order to identify new iodide transporters, we developed a PCR cloning strategy based on NIS sequence homologies. From a human kidney cDNA library, we characterized a gene, located on chromosome 12q23, that encodes a 610 amino acid protein sharing 46% identity (70% similarity) with the human NIS. Functional analysis of the protein expressed in mammalian cells indicates that it catalyzes a passive iodide transport. The protein product was immunohistochemically localized at the apical pole of the thyroid cells facing the colloid lumen. These results suggest that this new identified protein mediates iodide transport from the thyrocyte into the colloid lumen through the apical membrane. It was designated hAIT for human Apical Iodide Transporter.

Radioactive iodine (131I) is routinely used for the treatment of differentiated thyroid cancers. Attempts have been made to enlarge this therapeutic strategy to nonthyroid tumors by coupling radioactive iodide administration with transfer of the sodium iodide symporter (NIS) gene into target cells, for example with an adenoviral vector (AdNIS). Although efficient iodide uptake was achieved in the tumors treated with AdNIS, no therapeutic effect could be observed with 131I, most probably because the iodide retention time in the target cells was short. To circumvent this problem, we propose to organify the iodide taken up, as it occurs in the thyroid. We constructed a recombinant adenovirus encoding the human thyroperoxidase (TPO) gene under the control of the cytomegalovirus early promoter (AdTPO). Infection of nonthyroid tumor cells with this virus led to production of an enzymatically active protein. A significant increase in iodide organification could be observed in cells coinfected with both AdNIS and AdTPO in the presence of exogenous hydrogen peroxide. However, the levels of iodide organification obtained were too low to significantly increase the iodide retention time in the target cells.

Medullary thyroid carcinoma (MTC) arises from parafollicular or C cells of the thyroid that produce calcitonin. It accounts for 5–10% of all thyroid cancers. Hereditary MTC represents 20–30% of all MTCs. It can be transmitted with an autosomal dominant pattern, either as a single entity, familial MTC, or it can arise as part of a multiple endocrine neoplasia (MEN) syndrome type 2A or 2B. The identification of hereditary MTC has been facilitated in recent years by the direct analysis of the ret proto-oncogene.

The ISOBM TD-7 hCG Workshop was established to characterize the molecular epitope structure and specificities of a panel of diagnostically relevant monoclonal antibodies (MAbs) directed against human

Epigenetic control of gene expression plays a major influence in the development and progression of many cancer types. Aim of the present study was to investigate the expression of epigenetic regulators in a large cohort of medullary thyroid carcinomas (MTC), correlating the data with the clinical outcome and mutational status of the patients. Taqman Low Density Arrays (TLDAs) were used to analyze expression levels of several genes involved in the epigenetic control of transcription in a series of 54 MTCs. The patients cohort included 13 familial MTCs and 41 sporadic forms; 33 hosted a RET mutation and 13 a RAS somatic mutation. The expression profiling revealed in the more aggressive diseases (i.e. occurrence of metastases; persistent disease; disease-related death) a significant increase of EZH2 and SMYD3 gene expression. The increased levels of EZH2 and SMYD3 did not correlate significantly with mutational status of RET or RAS genes. Thus, the histone methyltransferases EZH2 and SMYD3 mRNA expression may represent useful prognostic biomarkers tailoring the most appropriate follow-up and timing of therapeutic approaches.

BACKGROUND: The study was conducted to evaluate whether the detection of serum molecular forms of inhibin (A and B) could be useful for the diagnosis, prognosis and follow-up of placental tumours. METHODS: A total of 17 patients with hydatidiform mole (n 13), invasive mole (n 1) or choriocarcinoma (n 3) were studied; serum concentrations of inhibins A and B, human chorionic gonadotrophin (HCG) and its free β subunit (HCGβ) were measured before chemotherapy (after mole evacuation for eight patients) and also during the course of chemotherapy (for 10 patients). RESULTS: After evacuation or before chemotherapy for refractory disease, serum inhibin A and B concentrations were found to be increased in 10/17 and 4/17 patients, when HCG and HCGβ were high in all patients. In 10 patients with a follow-up during treatment, nine had a high concentration of inhibin A which correlated with those of HCG and HCGβ. Normalization of inhibin A was faster than that of HCG and HCGβ for three and six patients respectively. There was no correlation between changes of inhibin B and HCGβ concentrations. CONCLUSIONS: Our results suggest that inhibins A and B are not useful markers and that HCG determination still remains the most useful marker for diagnosis and follow-up of placental tumours.

Recent studies performed with null mice suggested a role of either RXRα or PPARγ in murine placental development. We report here that both PPARγ and RXRα are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPARγ (but not for PPARα or PPARδ) increase both human CGβ transcript levels and the secretion of human CG and its free β-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPARγ ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPARγ/RXRα heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CGβ gene (β5) were found to bind RXRα and PPARγ from human cytotrophoblast nuclear extracts, suggesting that PPARγ/RXRα heterodimers directly regulate human CGβ transcription. Altogether, these data show that PPARγ/RXRα heterodimers play an important role in human placental development.

Thyroid cells synthesize thyroid hormones through a multistep process during which iodide is transported through the basolateral and the apical membranes of thyrocytes. Two genes that participate in these transports and the corresponding proteins, namely sodium iodide symporter (NIS) and pendrin, the product of the Pendred syndrome gene, have recently been characterized. We studied NIS and pendrin expression at the mRNA and protein levels by a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method and by single and double immunostaining in normal and pathological human thyroid tissues. In normal tissue, NIS and pendrin were detected in about 20% and 40%-60% of thyrocytes, respectively. The number of NIS- and pendrin-positive cells was much higher in hyperfunctioning tissue from Graves' disease or toxic adenoma. In hypofunctioning adenomas and carcinomas, the number of NIS- and pendrin-positive cells was low or nonexistent. Three types of follicular cells were observed in positive tissues: NIS-negative/pendrin-negative cells, NIS-positive/pendrin-positive cells, and NIS-negative/pendrin-positive cells. The first two types of cells appear to be resting and active cells, respectively, but the functional status of NIS-negative/pendrin-positive thyrocytes remains to be determined.

The large homolog of NADPH oxidase flavoprotein LNOX2, and probably LNOX1, are flavoproteins involved in the thyroid H2O2 generator. Western blot analysis of membrane proteins from normal human thyroid, using antipeptide antibodies, indicated that LNOX1,2 are 164-kDa glycoproteins and that N-glycosylated motifs account for at least 10–20 kDa of their total apparent molecular mass. Northern blot analysis of 23 different human tissues demonstrated that LNOX2 messenger RNA (mRNA) is strongly expressed only in the thyroid gland, although blast analysis of expressed sequence tags databases indicated that LNOX genes are also expressed in some nonthyroid cells. We investigated LNOX1,2 gene and protein expressions in normal and pathological human thyroid tissues using real-time kinetic quantitative PCR and antipeptide antibodies, respectively. In normal tissue, LNOX1,2 are localized at the apical pole of thyrocytes. Immunostaining for LNOX1,2 was heterogeneous, inside a given follicle, with 40–60% of positive fol...

Summary Since the development of the first immunoassay for circulating chromogranin A in 1984, a lot of studies have evaluated its clinical impact in neuroendocrin e tumors. Initially studied in pheochromocytoma patients, the clinical impact of chromogranin A has rapidly extended to most neuroendocrine tumours, sometimes in combination with other eutopic or ectopic secretions. In our experience, CgA demonstrates a variable sensitivity between NET primary and a high specificity. Our results suggest that CgA should be routinely screened in foregut-derived NET and abandoned in the routine screening of medullary thyroid carcinoma. In addition, in phaeochromocy toma and ileumNET patients, CgA demonstrates a comparable sensitivity with urinary reference markers and its impact on the followup will form a key point when recommending routine screening. Both tumor burden and secretory activity should be taken into account when interpreting CgA results. Neuroendocrine tumors (NET) constitute a tumor network scattered in the body. They are characterized by common features including secretion of hormones, association as part of hereditary syndrome, impact of functioning imaging and, in one subgroup, poor aggressiveness. Secretion of hormones is a main feature of neuroendocrine tumors (NET) with multiple consequences for the diagnosis and prognosis of these tumors. Since 1984, the chromogranin A (CgA), a glycoprotein of the core of storage vesicles, has emerged as a potential general marker of these tumors [1]. CgA plays major roles in the storage and secretion of several hormones within the neuroendocrine cell vesicles. Furthermore, its impact on the pathological diagnosis of NET is well established [2]. This review article summarizes our experience with CgA measurement in the diagnostic biological work-up of NET.

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.

Sex cord stromal tumors are gonadal neoplasms containing Sertoli, granulosa, Leydig, or thecal cells, which originate from cells derived from either the sex cords (Sertoli and granulosa cell tumors) or the specific mesenchymal stroma (Leydig and thecal cell tumors) of the embryonic gonad. Only granulosa and Sertoli cells produce anti-Müllerian hormone (AMH). Our purpose was to investigate whether AMH can be used as a specific marker of human granulosa or Sertoli cell origin in gonadal tumors, to distinguish them from other primary or metastatic neoplasms, using immunohistochemistry. We studied 7 juvenile and 6 adult-type granulosa cell tumors of ovarian localization and 3 extraovarian metastases, 20 other ovarian tumors, 6 testicular Sertoli cell tumors, 2 gonadoblastomas, and 13 extragonadal tumors. Granulosa cell tumors, both juvenile- and adult-type of either ovarian or metastatic localization, showed an heterogeneous pattern of AMH immunoreactivity: Areas containing intensely or weakly AMH-positive cells were intermingled with AMH-negative areas. Although in most cases AMH-positive areas represented a minor proportion of tumor cells, we found a positive reaction in all the cases examined. In testes, although normal prepubertal Sertoli cells were intensely positive, testicular Sertoli cell tumors showed large areas of negative reaction, with few positive cells scattered throughout the tumor. AMH was also reactive in most of the cells of sex-cord origin in gonadoblastomas. No AMH immunoreaction was observed in other gonadal and extragonadal tumors. We conclude that AMH expression is conserved in only a small proportion of tumor cells of granulosa or Sertoli cell origin; however, a positive reaction in a few cells helps to distinguish between granulosa or Sertoli cell tumors or gonadoblastomas and other gonadal tumors of different origin.

Little is known about the physiological secretion of the free beta-subunit of LH (LHbeta). The aim of this study was to compare in women the secretion of LHbeta, using sensitive and specific two-site immunoassays, with dimeric LH and the free common alpha-subunit (FAS). The LHbeta assay does not recognize the dimeric LH and cross-reacts only with free hCG beta-subunit (CGbeta). Thus, all of the plasma samples were also tested with a highly specific immunoradiometric assay for free CGbeta. Molar concentrations (i.e. picomoles per L) were used to compare the plasma levels of LH and its free subunits. Plasma LH, LHbeta, FAS, and CGbeta levels were measured in five normally cycling women during the early follicular phase and the ovulatory peak of LH. The pulsatile profiles of LH, LHbeta, FAS, and CGbeta were studied in five postmenopausal women before and 21 days after injection of a depot preparation of the GnRH agonist D-Trp6 (3.75 mg, im) and in five women with functional hypothalamic amenorrhea (FHA), i.e. low plasma LH levels, during pulsatile GnRH administration (20 microg/pulse, 90 min, sc). Afterward, one of the patients with FHA received a single sc injection of 1350 U recombinant human LH, and plasma LH, LHbeta, FAS, and CGbeta levels were measured and compared with the high plasma levels of one postmenopausal woman. In cycling women, basal plasma LHbeta and CGbeta levels were below the detection limit of the assays (1.34 and 0.65 pmol/L, respectively), and plasma FAS levels were 13.60 +/- 0.13 pmol/L. During the LH surge, there was a parallel increase in LH, LHbeta, and FAS. Plasma CGbeta levels remained undetectable. In normal postmenopausal women, basal plasma dimeric LH, LHbeta, and FAS levels were increased in parallel, and their pulsatile profiles were similar, without measurable plasma CGbeta levels. After D-Trp6 administration, plasma LH and LHbeta levels were completely suppressed, whereas plasma FAS levels increased, and plasma CGbeta remained below 0.65 pmol/L. In FHA women, basal plasma levels of LH and FAS were low, without detectable LHbeta and CGbeta levels. During pulsatile GnRH administration, LHbeta became detectable, and pulses were synchronous with those of LH and FAS. The secretion of LH and LHbeta was almost equimolar. Plasma CGbeta levels remained undetectable. In the patient with FHA, administration of recombinant human LH increased only plasma LH levels, whereas plasma LHbeta and FAS levels remained very low. In conclusion, when the production of dimeric LH increases, a concomitant, parallel, and almost equimolar hypersecretion of uncombined and biologically inactive LHbeta occurs. Like the alpha-subunit, LHbeta may be secreted in the dissociated free form. This can lead to pitfalls during clinical investigations if assays of free CGbeta display some cross-reaction with free LHbeta.

Only a few markers have been instrumental in the diagnosis of cancer. In contrast, tumor markers play a critical role in the monitoring of patients. The patient’s clinical status and response to treatment can be evaluated rapidly using the tumor marker half-life (t1/2) and the tumor marker doubling time (DT). This report reviews the interest of determining these kinetic parameters for prostate-specific antigen, human chorionic gonadotropin, α-fetoprotein, carcinoembryonic antigen, cancer antigen (CA) 125, and CA 15-3. A rise in tumor markers (DT) is a yardstick with which benign diseases can be distinguished from metastatic disease, and the DT can be used to assess the efficacy of treatments. A decline in the tumor marker concentration (t1/2) is a predictor of possible residual disease if the timing of blood sampling is soon after therapy. The discrepancies in results obtained by different groups may be attributable to the multiplicity of immunoassays, the intrinsic characteristics of each marker (e.g., antigen specificity, molecular heterogeneity, and associated forms), individual factors (e.g., nonspecific increases and renal and hepatic diseases) and methods used to calculate kinetics (e.g., exponential models and timing of blood sampling). This kinetic approach could be of interest to optimize patient management.

The expression of 4 thyroid tissue-specific genes [Na+/I- symporter (NIS), thyroid peroxidase (TPO), thyroglobulin (Tg), TSH receptor (TSH-R)] as well as of the glucose transporter type 1 (Glut1) gene was analyzed in 90 human thyroid tissues Messenger ribonucleic acids were extracted from 43 thyroid carcinomas (38 papillary and 5 follicular), 24 cold adenomas, 5 Graves' thyroid tissues, 8 toxic adenomas, and 5 hyperplastic thyroid tissues; 5 normal thyroid tissues were used as reference. A kinetic quantitative PCR method, based on the fluorescent TaqMan methodology and real-time measurement of fluorescence, was used. NIS expression was decreased in 40 of 43 thyroid carcinomas (10- to 1200-fold) and in 20 of 24 cold adenomas (2- to 700-fold); it was increased in toxic adenomas and Graves' thyroid tissues (up to 140-fold). TPO expression was decreased in thyroid carcinomas, but was normal in cold adenomas; it was increased in toxic adenomas and Graves' thyroid tissues Tg expression was decreased in thyroid carcinomas, but was normal in the other tissues. TSH-R expression was normal in most tissues studied and was decreased in only some thyroid carcinomas. In thyroid cancer tissues, a positive relationship was found between the individual levels of expression of NIS, TPO, Tg and TSH-R. No relationship was found with the age of the patient. Higher tumor stages (stages >I vs stage I) were associated with lower expression of NIS (P = 0.03) and TPO (P < 0.01). Expression of the Glut1 gene was increased in 1 of 24 adenomas and in 8 of 43 thyroid carcinomas. In 6 thyroid carcinoma patients, 131I uptake was studied in vivo; NIS expression was low in all samples; 3 patients with normal Glut-1 gene expression had 131I uptake in metastases, whereas the other 3 patients with increased Glut-1 gene expression had no detectable 131I uptake. In conclusion, this study shows 1) a reduced expression of NIS gene in most hypofunctioning benign and malignant thyroid tumors; 2) a differential regulation of the expression of thyroid-specific genes; 3) an increased expression of Glut-1 gene in some malignant tumors that may suggest a role for glucose derivative tracers to detect in vivo thyroid cancer metastases by positron emission tomography scanning.

Purpose: Understanding growth regulation in hormone-refractory prostate cancer may provide avenues for novel treatment interventions. This study was conducted to characterize the expression of the receptor (FSHR) for follicle-stimulating hormone (FSH) in androgen-independent prostate cancer cell lines and in human malignant prostate tissues.Materials and Methods: Western blotting, immunohistochemistry (IHC), and flow cytometric analysis were used to study the expression of FSHR. The effect of FSH on cell growth and clonogenicity was studied using proliferation and clonogenic assays.Results: Immunohistochemistry revealed expression of FSH in PC3 and Du145 cells. FSHR was identified in PC3 and Du145 cells, as well as in human adenocarcinoma of the prostate. The specificity of the FSHR detected on prostate cancer tissues or cells by IHC and Western blotting was confirmed by preabsorbing the antibodies with the immunizing antigens. Stimulation of these hormone-refractory cells with FSH triggered a pro...

Screening was performed in 130 consecutive patients with apparently sporadic neuroendocrine tumors (NET) to assess the prevalence of multiple endocrine neoplasia type 1 (MEN1) and hormonal production. Screening for MEN1 included measurement of serum calcium and PTH[ PTH-(1–84)], gastrin, PRL, and insulin-like growth factor type I (IGF-I) levels. MEN1 genetic testing was performed in patients with two components of the MEN1 syndrome. Screening for hormonal production included measurement of serum neuron-specific enolase (NSE), calcitonin (CT), glycoprotein α-subunit (GPα), hCG β-subunit (free hCGβ), and somatostatin levels. Twenty-four-hour urinary free cortisol (UFC) and 5-hydroxyindolacetic acid (5-HIAA) determinations were also performed. Four patients had hyperparathyroidism, none of whom had pituitary or familial disease. Hyperprolactinemia was compatible with a pituitary disease in one patient. No acromegalic feature or any increase in IGF-I was found. Hypergastrinemia, compatible with an associated ...

Antipeptide antibodies raised against the carboxyl-terminal region of the human sodium/iodide (Na+/I−) symporter (hNIS) were used to investigate by immunohistochemistry the presence and distribution of the hNIS protein in normal thyroid tissues, in some pathological nonneoplastic thyroid tissues, and in different histotypes of thyroid neoplasms. In normal thyroid tissue, staining of hNIS protein was heterogeneous and limited to a minority of follicular cells that were in close contact with capillary vessels. In positive cells, immunostaining was limited to the basolateral membrane. In contrast, in Graves’ disease the majority of follicular cells expressed the hNIS protein. In autoimmune thyroiditis, the number of hNIS-positive cells, was similar to that found in normal tissue. These positive cells were found essentially close to lymphocytic infiltrates. This observation supports the concept of hNIS as an autoantigen. In diffuse nodular hyperplasia, hNIS staining was heterogeneous, but the number of hNIS-positive cells exceeded that found in normal tissue. In well differentiated follicular or papillary carcinoma, the number of hNIS-positive cells was significantly lower than in normal tissue. In poorly differentiated follicular carcinoma, the number of hNIS-positive cells was less than that found in well differentiated carcinoma, or there were no positive cells. Interestingly, in all of these thyroid tissues, the number of follicular cells exhibiting TSH receptor (TSHR) immunoreactivity was greater than the number of hNIS-positive cells. As hNIS expression appears to be related to TSHR stimulation, the decreased number of TSHR-positive cells in cancers may contribute to the reduced capacity of neoplastic cells to concentrate iodide. In one patient with a follicular cancer with an absence of hNIS immunostaining, the total body 131I scan showed no uptake in metastatic tissue. In three cancers with positive hNIS cells, the 131I scan showed uptake in lymph node metastases. This suggests that immunodetection of hNIS could predict radioiodine uptake in thyroid cancers.

The monoclonal origin of gonadotropin-secreting pituitary adenomas has been well demonstrated but only few molecular abnormalities have so far been recognized in these tumors. For many years, several authors have suggested a role for GnRH and/or GnRH receptors (GnRH-R) in the development of these pituitary adenomas. To test the hypothesis that mutant genes encoding a constitutively activated GnRH-R might be involved in the pathogenesis of these tumors, the sequence of the GnRH-R gene was analyzed in tumoral pituitary tissue obtained from ten patients (six female, four male). The pituitary gonadotropin-secreting adenoma was associated with in vivo hypersecretion of FSH, LH and/or free alpha-subunit (n = 7) or was clinically silent (normal plasma levels of gonadotropins or free alpha-subunit, n = 3). In all cases, immunocytochemical studies of the removed adenoma confirmed their gonadotroph nature by revealing positivity for FSH, LH and/or alpha-subunit. Genomic DNA was extracted from the pathological tissue obtained at neurosurgery. Eight sequencing primers were used to amplify the three exons of the GnRH-R gene from tumoral DNA. The entire coding sequence of the GnRH-R gene was sequenced in the ten adenomas. No mutation was found in any of the tumor specimens examined. In conclusion, mutations in the GnRH receptor coding sequence occur infrequently if at all in gonadotropin-secreting pituitary adenomas.

An active recombinant glycoprotein hormone, porcine follicle-stimulating hormone (recFSH), has been produced for the first time in the methylotrophic yeast, Pichia pastoris. The yield of secreted recFSH (10 mg/l) was the highest ever reached. RecFSH displayed an apparent molecular mass of 41 kDa by SDS-PAGE and was found to bear only N-linked carbohydrates of the high-mannose type. Its in vitro binding and cell-stimulating activities were identical to those of pituitary porcine FSH. The large availability and the noncharged N-glycans of FSHrec should render it highly valuable for structural studies.

Both experimental and clinical studies suggest that inhibin plays a critical role in the development of granulosa cell tumors (GCT), a subgroup of malignant ovarian tumors. Inhibin has been proposed as a biological marker for the follow-up of patients bearing these particular tumors. Hitherto, there is no general agreement on the molecular form(s) of the inhibin family that are secreted by malignant granulosa cells. Using specific and sensitive immunoassays for activin A and for inhibins A and B, we investigated the production of these molecules in patients with either an adult GCT (n=13) or an epithelial ovarian cancer (n=11). Results showed that serum activin A level was increased in all patients, independently of their clinical status (progressive disease or remission) in comparison to that observed in the healthy pre- and postmenopausal women. Most of the patients also presented a moderate increase in serum inhibin A level compared to that in controls. Only one of eight patients with a progressive granulosa cell tumor had a high value of serum inhibin A. In contrast, serum inhibin B was dramatically increased in eight of nine patients with a granulosa cell tumor and its level correlated with the clinical status of the patients. No correlation was found between the level of serum inhibin B and that of serum antimüllerian hormone, a recently described specific and reliable marker for GCT. None of the patients with an epithelial ovarian cancer presented an increase of serum inhibin B. These observations demonstrate that inhibin B is the major molecular form of the inhibin family proteins produced by malignant granulosa cells.

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.

Circulating neuron-specific enolase (NSE) and chromogranin A (CgA) were measured in 128 patients with neuroendocrine tumours (NET) to compare their sensitivity and specificity, to investigate factors associated with elevated serum levels and to determine the usefulness of these markers in the follow-up of NET patients. NSE (Cispack NSE, Cis Bio International, Gif-sur-Yvette, France; normal

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.

Most clinically nonfunctioning pituitary adenomas (NFPA) are found to be gonadotropinomas when assessed by immunocytochemistry. However, they are rarely associated with increased basal plasma levels of FSH, LH and/or alpha-subunit. It has been claimed that the paradoxical free LHbeta response to TRH may be a useful clinical tool for determining the gonadotropic nature of NFPA. We used a very specific and sensitive immunoradiometric assay (IRMA) for free LHbeta measurement and another specific IRMA to check the absence of free CGbeta, to study normal subjects and 26 patients with NFPA. Basal plasma levels of LHbeta were undetectable in normal men and premenopausal women in the early follicular phase. In contrast, normal postmenopausal women had increased basal plasma LHbeta, parallel to dimeric LH and alpha-subunit levels. In healthy subjects, stimulation with GnRH elicited an increase in LHbeta while TRH was ineffective. In patients with NFPA, LHbeta hypersecretion was found basally and/or after stimulation with TRH in 3 of 16 men, 3 of 5 premenopausal women, and 1 of 5 postmenopausal women, i.e. 7 of 26 patients (26%). In 3 of these 7 cases, alpha-subunit and/or FSH levels were also increased. The LHbeta measurement was thus truly informative on the gonadotropic nature of NFPA in only 4 out of 26 cases (15%). In addition, increased LHbeta levels and/or a positive response of free LHbeta to TRH was observed in 3 patients with pure prolactinomas but in no patients with GH-secreting adenomas. Thus, using this very sensitive and specific IRMA, free LHbeta measurement is rarely helpful for determining the gonadotropic nature of NFPA.

As a glycoprotein hormone, human chorionic gonadotropic (hCG) is not a single molecular entity but this term rather comprises an array of molecular variants such as hCG, hCG beta, hCGn, hCG beta n, hCG beta cf, -CTPhCG, hCG beta CTP, deglyhCG, asialohCG, hCGav and the closely related molecules hLH, hLH beta and hLH beta ef. The advent of monoclonal antibodies (MCA), the availability of ultrasensitive detection systems and the recent determination of the crystal structure of hCG, made it possible to design special purpose diagnostic and clinical research immunoassays for hCG-like molecules. For more than a decade we and others have tried to refine epitope maps for hCG and related molecules by means of a large panel of MCA, naturally occurring metabolic variants of hCG (hCGn, hCG beta, hCG alpha, hCG beta cf, hCG beta CTP), homologous hormones and subunits of various species (e.g. hLH, hLH beta, hFSH, hTSH, oLH, rLH beta), chemically modified molecules (deglyhCG, asialohCG, tryptic and chymotryptic hCG beta and hCG alpha fragments) and synthetic peptides (octapeptides and longer). It appeared that all epitopes on molecular hCG-variants recognized by our MCA are determined by the protein backbone. Except for the two major epitopes on hCG beta CTP and parts of two antigenic domains on hCG alpha, epitopes on hCG-derived molecules are determined by the tertiary and quarternary structure. Operationally useful descriptive epitope maps were designed including information on assay suitability of antigenic determinants. On this basis we established ultrasensitive time-resolved fluoroimmuno-assays for hCG, hCG and hCGn, hCG beta and hCG beta n and hCG beta cf, hCG alpha and additional assays recognizing different spectra of hCG-variants. Such assay have been applied by us and others to the detection of pregnancy, early pregnancy loss, choriocarcinoma, testicular cancer, other cancers and prenatal diagnosis. However, as the molecular structure of many epitopes utilized in immunoassays of different laboratories was not resolved, comparability of results was not satisfactory. Consequently, attempts were made to compare schematic epitope maps from different research institutions. The situation has been much improved by solving the three-dimensional (3D) structure of hCG. It has been shown that hCG is a member of the structural superfamily of cystine knot growth factors like NGF, PDGF-B and TGF-beta. Each of its subunits is stabilized in its topology by three disulfide bonds forming a cystine knot. Moreover, it turned out that the disulfide bridges in their majority have previously been wrongly assigned. Computer molecular modeling of crystallographic coordinates of hCG and subsequent selective combined--PCR-based and immunological--mutational analyses of hCG beta expressed via the transmembrane region of a MHC molecule made it possible to more precisely localize epitopes on hCG-derived molecules. Although the entire surface of hCG has to be regarded as potentially immunogenic there seems to be hot spots where epitopes are clustered in antigenic domains. These are located on the first and third loops protuding from the cystine knots of both subunits and are possibly centered around the knot itself. Ultimate answers on epitope localizations will be given by the crystal structure determination of hCG complexed with different Fabs.

Resume L'auteur resume l'essentiel des caracteristiques immunochimiques de l'hCG et des formes moleculaires circulantes en vue d'eclairer les difficultes methodologiques et de preciser les indications des differents analytes.

Efflux transporters play an important role in the resistance of tumor cells against anticancer agents. Interaction between these transporters, including P-glycoprotein (P-gp), and drugs might influence their pharmacological properties and toxicities. The aim of this study was to investigate whether vandetanib (Caprelsa(®)), a small tyrosine kinase inhibitor, could interact with the multidrug transporter P-gp. Interaction of vandetanib with the P-gp was investigated using the parental cell line (IGROV1) and the P-gp doxorubicin-resistant (IGROV1-DXR) cell line, derived from the parental drug-sensitive IGROV1 cells. Cytotoxicity tests were assessed in both cell lines to examine the impact of P-gp on the cell survival after a vandetanib treatment. The effects of P-gp on vandetanib intracellular pharmacokinetics were investigated. To this aim, we developed a quantitative liquid chromatography tandem mass spectrometry to quantify vandetanib in cell medium. Results showed that overexpression of P-gp confers resistance to vandetanib in the IGROV1-DXR cell line. Using a LC-MS/MS assay validated in cell medium, cellular pharmacokinetic studies revealed that in cells overexpressing the P-gp intracellular concentrations of vandetanib were decreased compared to parental cell line. For the first time, vandetanib is described as a substrate of P-gp. In tumor cells, P-gp could be responsible for cellular resistance to vandetanib. It may be relevant to the clinical efficacy of vandetanib. Moreover, interaction of vandetanib with P-gp could modify the pharmacodynamics of other conventional chemotherapeutics, substrates of P-gp. It could impact on the overall response to anticancer therapy.

Context The placenta plays an essential role in the fetomaternal exchanges of iodine and thyroid hormones. Propylthiouracil (PTU) is presently considered to be the treatment of choice for hyperthyroidism during the first trimester of pregnancy. Little is known on the expression of iodide transporters in invasive human trophoblast and the possible effect of PTU on this early phase of human placental development. Objective To analyze during early pregnancy expression of sodium/iodide symporter (NIS) and pendrin at the feto–maternal interface in situ in first trimester placentas, in vitro during human trophoblastic cell differentiation in presence or not of PTU. Design NIS and pendrin immunodetection were performed on 8–10 WG placental tissue sections and in primary cultures of first trimester placenta trophoblastic cells, which differentiate in vitro into syncytiotrophoblast or invasive extravillous cytotrophoblasts (EVCT). The effect of PTU (1 mM) was tested in EVCT on iodide transporters expression, cell invasion, and hCG secretion. Results NIS and pendrin were present in early human trophoblast at the maternofetal interface. Their expression was modulated with in vitro trophoblast differentiation. Early invasive EVCT were characterized by higher expression of NIS than pendrin. In vitro PTU did modify significantly neither EVCT iodide transporters expression nor EVCT biological functions: i.e. invasive properties and hCG secretion. Conclusion This study reveals that NIS is highly expressed in early human trophoblast at the feto–maternal interface. PTU has no effect on early human trophoblast invasion.

The molecular basis for antigenic determinants on the free β-subunit of human chorionic gonadotrophin (hCGβ), its carboxyl-terminal peptide (hCGβCTP) and the hCGβcore fragment (hCGβcf) was elucidated by means of monoclonal antibodies (MCAs). The objective of the present study was to resolve the antigenic topography of these three molecules in terms of epitope identification at different levels of structural organization as well as analysis of their spatial arrangement. An hCGβcf preparation, a synthetic peptide corresponding to the hCGβCTP (β109–145), overlapping synthetic peptides spanning the entire amino acid sequence of hCGβ, and a reduced and alkylated hCGβ preparation were assayed in a solid-phase one-site enzyme-linked immunoassay and in a solublephase direct-binding radioimmunoassay (RIA) or competitive RIA. The antigenic topography was mapped by incorporating the MCAs into two-site binding assays. On the surface of free hCGβ, nine different epitopes (β1–β9), arranged in three spatially distinct domains, could be distinguished. Epitopes β1–β7 were located in a single large domain on both hCGβ and the hCGβcf whereas hCGβCTP contained two topographically distant determinants, designated β8 and β9 respectively. All but the two epitopes located on hCGβCTP (β8 and β9) were destroyed by reducing and alkylating hCGβ, suggesting that most antigenic determinants are predominantly non-contiguous and require an intact tertiary structure whereas the molecular structure of hCGβCTP is linear. At a molecular level, amino acid residues spanning hCGβ 45–52, hCGβ 137–144 and hCGβ 113–116 contributed to the formation of epitopes β5, β8 and β9 respectively. We have also shown that the hCGβcf represents the immunodominant part of the free β-subunit of hCG, containing seven mainly conformationally determined epitopes, one of which has a share of the sequence β45–52. The hCGβCTP does not play a critical role in the immunologically important tertiary structure of hCGβ and was itself found to be a predominantly continuous sequence also within the native hormone, expressing two spatially distant antigenic determinants located within residues β113–116 and β137–144 respectively. Journal of Endocrinology (1994) 141, 153–162